WO2018095314A1 - 鉴定hd-hook效应样本和免疫测定的方法、系统、试剂盒及装置 - Google Patents

鉴定hd-hook效应样本和免疫测定的方法、系统、试剂盒及装置 Download PDF

Info

Publication number
WO2018095314A1
WO2018095314A1 PCT/CN2017/112145 CN2017112145W WO2018095314A1 WO 2018095314 A1 WO2018095314 A1 WO 2018095314A1 CN 2017112145 W CN2017112145 W CN 2017112145W WO 2018095314 A1 WO2018095314 A1 WO 2018095314A1
Authority
WO
WIPO (PCT)
Prior art keywords
sample
tested
antibody
antigen
reading
Prior art date
Application number
PCT/CN2017/112145
Other languages
English (en)
French (fr)
Inventor
杨阳
张向辉
练子富
刘贵东
吴栋杨
赵卫国
刘宇卉
李临
Original Assignee
北京科美生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201611034237.7A external-priority patent/CN108132344B/zh
Priority claimed from CN201611026623.1A external-priority patent/CN108152505B/zh
Priority claimed from CN201611034252.1A external-priority patent/CN108204959B/zh
Priority claimed from CN201710695530.6A external-priority patent/CN109406498B/zh
Application filed by 北京科美生物技术有限公司 filed Critical 北京科美生物技术有限公司
Priority to US16/462,968 priority Critical patent/US20190353664A1/en
Priority to EP17874359.7A priority patent/EP3546937A4/en
Priority to KR1020197014563A priority patent/KR102220361B1/ko
Priority to JP2019547751A priority patent/JP6980800B2/ja
Publication of WO2018095314A1 publication Critical patent/WO2018095314A1/zh
Priority to ZA2018/05983A priority patent/ZA201805983B/en

Links

Images

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B25/00—ICT specially adapted for hybridisation; ICT specially adapted for gene or protein expression
    • G16B25/10—Gene or protein expression profiling; Expression-ratio estimation or normalisation

Definitions

  • the invention relates to the field of photoexcited chemiluminescence technology, in particular to a method for identifying a HD-HOOK effect sample, a system, a kit and a device for identifying an HD-HOOK effect in an immunoassay, an immunoassay method, A system, kit and device for identifying immunoassays.
  • Immunological detection is based on the principle of antigen-antibody-specific reaction. Because it can display or signal the analyte by means of isotopes, enzymes, chemiluminescent substances, etc., it is often used to detect trace organisms such as proteins and hormones. Active substance.
  • Chemiluminescence immunoassay is a non-radioactive immunoassay technology that has developed rapidly in recent years. Its principle is to use a chemiluminescent substance to amplify the signal, and to directly measure the immunological binding process by means of its luminescence intensity. This method has become an immunology. One of the important directions of detection.
  • Photoexcimer chemiluminescence is one of the commonly used methods of chemiluminescence analysis technology. It can be used to study the interaction between biomolecules, and is mainly used for the detection of diseases in clinic.
  • the technology integrates research in the fields of polymer microparticle technology, organic synthesis, protein chemistry and clinical testing. It combines the photosensitive particles and the luminescent particles in a certain range to generate the energy of the ion oxygen energy, and emits an optical signal, thereby detecting the sample to be tested.
  • the photosensitive particles are filled with a photosensitive compound
  • the luminescent particles are filled with a luminescent compound and a lanthanoid.
  • the photosensitive particles Under the excitation of a red laser (600-700 nm), the photosensitive particles release a single-state oxygen ion (4 ⁇ S) in a high-energy state with a propagation distance of about 200 nm.
  • the singlet oxygen ions released by the photosensitive particles can reach the luminescent particles, and emit a high-energy 520-620 nm light through a series of chemical reactions, which is detected by the instrument.
  • the concentration of the particles is low, the collision probability is small, and the background signal is weak. Only when the photosensitive particles and the luminescent particles are combined by an immune reaction will they emit significant light, so the sensitivity of the system is high.
  • the commonly used detection mode consists of three to four components: luminescent particles coated with antigen or antibody, biotin or digoxigenin-labeled antigen or antibody, avidin or anti-digoxigen-coated sensitization Microparticles, neutralizing antigens or antibodies, etc.
  • the above components are combined with the antigen or antibody to be tested by two or more incubation reactions, and the samples to be tested are qualitatively or quantitatively detected by the intensity of the chemiluminescence.
  • it Compared with traditional enzyme-linked immunoassay methods, it has the characteristics of homogeneous, high sensitivity, easy operation and automation. Therefore, its application prospects are very broad.
  • the high-dose-hook effect refers to the high-dose section of the dose-response curve in the double-site sandwich immunoassay.
  • the linear trend is not a plate-like infinite retardation, but a downward curve, like a Only hooks lead to false negatives.
  • the HD-HOOK effect occurs frequently in immunoassays, and its incidence accounts for about 30% of positive samples. Due to the presence of the HD-HOOK effect, the sample to be tested cannot be correctly distinguished because its concentration exceeds the linear range of the detection kit or the concentration itself is such a value that the experimental misdiagnosis, especially leading to an increase in the false negative rate.
  • the high dose-hook effect may result in a low detection signal, and the sample is thus interpreted as a low concentration.
  • the previous solution was to increase the composition of the reagent, to dilute the sample to be tested, or to perform a two-step test.
  • the detection range was broadened mainly by optimizing antibodies or increasing antibodies. Wai.
  • the routine detection procedure has the following five steps: adding the analyte and reagent to the reaction well, incubating the first step, adding the universal solution, the second step of incubation, and reading.
  • the detection method of the invention is based on a conventional detection process, and the signal value is read multiple times in the reaction process without interrupting the reaction, and the true concentration of the sample is judged by observing the change of the signal.
  • the object of the present invention is to provide an immunoassay method.
  • the method of the present invention broadens the detection range by two readings without interrupting the reaction, and can accurately determine whether the sample to be inspected is There is a HOOK effect and the concentration of the analyte in the sample to be tested is calculated simply and quickly.
  • a first aspect of the invention provides a method for identifying a HD-HOOK effect sample, the method comprising the steps of: chemiluminescent immunization of a calibrator, a peak calibrator, a sample to be tested containing the antigen (or antibody) to be tested Reaction, excitation and recording of the first and second readings of chemiluminescence, recording the increase A of the difference between the second and first readings of the peak calibrator as R0, comparing the second sum of the sample to be tested Whether the increase A' between the first readings is greater than R0, if the sample is larger than R0, the sample has the HD-HOOK effect, and if it is less than R0, it does not have the HD-HOOK effect.
  • the method comprises the following steps:
  • peak calibrator refers to a sample containing a specific concentration of a test substance, wherein the high dose section of the dose response curve of the analyte of the double-antibody sandwich immunoassay, when the linear orientation begins to bend downward
  • concentration is the concentration of the analyte in the peak calibrator.
  • the two reading A' values of the sample to be tested are compared with R0. If A' is greater than or equal to R0, the sample to be tested is an HD-HOOK effect sample, which needs to be diluted; if A' is smaller than R0, the sample concentration is directly calculated using the calibration curve;
  • the calibration curve is a curve based on the first reading of the calibrator and the concentration of the calibrator.
  • the luminescent particles refer to polymer particles filled with a luminescent compound and a lanthanide compound; the photosensitive particles are polymer particles filled with a photosensitive compound, which can generate a single line under the excitation of a red laser. Oxygen ions.
  • red light is irradiated with 600 to 700 nm to detect the amount of light emitted from the reaction solution; and the detection wavelength of the emitted light is 520 to 620 nm.
  • the antigen refers to a substance having immunogenicity
  • the antibody refers to an immunoglobulin produced by the body capable of recognizing a specific foreign substance
  • the first antibody and the second antibody means An antibody that specifically binds to the antigen of interest
  • the first antigen and the second antigen refer to an antigen that specifically binds to the antibody of interest.
  • a second aspect of the invention provides a system for identifying an HD-HOOK effect in an immunoassay, the system comprising:
  • An immunoreaction device for performing a chemiluminescent immune response An immunoreaction device for performing a chemiluminescent immune response
  • Chemiluminescence immune response excitation and counting device for exciting and recording the first and second readings of chemiluminescence
  • a processor for determining the presence of an HD-HOOK effect sample based on a difference A' between the second and first readings of the sample to be tested.
  • the system comprises:
  • An immunoreaction device for performing a chemiluminescent immune response An immunoreaction device for performing a chemiluminescent immune response
  • Chemiluminescence immune response excitation and counting device for exciting and recording the first and second readings of chemiluminescence
  • a processor for comparing whether the difference A' between the second and first readings of the sample to be tested is greater than the increase R0 of the difference between the second and first readings of the peak calibrator, if If it is larger than R0, the sample has the HD-HOOK effect, and if it is smaller than R0, it does not have the HD-HOOK effect.
  • the second reading of chemiluminescence is obtained by re-exciting and reading after a period of time between the same immune response.
  • the system for identifying an immunoassay of the present invention comprises an immunoreactive device, such as a container for holding a solution; a chemiluminescent immunoreactive excitation and counting device, such as a photon counting module and a light emitting diode; and a processor,
  • an immunoreactive device such as a container for holding a solution
  • a chemiluminescent immunoreactive excitation and counting device such as a photon counting module and a light emitting diode
  • a processor For example, a computer processes and plots the readings.
  • a system for identifying an immunoassay can be referred to, for example, the applicant's utility model patent CN201532646U, which is incorporated herein by reference.
  • the method of using the system comprises the following steps:
  • the two reading A' values of the sample to be tested are compared with R0. If A' is greater than or equal to R0, the sample to be tested is an HD-HOOK effect sample, which needs to be diluted; if A' is smaller than R0, it is directly used.
  • the calibration curve calculates the sample concentration
  • the calibration curve is a curve based on the first reading of the calibrator and the concentration of the calibrator.
  • a third aspect of the invention provides a kit comprising a calibrator, a peak calibrator, a first antibody (or antigen) coated luminescent particle, a labeled second antibody (or antigen), a label specific conjugate Labeled photosensitive particles, characterized in that the method of using the kit comprises the steps of: performing a chemiluminescence immunoassay, excitation and recording on a calibrator, a peak calibrator, a sample to be tested containing the antigen (or antibody) to be tested. The first and second readings of chemiluminescence determine the presence of the HD-HOOK effect sample based on the difference A' between the second and first readings of the sample to be tested.
  • the method of using the kit comprises the steps of: performing a chemiluminescence immunoassay, excitation and recording on a calibrator, a peak calibrator, a sample to be tested containing the antigen (or antibody) to be tested.
  • the first and second readings of chemiluminescence compared to the difference between the second and first readings of the sample to be tested, is the difference between the second and first readings of the peak calibrator?
  • the increase in value R0 if greater than R0, has a HD-HOOK effect, and if it is less than R0, it does not have an HD-HOOK effect.
  • the method of using the kit comprises the following steps:
  • the two reading A' values of the sample to be tested are compared with R0. If A' is greater than or equal to R0, the sample to be tested is an HD-HOOK effect sample, which needs to be diluted; if A' is smaller than R0, the sample concentration is directly calculated using the calibration curve;
  • the calibration curve is a curve based on the first reading of the calibrator and the concentration of the calibrator.
  • the above method is a method for non-disease diagnosis purposes, and the method is for selecting a HD-HOOK effect sample simply and quickly in the process of double antibody sandwich immunoassay or double antigen sandwich immunoassay. To prevent misclassification of high concentrations of antigen (or antibody) samples to low concentrations of antigen (or Antibody) sample.
  • the antigen refers to a substance that is immunogenic.
  • proteins peptides.
  • Representative antigens include, but are not limited to, cytokines, tumor markers, metalloproteins, cardiovascular diabetes related proteins, and the like.
  • the antibody refers to an immunoglobulin produced by the body that recognizes a specific foreign substance.
  • the antigen or antibody is selected from the group consisting of hepatitis B surface antigen (HBsAg), hepatitis B surface antibody (HBsAb), cancer antigen 125 (CA125), ferritin (Ferr), and C peptide (CP).
  • HBsAg hepatitis B surface antigen
  • HBsAb hepatitis B surface antibody
  • CA125 cancer antigen 125
  • Fer ferritin
  • CP C peptide
  • the sample which can be detected by the method of the present invention is not particularly limited and may be any sample containing the antigen (or antibody) to be tested, and representative examples may include serum samples, urine samples, saliva samples and the like.
  • a preferred sample of the invention is a serum sample.
  • the first antibody and the second antibody refer to an antibody that specifically binds to the antigen.
  • the corresponding first antibody and second antibody may be the same or different and may bind to the antigen at the same time.
  • the first antigen and the second antigen refer to an antigen that can specifically bind to the antibody of interest.
  • the corresponding first antigen and second antigen may be the same or different and may bind to the antibody simultaneously.
  • the label is capable of specifically binding to a marker-specific conjugate.
  • the marker is biotin and the marker specific binder is streptavidin.
  • the luminescent particles refer to polymer particles filled with a luminescent compound and a lanthanide compound.
  • the luminescent compound may be a derivative of Dioxene (dioxene) or thioxene (dimethylthiophene), and the lanthanide compound may be Eu(TTA)3/TOPO or Eu(TTA)3/Phen, etc.
  • Microparticles are commercially available.
  • the surface functional group of the luminescent particles may be any group capable of linking a protein, such as a carboxyl group, an aldehyde group, an amine group, an epoxy group or a halogenated alkyl group, and the like, and various known linkable protein functional groups.
  • the photosensitive particles are polymer particles filled with a photosensitive compound, and under the excitation of a red laser, singlet oxygen ions can be generated.
  • the single-line oxygen ions are transmitted to the luminescent particles to react with the luminescent compounds in the luminescent particles to generate ultraviolet light, which further excites the lanthanide compound to generate photons of a certain wavelength.
  • the photosensitive compound may be a phthalocyanine dye or the like, which is also commercially available.
  • red excitation light is irradiated at 600 to 700 nm, and the amount of emitted light of the reaction solution is detected.
  • the detection wavelength of the emitted light is 520 to 620 nm.
  • the red laser (600-700 nm) illuminates the photosensitive particles, and the singlet oxygen ions released by the photosensitive particles, and a part of the singlet oxygen ions are received by the luminescent particles, thereby emitting light of a high energy level of 520 to 620 nm.
  • the concentration of the target antigen to be tested is expressed as the number of double antibody sandwich complexes, and is proportional to the number of photons; however, when the target antigen concentration is too high, part of the antigen to be tested is combined with a single antibody, resulting in The double-antibody sandwich complex is reduced, and the light signal is low, which does not reflect the true concentration of the target antigen to be tested.
  • the concentration of the antibody to be tested is expressed as the number of double antigen sandwich complexes, and is proportional to the number of photons; however, when the concentration of the target antibody to be tested is too high, some of the antibodies to be tested are respectively associated with a single antigen. The combination results in a decrease in the double antigen sandwich complex and a low light signal, which does not reflect the true concentration of the antibody to be tested.
  • the method of the present invention compares the relationship between the increase of the signal values obtained by two readings by two readings, thereby functioning to broaden the detection range and distinguish the HD-HOOK effect samples.
  • the difference between the two readings is determined by the following three aspects:
  • the photosensitive particles when the first reading is performed, are irradiated with a red laser (600-700 nm) to release singlet oxygen ions. After a part of the singlet oxygen ions are transmitted to the luminescent particles, a series of chemical reactions are used to emit light of a high energy level of 520 to 620 nm; and a part of the singlet oxygen ions are combined with the target antigen to be tested which is not bound by the antibody (or antigen) ( Or antibody) reacts to reduce the concentration of the antigen (or antibody) to be tested.
  • a red laser 600-700 nm
  • the double-antibody sandwich complex decreases, and the second reading signal value decreases; and for the high concentration HD-HOOK effect sample, the target antigen to be tested ( After the concentration of the antibody or antibody is decreased, the double-antibody sandwich complex increases, and the second reading signal value increases.
  • the photosensitive particles are irradiated by a red laser (600-700 nm) during the first reading. After releasing the single-line oxygen ions, the energy is lost, and the second reading signal is lowered. .
  • the antigen-antibody reaction has not reached equilibrium in the first reading, and the reaction will still proceed in the positive direction at the interval between the two readings, and the second reading signal will increase.
  • the present invention performs the first reading when the reaction does not reach equilibrium, and the photosensitive particles are irradiated by the excitation light to release singlet oxygen, a part of which is transmitted to the luminescent particles, and a part of which can react with the unbound target antigen or antibody to be detected.
  • the photosensitive particles are depleted after being excited once, and when the second reading is performed, the signal value of the sample with a low target antigen or antibody concentration is lowered; and the double-antibody sandwich complex with the high concentration of the sample and the photosensitive particles are The combination is far from reaching equilibrium when the first reading is taken, and the reaction will move toward the positive reaction direction in the second reading, so the signal will increase, and the second photoexcitation will increase as the concentration of the target antigen (or antibody) is increased.
  • the increase in the signal value of the light and the value of the first signal also increases.
  • the increase of the signal is positively correlated with the sample concentration. Comparing the increase of the two signals can indicate that the sample with a low signal value and a high increase is the HD-HOOK effect.
  • a fourth aspect of the present invention provides an apparatus for identifying an HD-HOOK effect sample, comprising:
  • a reading unit for recording a chemiluminescent immunoreaction and performing multiple readings on the mixed mixture after incubation
  • a processing unit coupled to the reading unit, the processing unit determining whether the immunoassay has an HD-HOOK risk based on the reading of the reading unit.
  • a moving mechanism is also included for moving the incubated mixture to a reading unit for reading.
  • the device further comprises an incubator for providing a suitable ambient temperature for the chemiluminescent immunoreaction.
  • the apparatus further includes a reset mechanism for resetting the mixed liquid after completion of the reading to the incubator for re-incubation.
  • the moving mechanism is a pushing mechanism
  • the reset mechanism is a pushback mechanism
  • the mixed liquid is held by a slat.
  • the reading unit is used to record a chemiluminescent immunoreaction and to perform two readings of the mixed mixture after incubation.
  • the incubator includes a first incubator and a second incubator, and the pushing mechanism is configured to push the mixed solution in the first incubator to the second incubator for incubation, and The pushing mechanism is configured to push the mixed solution in the second incubator to the reading unit for the first reading;
  • the pushback mechanism is configured to push the mixed liquid after the completion of the first reading back to the second incubator for re-incubation;
  • the pushing mechanism is further configured to push the re-incubated mixture in the second incubator to the reading unit for a second reading;
  • the pushback mechanism includes:
  • a cupping mechanism disposed on the rail, the cupping mechanism for carrying a slat;
  • a driving device for driving the cupping mechanism to move along the guide rail
  • Photoelectric sensors disposed at both ends of the bottom plate, the photoelectric sensors being used to detect a position of the cupping mechanism
  • a position adjustment mechanism coupled to the photosensor, the position adjustment mechanism being capable of adjusting a position of the cupping mechanism based on a position signal emitted by the photosensor.
  • the rail is a straight rail or a rail.
  • the apparatus further includes a slat-loading tray disposed on one side of the incubator for completing mixing of the sample to be tested and reagents, and disposed on the other side of the incubator In the refrigerated area where the reagents are stored.
  • the apparatus further includes a blank slat stack and a loading mechanism disposed on a side of the slat proofing tray, the blank slat stack and loading mechanism for blanking the board Push the strip to the slat sample plate.
  • the apparatus further includes a sample tube carrier for carrying the sample tube.
  • the apparatus further includes a dilution plate oscillator disposed on a side of the sample tube carrier adjacent to the blank slat stack and the loading mechanism, the dilution plate oscillator being used for The dilution plate is diluted.
  • the device further includes a mechanical arm, and the mechanical arm is provided with a sampling needle;
  • the mechanical arm includes a first mechanical arm and a second mechanical arm
  • the first mechanical arm is configured to suck a sample from the sample tube carrier area and distribute it into a slat of the slat proofing tray
  • a second robotic arm is used to draw reagent from the reagent refrigerated area and dispense it into the slats of the slat proofing tray.
  • the apparatus further includes a first cleaning mechanism and a second cleaning mechanism, the first cleaning mechanism is for cleaning the loading needle on the first robot arm, and the second cleaning The mechanism is used to clean the needle on the second robot arm.
  • the sample needs to go through the following steps before entering the incubator:
  • the blank slat is pushed to the slat loading tray position D0 by the blank slat stack and the loading mechanism;
  • the slat is rotated 90 degrees clockwise to the slat tray position D3. At this position, the reagent is drawn from the reagent refrigerating zone by the second robot arm and dispensed into the slats at the location.
  • the slat-loading tray is rotated from the D3 position to D0 (this process should wait for the slats at the D1 position)
  • the slat-loading tray is rotated from the D3 position to D0 (this process should wait for the slats at the D1 position)
  • the sample allocation complete one cycle of the slat sample plate.
  • the D0 position of the slat proofing plate is loaded with blank slats
  • the D1 position is for sample distribution
  • the D2 position is waiting
  • the D3 position is for reagent dispensing
  • the slats are distributed after reagent dispensing.
  • Push to the incubator The rotation of the slat sample loading tray should wait for all the movements in the four areas D0 to D3 to be completed before the rotation can be performed.
  • the slats push the slats to the second incubator.
  • the marker specific conjugate is also added to the slab containing the sample and the reagent to be tested.
  • the labeled photosensitive particles are then infiltrated into the second incubator 12 for incubation. After the incubation, the slats are pushed by the push mechanism to the reading unit for excitation light illumination and the amount of emitted light is detected, and the first reading is recorded. After the first reading is completed, the slats are pushed back to the second incubator for re-incubation.
  • the processing unit processes the two readings.
  • the second reading and the first reading increase more than the maximum value of the standard curve, it is determined that the immunoassay has a HOOK risk.
  • One method is that the device qualitatively gives a HOOK prompt, and the operator can dilute the sample before performing the measurement; the second method is that the device directly gives a quantitative result, but the result is much higher than the linear range.
  • the device sets the reading unit so that the reading unit performs two or even multiple readings on the mixed mixture, and processes the reading of the reading unit through the processing unit to determine whether the immunoassay exists.
  • HOOK risk to avoid the HOOK effect caused by the test sample can not be correctly distinguished because its concentration exceeds the linear range of the test kit or its own concentration is this value, thus avoiding experimental misdiagnosis.
  • a fifth aspect of the present invention provides an immunoassay method comprising the steps of: (1) performing a chemiluminescence immunoassay on a sample to be tested containing an antigen (or antibody) to be tested, and exciting and recording chemiluminescence.
  • First and second readings, and the difference between the second and first readings is recorded as A, (2) based on a known series of reference materials containing the antigen (or antibody) of the target to be tested
  • the difference between the two readings is increased by A' as a standard curve and/or based on the difference A of the two readings of a known standard substance containing the antigen (or antibody to be tested);
  • the difference A between the second and first readings of the sample to be tested containing the antigen (or antibody) to be tested is compared to the standard curve and/or standard.
  • reaction conditions for detecting the difference between the difference in the sample to be tested and the increase in the difference between the known standard materials are consistent.
  • the difference A between the second and first readings of the sample to be tested containing the antigen (or antibody) to be tested is compared to the standard curve.
  • the concentration of the known standard substance is lower than the concentration at which the HOOK effect is produced, and the known standard substance is a positive control.
  • the method further comprises the step (4): if the increase A of the two readings of the sample to be tested containing the antigen (or antibody) to be tested is greater than the maximum value of the standard curve, The sample is then diluted and then measured.
  • the method includes the following steps:
  • step (a2) the first reading: in the mixture of step (a1), the labeling specific conjugate labeled photosensitive particles are added, after the incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter is read as RLU1;
  • the difference A between the second and first readings of the sample to be tested containing the antigen (or antibody) to be tested is compared to the standard, and the standard Recorded as a cut-off value; and/or the known standard substance is a positive control.
  • the method further comprises the step (4): if the increase A of the two readings of the sample to be tested containing the antigen (or antibody) to be tested is greater than the critical value, The concentration of the test sample is higher than the concentration of the known standard substance.
  • the method comprises the steps of:
  • step (c2) the first reading: in the mixture of step (c1), the labeling specific conjugate labeled photosensitive particles are added, after the incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter is read as RLU1;
  • step (c3) second reading after the first reading of the reaction solution in step (c2) is further incubated, then the excitation light is irradiated and the amount of emitted light is detected, the photon counter reading is counted as RLU2;
  • the method further comprises the step (4): if the increase A of the two readings of the sample to be tested containing the antigen (or antibody) to be tested is greater than the threshold, and at the same time If the first reading of the sample to be tested is lower than the known standard substance, the sample is diluted and then measured.
  • the method comprises the steps of:
  • step (d2) First reading: additional marker specific binding label in the mixture of step (d1) Photosensitive particles, after incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter reading is counted as RLU1;
  • step (d3) second reading after the first reading of the reaction solution in step (d2) is further incubated, then the excitation light is irradiated and the amount of emitted light is detected, the photon counter reading is counted as RLU2;
  • the difference A between the second and first readings of the sample to be tested containing the antigen (or antibody) to be tested is compared to the standard curve;
  • the method also includes the step (4) of determining the concentration of the sample.
  • the method comprises the steps of:
  • step (b2) the first reading: in the mixture of step (b1), the labeling specific conjugate labeled photosensitive particles are added, after the incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter reading is calculated as RLU1;
  • the luminescent particles refer to polymer particles filled with a luminescent compound and a lanthanide compound;
  • the photosensitive particles are polymer particles filled with a photosensitive compound, which can generate singlet oxygen ions under excitation by a red laser. .
  • red light is irradiated with excitation light of 600 to 700 nm, and the amount of emitted light of the reaction solution is detected; the detection wavelength of the emitted light is 520 to 620 nm.
  • the antigen refers to a substance having immunogenicity
  • the antibody refers to an immunoglobulin produced by the body capable of recognizing a specific foreign substance
  • the first antibody and the second antibody finger can specifically bind to the same An antibody of the target antigen
  • the first antigen and the second antigen are antigens that specifically bind to the antibody of interest.
  • a sixth aspect of the invention provides a system for identifying an immunoassay, the system comprising:
  • An immunoreaction device for performing a chemiluminescent immune response An immunoreaction device for performing a chemiluminescent immune response
  • a chemiluminescent immunoreactive excitation and counting device for exciting and recording the first and second readings of chemiluminescence, and recording the difference between the second and first readings as A;
  • the system for identifying an immunoassay of the present invention comprises an immunoreactive device, such as a container for holding a solution; a chemiluminescent immunoreactive excitation and counting device, such as a photon counting module and a light emitting diode; and a processor,
  • an immunoreactive device such as a container for holding a solution
  • a chemiluminescent immunoreactive excitation and counting device such as a photon counting module and a light emitting diode
  • a processor For example, a computer processes and plots the readings.
  • a system for identifying an immunoassay can be referred to, for example, the applicant's utility model patent CN201532646U, which is incorporated herein by reference.
  • the processor is configured to perform a standard curve based on an increase A of two readings of a known series of standard substances containing the antigen (or antibody) to be tested, wherein the concentration of the standard substance is low The concentration at which the HOOK effect is produced; if the increase A of the two readings of the sample to be tested containing the antigen (or antibody) to be tested is greater than the maximum value of the standard curve, the sample is diluted and then measured.
  • the method of using the system includes the following steps:
  • Second reading further incubate the reaction solution after the first reading in step (2) After that, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter reading is counted as RLU2;
  • the processor is configured to compare an increase A of two readings of a sample to be tested containing the antigen (or antibody) to be tested with a threshold value, if the antigen (or antibody) to be tested is contained
  • the increase A of the two readings of the sample to be tested is greater than the critical value, and the concentration of the sample to be tested is higher than the concentration of the known standard substance.
  • the method of using the system includes the following steps:
  • the processor is configured to compare an increase A of two readings of a sample to be tested containing the antigen (or antibody) to be tested with a threshold value, if the antigen (or antibody) to be tested is contained.
  • the increase A of the two readings of the sample to be tested is greater than the critical value, and at the same time, the signal value obtained by the first reading of the sample to be tested is lower than the known standard substance, and then the sample is diluted and then performed. Determination.
  • the method of using the system includes the following steps:
  • the processor is configured to perform a standard curve according to a first reading of a known series of standard substances containing the antigen (or antibody) to be tested and an increase A of two readings, respectively.
  • the first reading of the sample to be tested containing the antigen (or antibody) to be tested and the increase A of the two readings are compared with a standard curve to determine the concentration of the sample.
  • the method of using the system includes the following steps:
  • the calibration curve is a curve based on the first reading of a known series of standard substances containing the antigen (or antibody) to be tested and the concentration of a known series of standard substances.
  • the luminescent particles refer to polymer particles filled with a luminescent compound and a lanthanide compound;
  • the photosensitive particles are polymer particles filled with a photosensitive compound, which can generate singlet oxygen ions under excitation by a red laser. .
  • red light is irradiated with excitation light of 600 to 700 nm, and the amount of emitted light of the reaction solution is detected; the detection wavelength of the emitted light is 520 to 620 nm.
  • the antigen refers to a substance having immunogenicity
  • the antibody refers to an immunoglobulin produced by the body capable of recognizing a specific foreign substance
  • the first antibody and the second antibody finger can specifically bind to the same An antibody of the target antigen
  • the first antigen and the second antigen are antigens that specifically bind to the antibody of interest.
  • a seventh aspect of the present invention provides a kit comprising a first antibody (or antigen)-coated luminescent particle, a label-labeled second antibody (or antigen), and a label-specific conjugate-labeled photosensitive particle
  • the method of using the kit includes the following steps: (1) performing a chemiluminescence immunoassay on a sample to be tested containing the antigen (or antibody) to be tested, and exciting and recording the first and second times of chemiluminescence Reading, and the difference between the second and first readings is recorded as A, (2) the difference between the two readings of a known series of standard substances containing the antigen (or antibody) to be tested.
  • Amplification A' is used as a standard curve and/or based on the difference A of the two readings of a known standard substance containing the antigen (or antibody) to be tested as a standard; (3) will contain the target antigen to be tested (or The difference A between the second and first readings of the test sample of the antibody is compared to the standard curve and/or standard.
  • the difference A between the second and first readings of the sample to be tested containing the antigen (or antibody) to be tested is compared to the standard curve.
  • the concentration of the known standard substance is lower than the concentration at which the HOOK effect is produced, and the known standard substance is a positive control.
  • the method of using the kit further comprises the step (4): if the increase A of the two readings of the sample to be tested containing the antigen (or antibody) to be tested is greater than the standard curve The maximum value is then diluted after the sample is measured.
  • the method of using the kit comprises the steps of:
  • step (a2) the first reading: in the mixture of step (a1), the labeling specific conjugate labeled photosensitive particles are added, after the incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter is read as RLU1;
  • the difference A between the second and first readings of the sample to be tested containing the antigen (or antibody) to be tested is compared to the standard, and the standard Recorded as a cut-off value; and/or the known standard substance is a positive control.
  • the method of using the kit further comprises the step (4): if the increase A of the two readings of the sample to be tested containing the antigen (or antibody) to be tested is greater than the critical value, Then, the concentration of the sample to be tested is higher than the concentration of the known standard substance.
  • the method of using the kit comprises the steps of:
  • step (c2) the first reading: in the mixture of step (c1), the labeling specific conjugate labeled photosensitive particles are added, after the incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter is read as RLU1;
  • step (c3) second reading after the first reading of the reaction solution in step (c2) is further incubated, then the excitation light is irradiated and the amount of emitted light is detected, the photon counter reading is counted as RLU2;
  • the method of using the kit further comprises the step (4): if the increase A of the two readings of the sample to be tested containing the antigen (or antibody) to be tested is greater than the critical value, And at the same time, the first reading of the sample to be tested is lower than the known standard substance, and then the sample is diluted and then measured.
  • the method of using the kit comprises the steps of:
  • step (d2) the first reading: in the mixture of step (d1), the labeling specific conjugate labeled photosensitive particles are added, after the incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter reading is calculated as RLU1;
  • step (d3) second reading after the first reading of the reaction solution in step (d2) is further incubated, then the excitation light is irradiated and the amount of emitted light is detected, the photon counter reading is counted as RLU2;
  • the method of using the kit further comprises the step (4) of: increasing the first reading and the two readings of the sample to be tested containing the antigen (or antibody) to be tested; The standard curves are compared to determine the concentration of the sample.
  • the method of using the kit comprises the steps of:
  • step (b2) the first reading: in the mixture of step (b1), the labeling specific conjugate labeled photosensitive particles are added, after the incubation, the excitation light is irradiated and the amount of emitted light is detected, and the photon counter reading is calculated as RLU1;
  • the calibration curve is a curve based on the first reading of a known series of standard substances containing the antigen (or antibody) to be tested and the concentration of a known series of standard substances. .
  • the above method is a non-disease diagnostic purpose method for widening the detection range by two readings during the double antibody sandwich immunoassay or the double antigen sandwich immunoassay detection process, and It can accurately determine whether the sample to be tested has a HOOK effect and calculate the concentration of the analyte in the sample to be tested simply and quickly.
  • the antigen refers to a substance that is immunogenic.
  • proteins peptides.
  • Representative antigens include, but are not limited to, cytokines, tumor markers, metalloproteins, cardiovascular diabetes related proteins, and the like.
  • the antibody refers to an immunoglobulin produced by the body that recognizes a specific foreign substance.
  • the antigen or antibody is selected from the group consisting of insulin (INS), hepatitis B virus surface antibody (HBsAb), alpha fetoprotein (AFP), and thyrotropin (TSH).
  • INS insulin
  • HBsAb hepatitis B virus surface antibody
  • AFP alpha fetoprotein
  • TSH thyrotropin
  • the sample which can be detected by the method of the present invention is not particularly limited and may be any sample containing the antigen (or antibody) to be tested, and representative examples may include serum samples, urine samples, saliva samples and the like.
  • a preferred sample of the invention is a serum sample.
  • the first antibody and the second antibody refer to an antibody that specifically binds to the antigen.
  • the corresponding first antibody and second antibody may be the same or different and may bind to the antigen at the same time.
  • the first antigen and the second antigen refer to an antigen that can specifically bind to the antibody of interest.
  • the corresponding first antigen and second antigen may be the same or different and may bind to the antibody simultaneously.
  • the label is capable of specifically binding to a marker-specific conjugate.
  • the marker is biotin and the marker specific binder is streptavidin.
  • the luminescent particles refer to polymer particles filled with a luminescent compound and a lanthanide compound.
  • the luminescent compound may be a derivative of Dioxene (dioxene) or thioxene (dimethylthiophene), and the lanthanide compound may be Eu(TTA)3/TOPO or Eu(TTA)3/Phen, etc.
  • Microparticles are commercially available.
  • the surface functional group of the luminescent particles may be any group capable of linking a protein, such as a carboxyl group, an aldehyde group, an amine group, an epoxy group or a halogenated alkyl group, and the like, and various known linkable protein functional groups.
  • the photosensitive particles are polymer particles filled with a photosensitive compound, and under the excitation of a red laser, singlet oxygen ions can be generated.
  • the single-line oxygen ions are transmitted to the luminescent particles to react with the luminescent compounds in the luminescent particles to generate ultraviolet light, which further excites the lanthanide compound to generate photons of a certain wavelength.
  • the photosensitive compound may be a phthalocyanine dye or the like, which is also commercially available.
  • the detection wavelength of the emitted light is 520 to 620 nm.
  • the red laser (600-700 nm) illuminates the photosensitive particles, and the singlet oxygen ions released by the photosensitive particles, and a part of the singlet oxygen ions are received by the luminescent particles, thereby emitting light of a high energy level of 520 to 620 nm.
  • the concentration of the target antigen to be tested is expressed as the number of double antibody sandwich complexes, and is proportional to the number of photons; however, when the target antigen concentration is too high, part of the antigen to be tested is combined with a single antibody, resulting in The double-antibody sandwich complex is reduced, and the light signal is low, which does not reflect the true concentration of the target antigen to be tested.
  • the concentration of the antibody to be tested is expressed as the number of double antigen sandwich complexes, and is proportional to the number of photons; however, when the concentration of the target antibody to be tested is too high, some of the antibodies to be tested are respectively associated with a single antigen. The combination results in a decrease in the double antigen sandwich complex and a low light signal, which does not reflect the true concentration of the antibody to be tested.
  • the relationship between the increase of the signal values obtained by the two readings is compared by two readings, thereby playing the role of broadening the detection range.
  • the difference between the two readings is determined by the following three aspects:
  • the photosensitive particles are irradiated with a red laser (600-700 nm) to release singlet oxygen ions.
  • a series of chemical reactions are used to emit light of a high energy level of 520 to 620 nm; and a part of the singlet oxygen ions are combined with the target antigen to be tested which is not bound by the antibody (or antigen) ( Or antibody) reacts to reduce the concentration of the antigen (or antibody) to be tested.
  • the concentration of the target antigen (or antibody) decreases
  • the double-antibody sandwich complex decreases
  • the second reading signal value decreases.
  • the target antigen (or antibody) concentration decreases. After that, the double-anti-sandwich composite increased, and the second reading signal value increased.
  • the photosensitive particles are irradiated by a red laser (600-700 nm) during the first reading. After releasing the single-line oxygen ions, the energy is lost, and the second reading signal is lowered. .
  • the antigen-antibody reaction has not reached equilibrium in the first reading, and the reaction will still proceed in the positive direction at the interval between the two readings, and the second reading signal will increase.
  • the present invention performs the first reading when the reaction does not reach equilibrium, and the photosensitive particles are irradiated by the excitation light to release singlet oxygen, a part of which is transmitted to the luminescent particles, and a part of which can react with the unbound target antigen or antibody to be detected. Consuming part of the target antigen or antibody to be detected, so that the reaction equilibrium moves backwards.
  • the photosensitive particles are depleted after one excitation, and the signal value of the sample with low target antigen or antibody concentration when the second reading is performed. It will decrease; the combination of the high-resistance double-anti-sandwich composite and the photosensitive particles will not reach equilibrium in the first reading.
  • the signal When the second reading will move in the positive reaction direction, the signal will increase.
  • the concentration of the target antigen (or antibody) is increased, the signal value of the second photoexcitation light and the increase of the first signal value are also increased.
  • the increase of the signal is positively correlated with the sample concentration. Comparing the increase of the two signals can broaden the detection range to calculate the concentration quickly and easily during the detection process.
  • the method of the invention is based on the no-wash and uniformity of reaction of the photoexcited chemiluminescence platform (light-emitting oxygen channel), and can perform multiple signal measurement on one reaction without interrupting the immune reaction.
  • the optical signals at different reaction times are detected, and the size comparison of the two signals can distinguish HD-HOOK effect samples, and the method is not limited by the detection range, and effectively widens the detection range by more than 100 times.
  • the method of the invention can correctly identify the HD-HOOK effect sample in the double-anti-sand sandwich assay, which can significantly improve the accuracy of the double-antibody sandwich immunoassay and reduce the false negative of the double antibody sandwich immunoassay. rate.
  • the method of the present invention is simple in operation, widening the detection range by two readings, and calculating the concentration of the analyte in a simple and rapid manner during the detection process.
  • An eighth aspect of the invention provides an immunoassay device comprising:
  • a reading unit for recording a chemiluminescent immunoreaction and performing multiple readings on the mixed mixture after incubation
  • a processing unit coupled to the reading unit, the processing unit determining whether the immunoassay has a HOOK risk based on the reading of the reading unit.
  • the device further includes a moving mechanism for moving the incubated mixture to a reading unit for reading.
  • the device further comprises an incubator for providing a suitable ambient temperature for the chemiluminescent immunoreaction.
  • the apparatus further includes a reset mechanism for resetting the mixed liquid after completion of the reading to the incubator for re-incubation.
  • the moving mechanism is a pushing mechanism
  • the reset mechanism is a pushback mechanism
  • the mixed liquid is held by a slat.
  • the reading unit is used to record a chemiluminescent immunoreaction and to perform two readings of the mixed mixture after incubation.
  • the incubator includes a first incubator and a second incubator, and the pushing mechanism is configured to push the mixed solution in the first incubator to the second incubator for incubation, and The pushing mechanism is configured to push the mixed solution in the second incubator to the reading unit for the first reading;
  • the pushback mechanism is configured to push the mixed liquid after the completion of the first reading back to the second incubator for re-incubation;
  • the pushing mechanism is further configured to push the re-incubated mixture in the second incubator to the reading unit for a second reading;
  • the processing unit detects that the second reading and the increase in the first reading are greater than the maximum value of the standard curve, it is determined that the immunoassay has a HOOK risk.
  • the pushback mechanism includes:
  • a cupping mechanism disposed on the rail, the cupping mechanism for carrying a slat;
  • a driving device for driving the cupping mechanism to move along the guide rail
  • Photoelectric sensors disposed at both ends of the bottom plate, the photoelectric sensors being used to detect a position of the cupping mechanism
  • a position adjustment mechanism coupled to the photosensor, the position adjustment mechanism being capable of adjusting a position of the cupping mechanism based on a position signal emitted by the photosensor.
  • the rail is a straight rail or a rail.
  • the apparatus further includes a slat-loading tray disposed on one side of the incubator for completing mixing of the sample to be tested and reagents, and disposed on the other side of the incubator In the refrigerated area where the reagents are stored.
  • the apparatus further includes a blank slat stack and a loading mechanism disposed on a side of the slat proofing tray, the blank slat stack and loading mechanism for blanking the board Push the strip to the slat sample plate.
  • the apparatus further includes a sample tube carrier for carrying the sample tube.
  • the apparatus further includes a dilution plate oscillator disposed on a side of the sample tube carrier adjacent to the blank slat stack and the loading mechanism, the dilution plate oscillator being used for The dilution plate is diluted.
  • the device further includes a mechanical arm, and the mechanical arm is provided with a sampling needle;
  • the mechanical arm includes a first mechanical arm and a second mechanical arm
  • the first mechanical arm is configured to suck a sample from the sample tube carrier area and distribute it into a slat of the slat proofing tray
  • a second robotic arm is used to draw reagent from the reagent refrigerated area and dispense it into the slats of the slat proofing tray.
  • the apparatus further includes a first cleaning mechanism and a second cleaning mechanism, the first cleaning mechanism is for cleaning the loading needle on the first robot arm, and the second cleaning The mechanism is used to clean the needle on the second robot arm.
  • the sample needs to go through the following steps before entering the incubator:
  • the blank slat is pushed to the slat loading tray position D0 by the blank slat stack and the loading mechanism;
  • the slat is rotated 90 degrees clockwise to the slat tray position D3. At this position, the reagent is drawn from the reagent refrigerating zone by the second robot arm and dispensed into the slats at the location.
  • the slat-loading tray is rotated from the D3 position to D0 (this process should wait for the slats at the D1 position)
  • the slat-loading tray is rotated from the D3 position to D0 (this process should wait for the slats at the D1 position)
  • the sample allocation complete one cycle of the slat sample plate.
  • the D0 position of the slat proofing plate is loaded with blank slats
  • the D1 position is for sample distribution
  • the D2 position is waiting
  • the D3 position is for reagent dispensing
  • the slats are distributed after reagent dispensing.
  • Push to the incubator The rotation of the slat sample loading tray should wait for all the movements in the four areas D0 to D3 to be completed before the rotation can be performed.
  • the slats push the slats to the second incubator.
  • the marker specific conjugate is also added to the slab containing the sample and the reagent to be tested.
  • the labeled photosensitive particles are then infiltrated into the second incubator 12 for incubation. After the incubation, the slats are pushed by the push mechanism to the reading unit for excitation light illumination and the amount of emitted light is detected, and the first reading is recorded. After the first reading is completed, the slats are pushed back to the second incubator for re-incubation.
  • the processing unit processes the two readings.
  • the second reading and the first reading increase more than the maximum value of the standard curve, it is determined that the immunoassay has a HOOK risk.
  • One method is that the device qualitatively gives a HOOK prompt, and the operator can dilute the sample before performing the measurement; the second method is that the device directly gives a quantitative result, but the result is much higher than the linear range.
  • An advantage of the present invention over the prior art is that the device sets the reading unit such that the reading unit performs two or more readings on the incubated mixture and processes the readings of the reading unit through the processing unit. Furthermore, it is judged whether there is a risk of HOOK in the immunoassay, and the detected sample which is prevented from being caused by the HOOK effect cannot be correctly distinguished because the concentration exceeds the linear range of the detection kit or the concentration itself is the value, thereby avoiding the misdiagnosis of the experiment.
  • Fig. 1 is a view showing the internal structure of the apparatus for identifying the HD-HOOK effect sample and the internal structure of the immunoassay device according to the present invention.
  • FIG. 2 shows an assay device and an immunoassay device for identifying HD-HOOK effect samples according to the present invention.
  • the internal structure of the chassis is shown in Figure 2.
  • Fig. 3 is a view showing the first embodiment of the measuring device for identifying the HD-HOOK effect sample and the push-back mechanism of the immunoassay device according to the present invention.
  • Fig. 4 is a view showing the second embodiment of the measuring device for identifying the HD-HOOK effect sample and the pushing back mechanism of the immunoassay device according to the present invention.
  • Fig. 5 is a view showing the first embodiment of the measuring device and the immunoassay device for identifying HD-HOOK effect samples according to the present invention.
  • Fig. 6 is a view showing the second embodiment of the measuring device and the immunoassay device for identifying HD-HOOK effect samples according to the present invention.
  • Fig. 7 is a timing chart showing a complete test procedure of the assay device and the immunoassay device for identifying HD-HOOK effect samples according to the present invention.
  • Figure 8 A plot of signal values versus sample concentration for HCG+ ⁇ using conventional methods.
  • Figure 9 is a graph showing the relationship between signal value and A and sample concentration for HCG+ ⁇ using the method of the present invention.
  • Figure 10 Graph of the relationship between the signal value and sample concentration obtained by Ferrer using the conventional method.
  • Figure 11 is a graph showing the relationship between the signal value and A and the sample concentration obtained by the method of the present invention by Ferr.
  • Figure 12 A graph showing the relationship between signal value and sample concentration of a C-peptide using a conventional method.
  • Figure 13 is a graph showing the relationship between the signal value and A and the sample concentration obtained by the method of the present invention.
  • Figure 14 A plot of signal values versus sample concentration for a HBsAb using conventional methods.
  • Figure 15 is a graph showing the relationship between the signal value and A and the sample concentration of the HBsAb using the method of the present invention.
  • Figure 16 Graph of signal values obtained by INS using conventional detection methods and sample concentration.
  • Figure 17 is a graph showing the relationship between the first reading signal and the amplitude A and sample concentration obtained by the INS using the method of the present invention.
  • Figure 18 A plot of signal values obtained by conventional detection methods for HBsAb versus sample concentration.
  • Figure 19 A plot of the first reading signal and the amplitude A versus sample concentration for the HBsAb using the method of the invention.
  • Figure 20 A plot of signal values obtained by AFP using conventional detection methods versus sample concentration.
  • Figure 21 A plot of the first reading signal and the amplitude A versus sample concentration obtained by the AFP using the method of the present invention.
  • Figure 22 A plot of signal values obtained by conventional detection methods for TSH versus sample concentration.
  • Figure 23 Relationship between the first reading signal and the amplitude A and sample concentration obtained by TSH using the method of the present invention. Graph.
  • Figure 24 Graph of the relationship between the signal value and the sample concentration obtained by Ferrer using the conventional detection method.
  • Figure 25 Graph of the first reading signal and the amplitude A and sample concentration obtained by Ferr using the method of the present invention.
  • Figure 26 A plot of signal values obtained by conventional detection methods for C peptide versus sample concentration.
  • Figure 27 A graph of the first reading signal and the amplitude A versus sample concentration for the C peptide using the method of the present invention.
  • Figure 28 A plot of signal values obtained by conventional detection methods for HBsAb versus sample concentration.
  • Figure 29 A plot of the first reading signal and the amplitude A versus sample concentration for the HBsAb using the method of the invention.
  • the experimental methods, detection methods, and preparation methods disclosed in the present invention employ molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields conventional in the art. Conventional technology. These techniques are well described in the existing literature. For details, see Sambrook et al.
  • MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, and the like.
  • the inventors of the present invention have found through extensive and in-depth research that by setting two readings without interrupting the reaction and comparing the increase A of the two readings with the critical value, it can be judged whether the sample to be tested needs to be diluted before Perform the determination; or establish the relationship between the increase A of the two readings and the sample concentration, widen the detection range by two readings, to calculate the concentration quickly and easily during the test; or to study the increase of the two readings Whether the sample is a relationship between HD-HOOK effect samples can easily and effectively eliminate the false negative caused by the HD-HOOK effect in the double-antibody sandwich immunoassay, and improve the accuracy of the double-antibody sandwich immunoassay.
  • the inventors of the present invention also provide an assay device which can perform two or more readings on the mixed solution after the incubation, and the method for identifying the HD-HOOK effect sample can be implemented by using the device. And immunoassay methods.
  • FIG. 1 and FIG. 2 a schematic diagram of an internal structure of a measuring device and an immunoassay device for identifying a HD-HOOK effect sample according to the present invention is shown, including:
  • the incubator 1 is for providing a suitable ambient temperature for the chemiluminescent immune response, the incubator 1 comprising a first incubator 11 and a second incubator 12.
  • Reading unit 2 for recording a chemiluminescent immune reaction and performing two readings on the mixed mixture, the reading unit 2 may be a photomultiplier tube or a laser driver;
  • a pushing mechanism 3 disposed between the incubator 1 and the reading unit 2, the pushing mechanism comprising a first pushing mechanism 31 and a second pushing mechanism 32, the first pushing mechanism 31 crossing the incubator 1, the second pushing mechanism 32 is connected to the rear end of the first urging mechanism 31, and the second urging mechanism 32 is located inside the casing.
  • the first urging mechanism 31 and the second urging mechanism 32 cooperate to push the slats in the first incubator 11 to the reading unit 2 for the first reading, and to use the second illuminator 12 for the internal temperature.
  • the post-natal slats are pushed to reading unit 2 for a second reading.
  • the slats are a mixture of a sample to be tested containing the antigen (or antibody) to be tested, a luminescent particle coated with a first antibody (or antigen), and a second antibody (or antigen) labeled with a label. Photosensitive particles labeled with a marker-specific binder are also added to the mixed solution.
  • both pushback mechanisms can be used to push the slats after the first reading is returned to the second incubator 12 for re-incubation.
  • the slats after the incubation are irradiated with excitation light and the amount of emitted light is detected; in the second reading, the slats after re-incubation are irradiated with excitation light and the amount of emitted light is detected.
  • the processing unit when the second reading and the first reading increase is greater than the maximum value of the standard curve, the strip is diluted and then measured.
  • the standard curve is based on an increase in two readings of a known series of standard materials containing the antigen or antibody of interest to be tested, the concentration of the standard substance being lower than the concentration at which the HOOK effect is produced.
  • the processing unit may be a computer that processes and maps the readings.
  • the setting of the incubator as the first incubator and the second incubator is for facilitating the mechanical realization of the immunoassay device, and the invention is not limited thereto.
  • the moving mechanism can also be a mechanical arm grasping type
  • the resetting mechanism can also be a mechanical arm grasping type, which is not limited by the present invention.
  • the pushback mechanism includes:
  • the rail can also be a rail 422, as shown in FIG.
  • the driving device includes a stepping motor 441.
  • the stepping motor 441 is fixed to one end of the rail 2 through a motor fixing plate 442.
  • the output end of the stepping motor 441 is provided with a timing pulley 443.
  • the other end of the 2 is provided with an idler pulley 444, and the timing belt 443 and the idler pulley 444 are sleeved with a timing belt 445.
  • the shaft of the idler pulley 444 is threaded with an idler shaft 446 that is fixed to the idler plate 447.
  • a cupping mechanism 43 disposed on the rail, the cupping mechanism 43 is for carrying a slat, the cupping mechanism 43 includes a cup drag chain 431 and a movement connected to the cup drag chain 431 A cup connecting plate 432 is connected to the timing belt 445 via a timing belt pressing plate 448.
  • a photosensor 45 is disposed at both ends of the bottom plate 41, and the photosensor 45 is connected to the sensor blank 46, and the photosensor 45 detects the position of the cupping mechanism 43 through the sensor blank 46.
  • a position adjustment mechanism coupled to the photosensor 45 the position adjustment mechanism being capable of adjusting a position of the cupping mechanism 43 based on a position signal from the photosensor 45.
  • the position adjustment mechanism includes a position adjustment plate 46 and a controller (not shown), so the controller is divided into The position adjustment plate 46 is connected to the photosensor 45, and the controller controls the position adjustment plate 46 to adjust the position of the cupping mechanism 43 according to the position signal from the received photosensor 45.
  • the position adjusting mechanism is used for fine adjustment, and the position adjusting mechanism finely adjusts when the cupping mechanism 43 does not reach the designated position or the position of the deviation is deviated.
  • the apparatus further includes a rotatable slat proofing tray 5 disposed on one side of the incubator 1, the blank slats being loaded on the slats
  • the tray 5 completes mixing of a sample to be tested containing a target antigen (or antibody) to be tested, the reagent being a luminescent particle coated with a first antibody (or antigen), and a second antibody labeled with a label ( Or antigen).
  • the apparatus further includes a reagent refrigerated area 8 for placing a reagent disposed on the other side of the incubator 1.
  • a blank slat stack and loading mechanism 6 for pushing the blank slats to the slat proofing tray 5 on one side of the slat proofing tray 5 is also included.
  • a sample tube carrier 7 is also included, the sample tube carrier 7 being used to carry a sample tube.
  • a mechanical arm (not shown) is further included, and the mechanical arm is provided with a sampling needle;
  • the mechanical arm includes a first robot arm for sucking a sample from the sample tube carrier region, and a second robot arm for using the reagent refrigerating region Pipette reagents.
  • the first cleaning mechanism 9 is for cleaning the loading needle on the first mechanical arm
  • the second cleaning mechanism 10 is used to clean the needle on the second robot arm.
  • the length of the module is lengthened, so that the chassis (that is, the overall structure shown in FIG. 1 and FIG. 2) and the position of the pump 11 are relatively compact (as shown in FIG. 5), which leads to complicated assembly and disassembly.
  • the variable rail 422 can be designed as a curved guide rail, and the width of the module is widened, so that the position of the chassis and the second needle washing mechanism 10 is relatively compact (as shown in FIG. 6). ), but will not affect disassembly.
  • the sample needs to undergo the following steps before entering the incubator 1:
  • the blank slat is pushed to the slat proofing plate 5 position D0 by the blank slat stack and the loading mechanism 6;
  • the slat is rotated 90 degrees clockwise to the slat tray position D3 where the reagent is drawn from the reagent refrigerated area 8 by the second robot arm and dispensed into the slats at that location.
  • the slat-loading tray 5 is rotated from the D3 position to D0 (this process should wait for the slats at the D1 position to complete the sample assignment).
  • the D0 position of the slat proofing plate 5 is loaded with blank slats, the D1 position is for sample distribution, the D2 position is waiting, the D3 position is for reagent dispensing and the plate is dispensed after reagent dispensing. Push the strip to the incubator 1.
  • the rotation of the slat-loading plate 5 should wait for all the movements in the four areas D0 to D3 to be completed before the rotation can be performed.
  • the displacing mechanism 3 pushes the slats to the second incubator, in which the photosensitive particles labeled with the marker specific conjugate are added, after which the slats enter
  • the second incubator 12 is incubated, and the slats are pushed by the urging mechanism 3 to the reading unit 2 for excitation light irradiation and the amount of emitted light is detected, and the first reading is recorded.
  • the slats are pushed back from the straight rail pushing mechanism 4 or the rail changing mechanism 4' to the second incubator 12 for re-incubation. After the incubation, the slats are further moved by the pushing mechanism. 3 Push to the reading unit 2 to perform excitation light irradiation and detect the amount of emitted light, and record the second reading.
  • processing unit 2 After completing two readings, processing unit 2 processes the two readings when the second reading and the first reading increase more than the maximum value of the standard curve, and at the same time the first reading of the sample to be tested is lower than
  • the known standard substance one method is that the device qualitatively gives a Hook prompt, and the operator can perform dilution after the sample is diluted; the second method is that the device directly gives a quantitative result, but the result is more than The linear range is much higher.
  • Figure 7 shows a complete test flow sequence diagram, which is a test sequence diagram for a non-dilution process.
  • a, b, c, d and e represent the first batch of slats, the second batch of slats, the third slats, the fourth slats and the fifth slats, respectively.
  • the slats are dispensed at the D3 position, as shown by D3 in Figure 7, assuming that the reagent is dispensed using 1 sip, and the reagent R1 is dispensed immediately after the reagent R2 is dispensed, assuming that the time for dispensing the reagent for each slat is 30 seconds, then 8 slats total 480 seconds;
  • F in Figure 7 represents the second incubation time
  • G in Figure 7 indicates the time (including mechanical movement and discarding slat time) for the reading unit to read a slat.
  • the above process assigns two reagents R1 and R2 in the reagent dispensing stage. It is understood that three reagents R1, R2 and R3 can also be assigned, assuming that R1, R2 and R3 are added after sample distribution is completed, for example HBeAb, R3 For 50 ⁇ l of neutralizing e antigen, the operation was similar to the case of R1 and R2 only, except that one reagent was added during the reagent dispensing phase, and the order of distribution of R1, R2 and R3 was arbitrary.
  • R1, R2 and R3 are added before the sample is dispensed, for example CA19-9, R3 is 15 ⁇ l of sample dilution, and a second robotic arm is used to complete the dispensing of a reagent at the D1 position, and then The dispensing of the sample is done by the first robot arm, and the other processes are identical to the case of only R1 and R2.
  • R3 is a pre-dilution solution, and it is necessary to use a pre-dilution plate, such as HCV, 10 ⁇ l sample + 100 ⁇ l of the dilution solution, and then take 25 ⁇ l of the diluted sample to participate in the test.
  • a pre-dilution plate such as HCV
  • 10 ⁇ l sample + 100 ⁇ l of the dilution solution and then take 25 ⁇ l of the diluted sample to participate in the test.
  • the second robot arm distributes the diluent R3 to the pre-dilution plate
  • the first robot arm distributes the sample into the pre-dilution plate, and if necessary, can repeatedly apply the suction, wherein the diluent is dispensed.
  • the process can be combined with one suction and multiple injections, for example, five projects, one project requires pre-dilution, and the other four projects do not require pre-dilution.
  • the first robot arm sucks five samples and one one is distributed to the pre-dilution plate. Within, the other four shares are assigned to the blank slats. After that, the pre-dilution plate is oscillated.
  • the sample tube carrier 7 is provided with a dilution plate oscillator 11 on the side of the blank slab stack and the loading mechanism 6. After the oscillating treatment, the first arm The diluted sample is dispensed into the slats.
  • the subsequent process is consistent with the case of only R1 and R2, and will not be described here.
  • first antibody and second antibody refer to an antibody that specifically binds to an antigen, such as a tumor marker.
  • an antigen such as a tumor marker
  • the corresponding first antibody and second antibody may be different or identical and may bind to the antigen simultaneously.
  • first antigen and second antigen refer to an antigen that specifically binds to an antibody, such as a hepatitis B surface antibody.
  • the corresponding first antigen and second antigen may be different or identical and may bind to the antibody simultaneously.
  • antigen refers to a substance that is immunogenic, such as a protein, a polypeptide.
  • Representative antigens include, but are not limited to, cytokines, tumor markers, metalloproteins, cardiovascular diabetes related proteins, and the like.
  • tumor marker refers to a type of tumor that occurs in the process of tumorigenesis and proliferation, which is produced by the tumor cells themselves or which is produced by the body's reaction to tumor cells, and which reflects the presence and growth of tumors. substance.
  • Representative tumor markers in the art include, but are not limited to, alpha-fetoprotein (AFP), cancer antigen 125 (CA125), and the like.
  • the first antibody is immobilized on a solid phase carrier, and then the first antibody is reacted with an antigen, and then reacted with the labeled second antibody, and finally a chemiluminescence or enzyme-linked color reaction detection signal is performed.
  • the basic principles of photoexcited chemiluminescence are well known to those skilled in the art.
  • the conventional method is to combine the photosensitive particles and the luminescent particles in a certain range to generate the transfer of ion oxygen energy, and emit an optical signal to detect the sample to be tested.
  • the photosensitive particles are filled with a photosensitive compound
  • the luminescent particles are filled with a luminescent compound and a lanthanoid.
  • the photosensitive particles release a single-state oxygen ion (4 ⁇ S) in a high-energy state with a propagation distance of about 200 nm.
  • the singlet oxygen ions released by the photosensitive particles can reach the luminescent particles, and emit a high-energy 520-620 nm light through a series of chemical reactions, which is detected by the instrument.
  • the characteristics of the first antibody immobilized on the luminescent particles are fully utilized, and the biotin-labeled secondary antibody, streptavidin-coated photosensitive particles, serum samples or antigen standards are utilized.
  • the control solution is added to the reaction vessel in sequence or simultaneously with the first antibody-coated luminescent particles and the biotin-labeled secondary antibody, and then the streptavidin-labeled photosensitive particles are added, thereby causing the following reaction:
  • the second antibody binds to the corresponding antigen in the serum sample or the antigen standard control solution, and finally forms a "second antibody-antigen-first antibody-luminescent particle" double-antibody sandwich complex;
  • Biotin and streptavidin specifically bind, allowing the double-antibody sandwich complex to bind to the photoreceptor particles.
  • the distance between the photosensitive particles and the luminescent particles is less than 200 nm, and after the red laser (600 to 700 nm) illuminates the photosensitive particles, the released singlet oxygen can be received by the luminescent particles.
  • the red laser 600 to 700 nm
  • the luminescent particles Through a series of chemical reactions, high-energy light of 520-620 nm is emitted, and the sample to be tested is qualitatively or quantitatively detected by the intensity of the chemiluminescence.
  • the characteristics of the first antigen immobilized on the luminescent particles are fully utilized, and the biotin-labeled second antigen, streptavidin-coated photosensitive particles, serum sample or antigen standard are used.
  • the control solution is added to the reaction container in sequence or simultaneously with the first antigen-coated luminescent particles and the biotin-labeled second antigen, and then the streptavidin-labeled photosensitive particles are added, thereby causing the following reaction:
  • the first antigen on the luminescent particles is combined with the corresponding anti-volume in the serum sample or the antigen standard control solution to form an "antibody-first antigen-luminescent particle" ternary complex;
  • the second antigen binds to the corresponding antibody in the serum sample or the antigen standard control solution, and finally forms a "second antigen-antibody-first antigen-luminescent particle" double-antibody sandwich complex;
  • Biotin and streptavidin specifically bind, allowing the double-antibody sandwich complex to bind to the photoreceptor particles.
  • the distance between the photosensitive particles and the luminescent particles is less than 200 nm, and after the red laser (600 to 700 nm) illuminates the photosensitive particles, the released singlet oxygen can be received by the luminescent particles.
  • the red laser 600 to 700 nm
  • the luminescent particles Through a series of chemical reactions, high-energy light of 520-620 nm is emitted, and the sample to be tested is qualitatively or quantitatively detected by the intensity of the chemiluminescence.
  • the first antibody (or antigen) coated luminescent particles which are referred to as reagent 1, can be purchased from Boyang Biotechnology Co., Ltd.
  • the second antibody (or antigen) can be labeled with a variety of markers known in the art and their specific binder systems.
  • the second antibody (or antigen) is labeled by a biotin-avidin system.
  • the biotin-labeled secondary antibody (or antigen), designated as Reagent 2 can be purchased from Boyang Biotechnology Co., Ltd.
  • Streptavidin-coated photosensitive particles which are recorded as general-purpose liquids, can be purchased from Boyang Biotechnology. Limited company.
  • the known standard substance solution in a certain concentration range (the peak calibrator concentration is equal to the HD-HOOK effect concentration) is configured with the antigen (or antibody) to be tested.
  • After the incubation reaction add LiCA universal solution, continue the incubation for the first time (RLU1), and then incubate for a second time (RLU2).
  • concentration range of the standard substance solution can span the HD-HOOK effect concentration or lower than the HD-HOOK effect concentration as needed.
  • the sample which can be detected by the method of the present invention is not particularly limited and may be any sample containing an antigen (or antibody), and representative examples may include a serum sample, a urine sample, a saliva sample, and the like.
  • a preferred sample is a serum sample.
  • the A value of the two readings of the sample to be tested is compared with the A of the calibrator. If the sample A to be tested is larger than the A of the calibrator, the concentration of the sample is greater than the concentration of the calibrator; if the sample RLU1 is smaller than the RLU1 of the calibrator, It indicates that the low RLU1 of this sample is due to the HD-HOOK effect and requires dilution detection;
  • the A value of the two readings of the sample to be tested is substituted into the standard curve of the concentration of the calibrator A and the calibrator, and it is judged whether the concentration of the sample to be tested is in the rising range or the falling range of the RLU1, and then the RLU1 of the sample to be tested is substituted into the interval. Calculate the concentration of the sample to be tested in the calibration curve of the calibrator RLU1 and the concentration of the calibrator;
  • the sample is not the HD-HOOK effect sample, and the RLU1 of the sample to be tested is substituted into the calibrator RLU1 and the calibrator concentration.
  • the concentration of the sample to be tested is calculated in the calibration curve; if A is greater than or equal to R0, the sample is identified as a HD-HOOK effect sample, which requires dilution detection.
  • Example 1 Detection of human chorionic gonadotropin and ⁇ subunit (HCG+ ⁇ ) in human serum samples
  • the kit includes a calibrator 1 - calibrator 6 (ie, a known series of standard materials), a reagent 1 (a luminescent antibody, that is, an antibody-coated luminescent particle), and a reagent 2 (a biotin-labeled antibody, That is, biotinylated antibody).
  • a calibrator 1 - calibrator 6 ie, a known series of standard materials
  • a reagent 1 a luminescent antibody, that is, an antibody-coated luminescent particle
  • a reagent 2 a biotin-labeled antibody, That is, biotinylated antibody
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • streptavidin-labeled photosensitive particles which is an auxiliary reagent for the production of photoexcited chemiluminescence analysis system by Boyang Biotechnology Co., Ltd. It is used together with the instrument and the corresponding photochemiluminescence detection kit for the detection of antigens and antibodies.
  • Serum samples of 18 patients with HCG+ ⁇ concentration obtained by Roche test were tested by conventional methods and methods of the present invention.
  • Routine detection method sample to be tested, calibrator, reagent 1 (luminescent antibody, ie, mouse monoclonal antibody-coated luminescent particles) and reagent 2 (biotin-labeled antibody, ie biotin-labeled mouse monoclonal antibody) After adding the cuvette separately, incubate at 37 ° C for 15 min, add the universal solution (streptavidin-labeled photosensitive particles), incubate at 37 ° C for 10 min, read the photon counter, read the RLU, calculate the sample concentration, the results are as follows 1 is shown.
  • a sample to be tested a calibrator
  • a reagent 1 a luminescent antibody, that is, a murine monoclonal antibody-coated luminescent particle
  • a reagent 2 a biotin-labeled antibody, that is, a biotin-labeled Mouse monoclonal antibody, incubated at 37 °C for 15 min, added universal solution (streptavidin-labeled photomicroparticles), incubated at 37 °C for 3 min, read RLU1, continue incubation at 37 °C for 7 min, read RLU2, and calculate second
  • the increase in the secondary signal value A (RLU2 / RLU1-1) ⁇ 100%, and the results are shown in Table 1.
  • the detection range of routine detection of HCG+ ⁇ is 0-10000 mIU/ml, and the concentration exceeding the upper limit of detection shows a concentration of >10000 mIU/ml.
  • the Roche test results are shown in Table 1 and Figure 8.
  • the concentration rises to 54531 mIU/ml, and the signal value increases with the increase of the concentration, the concentration continues to increase, and the signal value increases with HCG+ ⁇ .
  • the degree is lowered and decreased, that is, the concentration is greater than 54531 mIU/ml, then HD-HOOK.
  • the detection range is 0-10000 mIU/ml, and the sample exceeding the detection upper limit shows a concentration of >10000 mIU/ml.
  • an ultra-high concentration sample is reported as a low concentration, as in sample 18. Therefore, in the conventional detection, it is impossible to distinguish whether the detection result of the sample to be tested is a true concentration or a low concentration of the ultra-high value sample which is affected by the HD-HOOK effect.
  • the method of the present invention uses two readings to identify samples with a low reported concentration resulting from the HOOK effect.
  • the increase A of samples 10-18 was greater than the increase of calibrator 6 (11.1%), and the A value continued to increase, indicating that the HCG+ ⁇ concentrations of samples 10-18 were all greater than 10000 mIU/ml, and the concentration continued to rise, which was related to Roche.
  • the concentration results are consistent, the sample 18 signal value is lower than the calibrator 6, and the conventional method detection concentration is 8713.02 mIU/ml, which can be identified as the HD-HOOK effect sample by the method of the present invention, and the dilution test is required.
  • the ferritin (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of ferritin (purchased from Fitzgerald, Catalog No: 30-AF10) in the sample.
  • the kit includes a calibrator 1 - calibrator 6 (ie, a known series of standard materials), a reagent 1 (a luminescent antibody, that is, an antibody-coated luminescent particle), and a reagent 2 (a biotin-labeled antibody, That is, biotinylated antibody).
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and the calibration curve is used to calculate the concentration of the analyte.
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • streptavidin-labeled photosensitive particles which is an auxiliary reagent for the production of photoexcited chemiluminescence analysis system by Boyang Biotechnology Co., Ltd. It is used together with the instrument and the corresponding photochemiluminescence detection kit for the detection of antigens and antibodies.
  • Routine detection method calibrator 1 - calibrator 6, sample 1-15 to be tested, reagent 1 (luminescent antibody, that is, mouse monoclonal antibody coated luminescent particles) and reagent 2 (biotin labeled antibody, ie After adding the biotin-labeled murine monoclonal antibody to the reaction cup, incubate at 37 ° C for 15 min, add LiCA universal solution (streptavidin-labeled photosensitive particles), incubate at 37 ° C for 10 min, read photon counter, read RLU, The results are shown in Table 2.
  • the detection range of routine detection of ferritin is 0-2000 ng/ml, and the concentration exceeding the upper limit of detection shows a concentration of >2000 ng/ml.
  • the signal value increases to 51000 ng/ml and the signal value increases with the increase of the concentration, the concentration continues to increase, and the signal value decreases with the increase of the Ferr concentration.
  • the conventional detection range is 0-2000 ng. /ml, above the detection limit, the sample shows a concentration of >2000 ng/ml.
  • the HD-HOOK effect sample concentration continues to rise, when the concentration rises to 2550000 ng/ml and the signal falls below the signal of calibrator 6, an ultra-high concentration sample is reported as a low concentration, as in sample 15. Therefore, in the conventional detection, it is impossible to distinguish whether the detection result of the sample to be tested is a true concentration or a low concentration of the ultra-high value sample which is affected by the HD-HOOK effect.
  • the method of the present invention uses two readings to identify samples with a low reported concentration resulting from the HOOK effect.
  • the increase A of samples 4-15 was greater than the increase of calibrator 6 (-5.5%), indicating that the fert concentrations of samples 4-15 were both greater than 2000 ng/ml. This is consistent with the actual concentration.
  • the sample 15 signal value is lower than the calibrator 6.
  • the conventional method has a detection concentration of 1860.97 ng/ml. By the method of the present invention, it can be identified as a sample whose concentration exceeds the detection range, and dilution detection is required.
  • the C-peptide (CP) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of C-peptide (purchased from Fitzgerald, Catalog No: 30-AC96) in the sample.
  • the kit includes calibrator 1 - calibrator 6 (ie, a known series of standard substances), reagent 1 (luminescent antibody, that is, antibody coated luminescent particles), reagent 2 (biotin labeled antibody, also That is, biotinylated antibody).
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • streptavidin-labeled photosensitive particles which is an auxiliary reagent for the production of photoexcited chemiluminescence analysis system by Boyang Biotechnology Co., Ltd. It is used together with the instrument and the corresponding photochemiluminescence detection kit for the detection of antigens and antibodies.
  • the high concentration of the C peptide antigen was subjected to gradient dilution, and the concentration values of the samples containing different concentrations of the C peptide were determined by the conventional detection method and the detection method of the present invention, respectively.
  • Routine detection method calibrator 1 - calibrator 6, sample 1-15 to be tested, reagent 1 (luminescent antibody, that is, mouse monoclonal antibody coated luminescent particles) and reagent 2 (biotin labeled antibody, ie After adding the biotin-labeled murine monoclonal antibody to the reaction cup, incubate at 37 ° C for 15 min, add LiCA universal solution (streptavidin-labeled photosensitive particles), incubate at 37 ° C for 10 min, read photon counter, read RLU, The results are shown in Table 3.
  • the detection range of conventional detection of C peptide is 0-30ng/ml, and the concentration exceeding the upper limit of detection shows a concentration of >30ng/ml.
  • the signal value increased to 10000 ng/ml, the concentration increased with the increase of concentration, the concentration continued to increase, and the signal value decreased with the increase of C-peptide concentration.
  • the conventional detection range was 0- 30ng/ml, above the upper limit of detection, the sample showed a concentration of >30ng/ml.
  • the concentration rises to At 33500000 ng/ml the signal drops below the signal of calibrator 6, and the ultra-high concentration sample is reported as a low concentration, such as samples 16, 17. Therefore, in the conventional detection, it is impossible to distinguish whether the detection result of the sample to be tested is a true concentration or a low concentration of the ultra-high value sample which is affected by the HD-HOOK effect.
  • the method of the present invention uses two readings to identify samples with a low reported concentration resulting from the HOOK effect.
  • the increase A of samples 5-17 was greater than the increase of calibrator 6 (-4.9%), indicating that the C-peptide concentrations of samples 5-17 were all greater than 30 ng/ml, exceeding the upper limit of detection. This is consistent with the actual concentration.
  • the signal values of the samples 16 and 17 are lower than the calibrator 6.
  • the detection concentration of the conventional method is 8.15 ng/ml and 0.76 ng/ml, respectively, and the sample can be identified as exceeding the upper limit of detection by the method of the present invention, and dilution is required. Detection.
  • Example 4 Detection of hepatitis B virus surface antigen (HBsAg) in human serum samples
  • HBsAg concentration in the sample was measured using a hepatitis B virus surface antigen (HBsAg) detection kit (photochemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd., which includes calibrator 1 - calibrator 6 (That is, a series of known standard substances), a reagent 1 (a luminescent antibody, that is, an antibody-coated luminescent particle), and a reagent 2 (a biotin-labeled antibody, that is, a biotin-labeled antibody).
  • HBsAg detection kit photochemiluminescence method
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and the concentration of the analyte is calculated as a standard curve.
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • streptavidin-labeled photosensitive particles which is an auxiliary reagent for the production of photoexcited chemiluminescence analysis system by Boyang Biotechnology Co., Ltd. It is used together with the instrument and the corresponding photochemiluminescence detection kit for the detection of antigens and antibodies.
  • the concentration of the 15 serum samples obtained by the method of the present invention is as shown in Table 4.
  • the sample exceeding the upper limit of detection (336.56 IU/mL) is distinguished by the comparison with the increase of the calibrator 6, that is, the A value is greater than 27%, and the HOOK effect is judged.
  • the sample concentration can be calculated directly using the calibration curve.
  • sample 1 - sample 15 were diluted 2 times and diluted 4 times, while the undiluted original sample was detected by conventional detection method, and 2 was diluted.
  • the sample and the 4-fold diluted sample were judged by the change of the concentration after dilution to determine whether the sample had a HOOK effect, that is, if the sample concentration was increased after dilution, it was a HOOK effect sample.
  • Non-HOOK effect samples will decrease in concentration after dilution.
  • Table 5 The results are shown in Table 5:
  • serum samples 1, 2, 3, 5, 6, 9, 12, 13, 14, 15 were diluted and detected. The degree is increased, which is proved to be a HD-HOOK effect sample with a concentration greater than 336.56 IU/mL. The concentration of serum samples 4, 7, 8, 10, 11 was reduced after dilution, which proved to be not a HD-HOOK effect sample, and was identical to the results of the method of the present invention.
  • CA125 carbohydrate antigen 125
  • the concentration of CA125 in the sample is detected by a carbohydrate antigen 125 (CA125) detection kit (photochemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd., and the kit includes a calibration product 1 - calibrator 6 A series of known standard substances), reagent 1 (luminescent antibody, that is, antibody-coated luminescent particles), and reagent 2 (biotin-labeled antibody, that is, biotin-labeled antibody).
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • streptavidin-labeled photosensitive particles which is an auxiliary reagent for the production of photoexcited chemiluminescence analysis system by Boyang Biotechnology Co., Ltd. It is used together with the instrument and the corresponding photochemiluminescence detection kit for the detection of antigens and antibodies.
  • the concentration of the 18 serum samples obtained by the method of the present invention is as shown in Table 6.
  • the sample exceeding the upper limit of detection is distinguished by the comparison with the increase of the calibrator 6, that is, the sample having the A value greater than 7.4% is judged to exceed the upper limit of the detection, and the dilution is recommended.
  • Post-test while A is less than 7.4%, it is a non-HOOK effect sample, and the sample concentration can be calculated directly using the calibration curve.
  • sample 1 - sample 18 were diluted 2 and diluted 4 times, while the undiluted original sample was detected by conventional detection method, and 2 was diluted.
  • the sample and the 4-fold diluted sample were judged by the change of the concentration after dilution to determine whether the sample had a HOOK effect, that is, if the sample concentration was increased after dilution, it was a HOOK effect sample. non-HOOK
  • the effect sample will decrease in concentration after dilution. The result is shown in 7:
  • the conventional method detects the original sample, and the serum samples 16, 17, and 18 are misjudged as the low concentration sample due to the HD-HOOK effect.
  • Example 6 Detection of hepatitis B virus surface antibody (HBsAb) in a sample
  • Hepatitis B virus surface antibody was detected by HBsAbHBsAb (photochemical chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. (purchased from Beijing Zhongke Jingda Biotechnology Co., Ltd.) Company, Clone No: M2201).
  • the kit includes a calibrator 1 - calibrator 6 (ie, a known series of standard materials), a reagent 1 (a luminescent antibody, that is, an antibody-coated luminescent particle), and a reagent 2 (a biotin-labeled antibody, That is, biotinylated antibody).
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and the concentration of the analyte is calculated as a standard curve.
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • streptavidin-labeled photosensitive particles which is an auxiliary reagent for the production of photoexcited chemiluminescence analysis system by Boyang Biotechnology Co., Ltd. It is used together with the instrument and the corresponding photochemiluminescence detection kit for the detection of antigens and antibodies.
  • the high concentration of HBsAb was subjected to gradient dilution, and the concentration values of the samples containing different concentrations of HBsAb were determined by the conventional detection method and the detection method of the present invention, respectively.
  • Routine detection method calibrate 1 - calibrator 6, sample 1-14, reagent 1 (HBsAg coated luminescent particles) and reagent 2 (biotin-labeled HBsAg) were added to the reaction cup, and incubated at 37 ° C for 15 min. LiCA universal solution (streptavidin-labeled photosensitive particles) was added, incubated at 37 ° C for 10 min, photon counter reading, and RLU was read. The results are shown in Table 8.
  • calibrator 1 - calibrator 6 sample 1-14 to be tested, reagent 1 (HBsAg coated luminescent particles) and reagent 2 (biotinylated HBsAg), incubated at 37 ° C for 15 min
  • reagent 1 HBsAg coated luminescent particles
  • reagent 2 biotinylated HBsAg
  • Add LiCA universal solution streptavidin-labeled photosensitive particles
  • read RLU1 continue incubation at 37 °C for 7 min
  • the detection range of routine detection of HBsAb is 0-1000 mIU/ml, and the concentration exceeding the upper limit of detection shows a concentration of >1000 mIU/ml.
  • the signal value increased to 10000 mIU/ml, the concentration increased with the concentration, the concentration continued to increase, and the signal value decreased with the increase of the concentration of HBsAb.
  • the routine detection range was 0-1000 mIU. /ml, above the detection limit, the sample shows a concentration of >1000mIU/ml.
  • the concentration rises to At 335,000 mIU/ml and above the signal drops below the signal of calibrator 6, and the ultra-high concentration sample is reported as a low concentration, such as samples 12, 13, and 14. Therefore, in the conventional detection, it is impossible to distinguish whether the detection result of the sample to be tested is a true concentration or a low concentration of the ultra-high value sample which is affected by the HD-HOOK effect.
  • the method of the present invention uses two readings to identify samples with a low reported concentration resulting from the HOOK effect.
  • the increase A of samples 8-14 was greater than the increase of calibrator 6 (35.9%), indicating that the concentrations of HBsAb of samples 8-14 were all greater than 1000 mIU/ml, exceeding the upper limit of detection. This is consistent with the actual concentration, samples 12, 13, and 14.
  • the signal value is lower than the calibrator 6.
  • the conventional method detects the concentration of 802.57 mIU/ml, 352.22 mIU/ml, and 147.9 mIU/ml, respectively.
  • the method of the present invention can identify the sample exceeding the upper limit of detection, and the dilution test is required.
  • Example 7 Application of the method of the invention in a qualitative kit Anti-HCV
  • the hepatitis C virus antibody detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of Anti-HCV in the sample.
  • the kit includes a reference product, a negative control, a positive control, a reagent 1 (a luminescent HCV antigen, that is, an HCV antigen-coated luminescent particle), and a reagent 2 (a biotin-labeled HCV antigen, that is, a biotin-labeled HCV antigen). ).
  • Reference product, negative control, positive control The reference product is a known concentration standard used as a reference to judge the positive test of the test sample; the negative control and the positive control are known concentration standards used to judge the validity of the test.
  • the high concentration of Anti-HCV was subjected to gradient dilution, and the signal values of the samples containing different concentrations of Anti-HCV were determined by the conventional detection method and the detection method of the present invention, respectively.
  • Routine detection method a series of anti-HCV samples diluted in gradient, reagent 1 (luminescent HCV antigen, ie, HCV antigen-coated luminescent particles) and reagent 2 (biotin-labeled HCV antigen, ie biotin-labeled HCV) After adding the reaction cup, incubate at 37 ° C for 15 min, add LiCA universal solution (streptavidin-labeled photosensitive particles), incubate at 37 ° C for 10 min, read photon counter, read RLU, knot As shown in Table 9.
  • reagent 1 luminance-labeled HCV antigen, that is, HCV antigen-coated luminescent particles
  • reagent 2 biotin-labeled HCV antigen, ie, biological Labeled HCV antigen
  • 37 ° C for 15 min added LiCA universal solution (streptavidin-labeled photosensitive particles)
  • read RLU1 continue to incubate for 7 min at 37 ° C, read RLU2, and calculate
  • the increase in the second signal value A (RLU2 / RLU1-1) ⁇ 100%, and the results are shown in Table 9.
  • the signal value decreases with increasing concentration, and the HOOK effect occurs.
  • concentration continues to rise to a certain value (such as sample 12)
  • the RLU falls below the reference cut off value.
  • the A value (-25%) determines the size relationship between the sample to be tested and the positive reference.
  • the A value of sample 12 (47%) is much higher than the A value of the positive control (-25%). It indicates that the secondary sample concentration is higher than the positive control and is a positive sample.
  • the signal is not rich because of the HOOK effect, and dilution verification should be performed.
  • Example 8 Detection of insulin in a sample (INS)
  • the insulin (INS) detection kit (chemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of insulin (purchased from Fitzgerald, Catalog No: 30R-2704) in the sample.
  • the high concentration of the insulin antigen was subjected to gradient dilution, and the signal values of the samples containing different concentrations of insulin were determined by the conventional detection method and the detection method of the present invention, respectively.
  • a sample of a sample to be tested a reagent 1 (a luminescent antibody, that is, a mouse monoclonal antibody-coated luminescent particle) and a reagent 2 (a biotin-labeled antibody, that is, a biotin label)
  • a reagent 1 a luminescent antibody, that is, a mouse monoclonal antibody-coated luminescent particle
  • a reagent 2 a biotin-labeled antibody, that is, a biotin label
  • the method of the invention broadens the detection range by two readings.
  • the signal value continued to increase to 10,000 ⁇ IU/ml with concentration, after which the signal value began to decrease with increasing concentration, but the increase A continued to increase with concentration.
  • the RLU1 and A calibration curves and the standard curve are respectively determined by the method of the present invention (Fig. 17), and as the concentration increases, A continues to rise, and RLU1 is divided into A rising range of 3 ⁇ IU/ml to 10,000 ⁇ IU/ml and a falling range of 10,000 ⁇ IU/ml to 1,000,000 ⁇ IU/ml.
  • the RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.
  • the concentration of the test substance is determined by the value of A to be in the rising range of 3 ⁇ IU/ml to 10,000 ⁇ IU/ml or the falling range of 10,000 ⁇ IU/ml to 1,000,000 ⁇ IU/ml, and then the RLU1 of the substance to be tested is substituted. Its corresponding calibration curve calculates the exact concentration.
  • Example 9 Detection of hepatitis B virus surface antibody (HBsAb) in a sample
  • Hepatitis B virus surface antibody detection kit (photochemiluminescence method) was used to detect hepatitis B virus surface antibody in samples ( purchased from Beijing Zhongke Jingda Biotechnology Co., Ltd.) using Boyang Biotechnology (Shanghai) Co., Ltd. , Clone No: M2201) concentration.
  • the high concentration of HBsAb was subjected to gradient dilution, and the signal values of the samples containing different concentrations of HBsAb were determined by the conventional detection method and the detection method of the present invention, respectively.
  • Routine detection method Gradient dilution of HBsAb sample, reagent 1 (HBsAg-coated luminescent particles) and reagent 2 (biotin-labeled HBsAg) were added to the reaction cup, incubated at 37 ° C for 15 min, and added LiCA universal solution (streptavidin) The labeled fluorescent particles were incubated at 37 ° C for 10 min and the photon counter read the RLU.
  • Table 11 Routine detection and detection results of the present invention
  • the signal value from 1 mIU/ml to 10000 mIU/ml increases with increasing concentration, the concentration continues to increase, and the signal value decreases with increasing HBsAb concentration, ie, the concentration is greater than 10,000 mIU/ml.
  • -HOOK in routine testing, samples with antigen concentrations above this detection range will report low concentrations (reported concentrations are less than 10,000 mIU/ml).
  • the method of the invention broadens the detection range by two readings.
  • the signal value continued to increase with concentration to 10,000 mIU/ml, after which the signal value began to decrease with increasing concentration, but the increase A continued to increase with concentration.
  • the calibration curves and standard curves of RLU1 and A are respectively determined by the method of the present invention (Fig. 19), and as the concentration increases, A continues to rise, and RLU1 points. It is a rising range of 1 mIU/ml to 10,000 mIU/ml and a falling range of 10,000 mIU/ml to 3,350,000 mIU/ml.
  • the RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.
  • the concentration of the test substance is determined by the value of A to be in the rising range of 1 mIU/ml to 10,000 mIU/ml or the falling range of 10,000 mIU/ml to 3,350,000 mIU/ml, and then the RLU1 of the substance to be tested is substituted into its corresponding calibration curve. Calculate the exact concentration.
  • Example 10 Detection of fetal gamma globulin (AFP) in a sample
  • the fetal thyroglobulin test kit (photochemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of AFP (purchased from Fitzgerald, Catalog No: 30-1370) in the sample.
  • the high concentration of AFP antigen was subjected to gradient dilution, and the signal values of the samples containing different concentrations of AFP were determined by the conventional detection method and the detection method of the present invention, respectively.
  • the conventional detection method and the method of the present invention are referred to in Example 8.
  • the test results are as follows:
  • Table 12 Routine detection and detection results of the present invention
  • the method of the invention broadens the detection range by two readings.
  • the signal value continued to increase to 10,000 ng/ml with concentration, after which the signal value began to decrease with increasing concentration, but the increase A continued to increase with concentration.
  • the calibration curves and standard curves of RLU1 and A are respectively determined by the method of the present invention (Fig. 21), and as the concentration increases, A continues to rise, and RLU1 points. It is a rising range of 5 ng/ml to 10,000 ng/ml and a falling range of 10,000 ng/ml to 1,000,000 ng/ml.
  • the RLU1, RLU2 and A of the sample to be tested are detected by the method of the present invention.
  • Example 11 Detection of thyrotropin (TSH) in a sample
  • the thyrotropin test kit (photochemiluminescence method) produced by Boyang Biotechnology (Shanghai) Co., Ltd. was used to detect the content of thyrotropin (purchased from Fitzgerald, Catalog No: 30R-AT009) in the sample.
  • the high concentration of TSH antigen was serially diluted, and the signal values of the samples containing different concentrations of TSH were determined by the conventional detection method and the detection method of the present invention, respectively.
  • the conventional detection method and the method of the present invention are referred to in Example 8.
  • the test results are as follows:
  • the method of the invention broadens the detection range by two readings.
  • the signal value continued to increase with concentration to 10,000 ⁇ IU/ml, after which the signal value began to decrease with increasing concentration, but the increase A continued to increase with concentration.
  • the calibration curves and standard curves of RLU1 and A are respectively determined by the method of the present invention (Fig. 23), and as the concentration increases, A continues to rise, and RLU1 points. It is a rising range of 1 ⁇ IU/ml to 10,000 ⁇ IU/ml and a falling range of 10,000 ⁇ IU/ml to 1,000,000 ⁇ IU/ml.
  • the RLU1 and RLU2 of the sample to be tested are detected by the method of the present invention. And A.
  • the concentration of the test substance is determined by the value of A to be in the rising range of 3 ⁇ IU/ml to 10,000 ⁇ IU/ml or the falling range of 10,000 ⁇ IU/ml to 1,000,000 ⁇ IU/ml, and then the RLU1 of the substance to be tested is substituted into its corresponding calibration curve. Calculate the exact concentration.
  • Example 12 Detection of hepatitis B virus surface antigen (HBsAg) in human serum samples
  • the concentration of HBsAg in the sample is detected by a kit according to an immunoassay method according to the present invention, and the kit includes a calibrator 1 - a calibrator 6, a peak calibrator, and a reagent 1 (a luminescent antibody, that is, an antibody pack) The luminescent particles), the reagent 2 (biotin-labeled antibody, that is, the biotin-labeled antibody).
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • streptavidin-labeled photosensitive particles which is an auxiliary reagent for the production of photoexcited chemiluminescence analysis system by Boyang Biotechnology Co., Ltd. It is used together with the instrument and the corresponding photochemiluminescence detection kit for the detection of antigens and antibodies.
  • the concentration of 15 serum samples obtained by the method of the present invention is as shown in Table 14.
  • the HOOK effect sample is distinguished by comparing with the increase R0 of the peak calibrator, that is, the A value is greater than 31%, and the HOOK effect sample is determined, and the dilution test is recommended. And A less than 31% is a non-HOOK effect sample, and the sample concentration can be calculated directly using the calibration curve.
  • sample 1 - sample 15 were diluted 2 times and diluted 4 times, while the undiluted original sample was detected by conventional detection method, and 2 was diluted.
  • the sample and the 4-fold diluted sample were judged by the change of the concentration after dilution to determine whether the sample had a HOOK effect, that is, if the sample concentration was increased after dilution, it was a HOOK effect sample.
  • Non-HOOK effect samples will decrease in concentration after dilution.
  • Table 15 The results are shown in Table 15:
  • Example 13 Detection of CA125 in human serum samples
  • the concentration of CA125 in the sample is detected by a kit according to an immunoassay method according to the present invention, and the kit includes a calibrator 1 - a calibrator 6, a peak calibrator, and a reagent 1 (a luminescent antibody, that is, an antibody pack) The luminescent particles), the reagent 2 (biotin-labeled antibody, that is, the biotin-labeled antibody).
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • the concentration of the 18 serum samples obtained by the method of the present invention is as shown in Table 16.
  • the HOOK effect sample is distinguished by comparing with the increase R0 of the peak calibrator, that is, the A value is greater than 18.7%, and the HOOK effect sample is judged, and the dilution test is recommended. And A less than 18.7% is a non-HOOK effect sample, and the sample concentration can be calculated directly using the calibration curve.
  • sample 1 - sample 18 were diluted 2 and diluted 4 times, while the undiluted original sample was detected by conventional detection method, and 2 was diluted.
  • the sample and the 4-fold diluted sample were judged by the change of the concentration after dilution to determine whether the sample had a HOOK effect, that is, if the sample concentration was increased after dilution, it was a HOOK effect sample.
  • Non-HOOK effect samples will decrease in concentration after dilution.
  • Table 17 The results are shown in Table 17:
  • a kit involved in an immunoassay method according to the present invention is used to detect the content of ferritin (purchased from Fitzgerald, Catalog No: 30-AF10) in a sample.
  • the kit includes a calibration product 1 - a calibrator 6 , a peak calibrator, a reagent 1 (a luminescent antibody, that is, an antibody-coated luminescent particle), and a reagent 2 (a biotin-labeled antibody, that is, a biotin-labeled antibody) .
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • the high concentration of ferritin antigen was subjected to gradient dilution, and the concentration values of the samples containing different concentrations of ferritin were determined by the conventional detection method and the detection method of the present invention, respectively.
  • Routine test method calibrator 1 - calibrator 6, peak calibrator and sample 1-15, reagent 1 (luminescent antibody, ie, mouse monoclonal antibody coated luminescent particles) and reagent 2 (biotin labeling)
  • the antibody that is, the biotin-labeled murine monoclonal antibody
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • photon counter reading The RLU is read and the results are shown in the table below.
  • the detection range of routine detection of ferritin is 0-2000 ng/ml, and the concentration exceeding the upper limit of detection shows a concentration of >2000 ng/ml.
  • the detection range is 0-2000 ng/ml, and the sample exceeding the detection limit shows a concentration of >2000 ng/ml.
  • an ultra-high concentration sample is reported as a low concentration, as in sample 15. Therefore, in the conventional detection, it is impossible to distinguish whether the detection result of the sample to be tested is a true concentration or a low concentration of the ultra-high value sample which is affected by the HD-HOOK effect.
  • the method of the invention identifies HOOK samples by two readings.
  • the signal value continued to increase to 51000 ng/ml with concentration, and then the signal value began to decrease with increasing concentration, but the increase A continued to increase with concentration. Therefore, the A value of the sample to be tested and the A value of the calibrator can be directly compared to determine the relationship between the concentration of the sample to be tested and the concentration of the calibrator.
  • the increase A of samples 10-15 is greater than the increase of peak calibrator R0 (13.9%), indicating that the concentration of Ferr in samples 10-15 is greater than 51000 ng/ml, which is a HD-HOOK sample. This is consistent with the actual concentration.
  • the sample 15 signal value is lower than the calibrator 6.
  • the conventional method has a detection concentration of 1860.97 ng/ml.
  • the method of the present invention can identify the HD-HOOK effect sample, and the dilution test is required.
  • a kit involved in an immunoassay method according to the present invention is used to detect the content of C-peptide (purchased from Fitzgerald, Catalog No: 30-AC96) in a sample.
  • the kit includes a calibration product 1 - a calibrator 6 , a peak calibrator, a reagent 1 (a luminescent antibody, that is, an antibody-coated luminescent particle), and a reagent 2 (a biotin-labeled antibody, that is, a biotin-labeled antibody) .
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • the high concentration of the C peptide antigen was subjected to gradient dilution, and the concentration values of the samples containing different concentrations of the C peptide were determined by the conventional detection method and the detection method of the present invention, respectively.
  • Routine test method calibrator 1 - calibrator 6, peak calibrator and sample 1-15, reagent 1 (luminescent antibody, ie, mouse monoclonal antibody coated luminescent particles) and reagent 2 (biotin labeling)
  • the antibody that is, the biotin-labeled murine monoclonal antibody
  • LiCA universal solution streptavidin-labeled photosensitive particles
  • the RLU was read and the results are shown in Table 19.
  • the detection range of conventional detection of C peptide is 0-30ng/ml, and the concentration exceeding the upper limit of detection shows a concentration of >30ng/ml.
  • the detection range is 0-30 ng/ml, and the sample exceeding the upper limit of detection shows a concentration of >30 ng/ml.
  • an ultra-high concentration sample is reported as a low concentration, such as samples 16, 17. Therefore, in the conventional detection, it is impossible to distinguish whether the detection result of the sample to be tested is a true concentration or a low concentration of the ultra-high value sample which is affected by the HD-HOOK effect.
  • Example 16 Detection of hepatitis B virus surface antibody (HbsAb) in a sample
  • the concentration of the hepatitis B virus surface antibody (purchased from Beijing Zhongke Jingda Biotechnology Co., Ltd., Clone No: M2201) in the sample was measured using a kit according to an immunoassay method according to the present invention.
  • the kit includes a calibration product 1 - a calibrator 6 , a peak calibrator, a reagent 1 (a luminescent antigen, that is, an antigen-coated luminescent particle), and a reagent 2 (a biotin-labeled antigen, that is, a biotin-labeled antigen) .
  • Calibrator 1 - Calibrator 6 A sample of known concentration in a conventional kit, the concentration is much smaller than the HOOK sample, and a calibration curve is used to calculate the concentration of the analyte.
  • the high concentration of HBsAb was subjected to gradient dilution, and the concentration values of the samples containing different concentrations of HBsAb were determined by the conventional detection method and the detection method of the present invention, respectively.
  • Routine test method After adding calibrator 1 - calibrator 6, peak calibrator and sample 1-14, reagent 1 (HBsAg coated luminescent particles) and reagent 2 (biotin labeled HBsAg) to the reaction cup, 37 Incubate for 15 min at °C, add LiCA universal solution (streptavidin-labeled photosensitive particles), incubate at 37 ° C for 10 min, read the photon counter, and read RLU. The results are shown in Table 20.
  • the detection range of routine detection of HBsAb is 0-1000 mIU/ml, and the concentration exceeding the upper limit of detection shows a concentration of >1000 mIU/ml.
  • the detection range is 0-1000 mIU/ml, and the sample exceeding the upper limit of detection shows a concentration of >1000 mIU/ml.
  • an ultra-high concentration sample is reported as a low concentration, such as samples 12, 13, and 14. Therefore, in the conventional detection, it is impossible to distinguish whether the detection result of the sample to be tested is a true concentration or a low concentration of the ultra-high value sample which is affected by the HD-HOOK effect.
  • the method of the invention identifies HOOK samples by two readings.
  • the signal value continued to increase with concentration to 10000 mIU/ml, after which the signal value began to decrease with increasing concentration, but the increase A continued to increase with concentration. Therefore, the A value of the sample to be tested and the A value of the calibrator can be directly compared to determine the relationship between the concentration of the sample to be tested and the concentration of the calibrator.
  • the increase A of samples 10-14 was greater than the increase of peak calibrator R0 (37.5%), indicating that the concentrations of HBsAb of samples 10-14 were all greater than 10000 mIU/ml, which were HD-HOOK samples. This is consistent with the actual concentration.
  • the signal values of samples 12, 13, and 14 are lower than that of calibrator 6.
  • the conventional methods have detection concentrations of 802.57 mIU/ml, 352.22 mIU/ml, and 147.9 mIU/ml, respectively, which can be identified as HD by the method of the present invention. -HOOK effect sample, dilution test is required.

Abstract

一种鉴定HD-HOOK效应样本的方法、一种鉴定免疫测定中的HD-HOOK效应的系统、试剂盒和装置、一种免疫测定方法、一种用于鉴定免疫测定的系统、试剂盒和装置。该鉴别HD-HOOK效应样本的方法包括：对校准品、峰值校准品、含待测目标抗原（或抗体）的检测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0，对比待测样本的第二次和第一次读数之间的增幅A'是否大于R0，如果大于R0则样本具有HD-HOOK效应，如果小于R0则不具有HD-HOOK效应。

Description

鉴定HD-HOOK效应样本和免疫测定的方法、系统、试剂盒及装置
相关申请的交叉引用
本申请要求享有于2016年11月22日提交的名称为“免疫测定方法、用于鉴定免疫测定的系统和试剂盒”的中国专利申请CN201611026623.1的优先权，该申请的全部内容通过引用并入本文中。
本申请要求享有于2016年11月22日提交的名称为“免疫测定方法、用于鉴定免疫测定的系统和试剂盒”的中国专利申请CN201611034237.7的优先权，该申请的全部内容通过引用并入本文中。
本申请要求享有于2016年11月22日提交的名称为“鉴别HD-HOOK效应样本的方法和鉴定免疫测定中的HD-HOOK效应的系统”的中国专利申请CN201611034252.1的优先权，该申请的全部内容通过引用并入本文中。
本申请要求享有于2017年8月15日提交的名称为“一种免疫测定装置”的中国专利申请CN201710695530.6的优先权，该申请的全部内容通过引用并入本文中。
技术领域
本发明涉及光激化学发光技术领域，具体涉及一种鉴定HD-HOOK效应样本的方法、一种用于鉴定免疫测定中的HD-HOOK效应的系统、试剂盒和装置、一种免疫测定方法、一种用于鉴定免疫测定的系统、试剂盒和装置。
背景技术
免疫学检测是基于抗原抗体特异性反应的原理进行的，由于其可以利用同位素、酶、化学发光物质等对被测物进行显示或信号的放大，因此常被用于检测蛋白质、激素等微量生物活性物质。
化学发光免疫分析则是近年来发展较迅速的非放射性免疫检测技术，其原理是利用化学发光物质进行信号的放大，并借助其发光强度，对免疫结合过程进行直接测定，该法已成为免疫学检测的重要方向之一。
光激化学发光法是化学发光分析技术的常用方法之一，可用于研究生物分子间的相互作用，临床上主要用于疾病的检测。该技术整合了高分子微粒技术、有机合成、蛋白质化学及临床检测等相关领域的研究。它通过感光微粒和发光微粒在一定范围内结合，产生离子氧能量的传递，发出光信号，从而对待测样本进行检测。其中，感光微粒内部填充有感光化合物，而发光微粒内部填充有发光化合物和镧系元素。在红色激光(600～700nm)的激发下，感光微粒释放出高能态的单线态氧离子(4μS)，其传播距离约为200nm。当感光微粒和发光微粒的距离足够接近时，感光微粒释放的单线态氧离子能到达发光微粒，并通过一系列的化学反应，发射出520～620nm高能级的光，而被仪器检测到。在本反应体系中，微粒的浓度很低，碰撞几率较小，本底信号微弱。只有在感光微粒和发光微粒通过免疫反应结合以后，才会发射出明显的光，因此系统的灵敏度很高。在疾病诊断中，常用的检测模式包含三到四个组分：包被抗原或抗体的发光微粒、生物素或地高辛标记的抗原或抗体、亲和素或抗地高辛包被的感光微粒，中和抗原或抗体等。以上各组份通过两步以上温育反应与待测抗原或抗体结合，并通过化学发光量的强弱对待测样本进行定性或定量检测。与传统的酶联免疫分析方法相比，它具有均相、灵敏度高和操作简便易于自动化等特点。因此，其应用前景十分广阔。
对于双抗夹心的检测模式中，当待检测物质浓度高到一定浓度时，会因为不能形成双抗夹心复合物从而信号值偏低的现象，称为高剂量-钩状效应(HD-HOOK效应)。也就是说，高剂量-钩状效应是指在双位点夹心免疫实验中，其剂量反应曲线的高剂量区段，线性走向不是呈平台状无限后延，而是向下弯曲状，似一只钩子，导致产生假阴性的现象。
HD-HOOK效应在免疫检测中经常发生，其发生率占阳性样本30％左右。由于HD-HOOK效应的存在导致被检测样本不能被正确区分为是由于其浓度超出检测试剂盒的线性范围还是本身浓度就是该值，以至于实验误诊，尤其是导致假阴性率上升。
具体来说，一方面，在检测高浓度的样本时，高剂量-钩状效应可能导致检测信号偏低，样本也因此被判读为偏低浓度。既往的解决办法是增加试剂的组分，对待测样本进行稀释或进行两步法检测等。
另一方面，因为高剂量-钩状效应，当样本浓度的升高到一定值时，信号并不能持续升高，限制了检测范围。既往主要通过优化抗体或提高抗体来拓宽检测范 围。
常规检测流程有以下5个步骤：反应孔中加入待测物及试剂、第一步温育、添加通用液、第二步温育和读数。
本发明的检测方法是基于常规检测流程，在不中断反应的前提下，在反应过程多次读取信号值，通过观察信号的变化来判断样本的真实浓度。
发明内容
针对现有技术中存在的缺陷，本发明的目的在于提供一种免疫测定方法，本发明方法在不中断反应的前提下，通过两次读数来拓宽检测范围，并可以准确判断出待检样本是否存在HOOK效应及简便快速地计算出待测样本中待测物的浓度。
为了实现上述目的及其他相关目的，本发明采用如下技术方案：
本发明第一方面提供了一种鉴别HD-HOOK效应样本的方法，所述方法包括如下步骤：对校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0，对比待测样本的第二次和第一次读数之间的增幅A’是否大于R0，如果大于R0则样本具有HD-HOOK效应，如果小于R0则不具有HD-HOOK效应。
需要说明的是，检测待测样本的差值增幅和峰值校准品的差值增幅的反应条件和反应之间均一致。根据本发明一个优选的实施方式，所述方法包括如下步骤：
(1)将校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(5)将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0；
(6)将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则鉴定此样本为HD-HOOK效应样本。
在本发明上下文中，术语“峰值校准品”是指含特定浓度的待测物的样本，其中双抗夹心免疫实验的待测物剂量反应曲线的高剂量区段，线性走向开始向下弯曲时的浓度为峰值校准品中的待测物浓度。
根据本发明一个优选的实施方式，将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则待测样本为HD-HOOK效应样本，需要稀释；如果A’小于R0，则直接用校准曲线计算出样本浓度；
所述的校准曲线为根据校准品的第一次读数与校准品的浓度所做的曲线。
根据本发明一个优选的实施方式，发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
根据本发明一个优选的实施方式，步骤(2)和(3)中，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
根据本发明一个优选的实施方式，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
本发明的第二方面提供一种用于鉴定免疫测定中的HD-HOOK效应的系统，所述系统包括：
免疫反应装置，其用于实施化学发光免疫反应，
化学发光免疫反应激发和计数装置，其用于激发和记录化学发光的第一次和第二次读数，
处理器，其用于根据待测样本的第二次和第一次读数之间的差值增幅A’来确定HD-HOOK效应样本的存在。
根据本发明一个优选的实施方式，所述系统包括：
免疫反应装置，其用于实施化学发光免疫反应，
化学发光免疫反应激发和计数装置，其用于激发和记录化学发光的第一次和第二次读数，
处理器，其用于对比待测样本的第二次和第一次读数之间的差值增幅A’是否大于峰值校准品的第二次和第一次读数之间差值的增幅R0，如果大于R0则样本具有HD-HOOK效应，如果小于R0则不具有HD-HOOK效应，
其中化学发光的第二次读数是针对同一免疫反应间隔一段时间后再次激发和读数得到的。
在一个具体的实施方式中，本发明用于鉴定免疫测定的系统包括免疫反应装置，例如盛放溶液的容器；化学发光免疫反应激发和计数装置，例如光子计数模块和发光二极管；以及处理器，例如电脑，对所述读数进行处理和作图等。这种用于鉴定免疫测定的系统可以例如参考本申请人的实用新型专利CN201532646U，其以引用方式引入本申请。
根据本发明一个优选的实施方式，所述系统的使用方法包括如下步骤：
(1)将校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(5)将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0；
(6)将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则鉴定此样本为HD-HOOK效应样本。
根据本发明，将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则待测样本为HD-HOOK效应样本，需要稀释；如果A’小于R0，则直接用校准曲线计算出样本浓度；
所述的校准曲线为根据校准品的第一次读数与校准品的浓度所做的曲线。
本发明的第三方面提供一种试剂盒，包括校准品、峰值校准品、第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)、标记物特异结合物 标记的感光微粒，其特征在于，所述试剂盒的使用方法包括如下步骤：对校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，根据待测样本的第二次和第一次读数之间的差值增幅A’来确定HD-HOOK效应样本的存在。
根据本发明一个优选的实施方式，所述试剂盒的使用方法包括如下步骤：对校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，对比待测样本的第二次和第一次读数之间的差值增幅A’是否大于峰值校准品的第二次和第一次读数之间差值的增幅R0，如果大于R0则样本具有HD-HOOK效应，如果小于R0则不具有HD-HOOK效应。
根据本发明一个优选的实施方式，所述试剂盒的使用方法包括如下步骤：
(1)将校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(5)将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0；
(6)将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则鉴定此样本为HD-HOOK效应样本。
根据本发明一个优选的实施方式，将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则待测样本为HD-HOOK效应样本，需要稀释；如果A’小于R0，则直接用校准曲线计算出样本浓度；
所述的校准曲线为根据校准品的第一次读数与校准品的浓度所做的曲线。
在此，需要特别说明的是，上述方法为非疾病诊断目的的方法，所述方法用于在双抗体夹心免疫法或者双抗原夹心免疫法检测过程中，简便快速地挑选出HD-HOOK效应样本，以防止将高浓度抗原(或抗体)样本误判为低浓度抗原(或 抗体)样本。
优选地，所述抗原是指具有免疫原性的物质。例如蛋白质、多肽。代表性的抗原包括(但不限于)：细胞因子、肿瘤标志物、金属蛋白类、心血管糖尿病相关蛋白等。
所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
本发明实施例中，所述抗原或抗体选自乙肝表面抗原(HBsAg)、乙肝表面抗体(HBsAb)、癌抗原125(CA125)、铁蛋白(Ferr)和C肽(CP)。
可用本发明方法检测的样本没有特别限制，可以是任何含有待测目标抗原(或抗体)的样本，代表性的例子可包括血清样本、尿液样本、唾液样本等。本发明优选的样本是血清样本。
优选的，所述第一抗体和第二抗体指可特异性结合于所述抗原的抗体。
对于同一抗原而言，相应的第一抗体和第二抗体可以是相同的也可以是不同的，并且可同时结合于所述的抗原。
所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
对于同一抗体而言，相应的第一抗原和第二抗原可以是相同的也可以是不同的，并且可同时结合于所述的抗体。
优选的，所述标记物与标记物特异结合物之间能够特异性结合。
更优选的，所述标记物为生物素，所述标记物特异结合物为链霉亲和素。
优选的，所述发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒。发光化合物可以是Dioxene(二氧杂环己烯)或thioxene(二甲基噻吩)的衍生物等，镧系元素化合物可以是Eu(TTA)3/TOPO或Eu(TTA)3/Phen等，该微粒可由市场上购得。发光微粒的表面官能团可以是任何能联接蛋白质的基团，如羧基，醛基，胺基，环氧乙基或卤代烷基等各种已知的可连接蛋白质的官能团。
优选的，所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。当其与发光微粒距离足够近的情况下，单线氧离子传递到发光微粒，与发光微粒中的发光化合物反应，产生紫外光，紫外光再进一步激发镧系元素化合物，产生一定波长的光子。感光化合物可以是酞菁染料等，该微粒也可由市场上购得。
优选的，步骤(2)和(3)中，以600～700nm的红色激发光照射，检测反应溶液的发射光量。发射光的检测波长为520～620nm。
进一步的，红色激光(600～700nm)照射感光微粒，感光微粒释放的单线态氧离子，一部分单线态氧离子被发光微粒接收，从而发射出520～620nm高能级的光。
在检测范围内，待测目标抗原的浓度表现为双抗体夹心复合物的数量，并与光子数成正比；但当待测目标抗原浓度过高时，部分待测抗原分别与单个抗体结合，导致双抗夹心复合物减少，光信号偏低，不能反映待测目标抗原的真实浓度。
同理，在检测范围内，待测目标抗体的浓度表现为双抗原夹心复合物的数量，并与光子数成正比；但当待测目标抗体浓度过高时，部分待测抗体分别与单个抗原结合，导致双抗原夹心复合物减少，光信号偏低，不能反映待测目标抗体的真实浓度。
本发明的方法，通过两次读数，比较两次读数所得信号值增幅之间的关系，从而可以起到拓宽检测范围和区分HD-HOOK效应样本的作用。两次读数的差异由以下三个方面决定：
第一方面，第一次读数时，感光微粒受红色激光(600～700nm)照射后，释放出单线态氧离子。一部分单线态氧离子传递至发光微粒后，通过一系列的化学反应，发射出520～620nm高能级的光；而一部分单线态氧离子则与未被抗体(或抗原)结合的待测目标抗原(或抗体)反应，使得待测目标抗原(或抗体)的浓度降低。对于低浓度的样本，待测目标抗原(或抗体)浓度下降后，双抗夹心复合物减少，第二次读数信号值会降低；而对于高浓度的HD-HOOK效应样本，待测目标抗原(或抗体)浓度降低后，双抗夹心复合物增多，第二次读数信号值反而升高。
第二方面，对于低浓度样本而言，感光微粒在第一次读数过程中受红色激光(600～700nm)照射，释放单线态氧离子后，其能量有所损耗，第二次读数信号会降低。
第三方面，对于HD-HOOK效应而言，第一次读数时，抗原抗体反应尚未达到平衡，在两次读数的间隔时间，反应仍会朝正方向进行，第二次读数信号会增高。
综上所述，本发明在反应未达到平衡时进行第一次读数，感光微粒受激发光照射释放单线态氧，一部分传递到发光微粒，一部分能与未结合的待检测目标抗原或抗体反应，消耗部分待检测目标抗原或抗体，使得反应平衡逆向移动，另一 方面感光微粒在激发过一次后，有所损耗，当第二次读数时，待测目标抗原或抗体浓度低的样本的信号值会降低；而浓度高样本的双抗夹心复合物与感光微粒的结合在第一次读数时远未到达平衡，第二次读数时反应会朝正反应方向移动，故而信号会增高，随着待测目标抗原(或抗体)浓度的升高，第二次光激发光的信号值与第一次信号值的增高幅度也增高。信号的增幅与样本浓度正相关，比较两次信号的增幅可以提示一个信号值低而增幅高的样本是HD-HOOK效应。
本发明第四方面提供了一种鉴别HD-HOOK效应样本的测定装置，包括：
读数单元，用于记录化学发光免疫反应并对温育后的混合液进行多次读数；
与所述读数单元连接的处理单元，所述处理单元根据所述读数单元的读数判断免疫测定是否存在HD-HOOK风险。
在本发明的一些实施方式中，还包括移动机构，用于将温育后的混合液移动至读数单元进行读数。
在本发明的另一些实施方式中，所述装置还包括温育器，用于为化学发光免疫反应提供合适的环境温度。
在本发明的一些实施方式中，所述装置还包括复位机构，用于将完成读数后的混合液复位至所述温育器进行再温育。
在本发明的一些具体实施方式中，所述移动机构为推移机构，所述复位机构为推回机构，所述混合液采用板条盛放。
在本发明的另一些具体实施方式中，所述读数单元用于记录化学发光免疫反应并对温育后的混合液进行两次读数。
在本发明的一些具体实施方式中，所述温育器包括第一温育器和第二温育器，所述推移机构用于将第一温育器内温育后的混合液推送至第二温育器进行温育，且所述推移机构用于将第二温育器内温育后的混合液推送至读数单元进行第一次读数；
所述推回机构用于将完成第一次读数后的混合液推回至第二温育器进行再温育；
所述推移机构还用于将第二温育器内再温育后的混合液推送至读数单元进行第二次读数；
当所述处理单元检测到第二次读数和第一次读数的增幅大于标准曲线的最 大值时，则判断免疫测定存在HOOK风险。
在本发明的另一些具体实施方式中，所述推回机构包括：
底板；
设置在所述底板上的导轨；
设置在所述导轨上的移杯机构，所述移杯机构用于承载板条；
驱动装置，用于带动所述移杯机构沿所述导轨移动；
设置在所述底板两端的光电传感器，所述光电传感器用于检测所述移杯机构的位置；
与所述光电传感器连接的位置调节机构，所述位置调节机构能够根据所述光电传感器发出的位置信号对所述移杯机构的位置进行调整。
在本发明的一些具体实施方式中，所述导轨为直轨或变轨。
在本发明的一些具体实施方式中，所述装置还包括设置在所述温育器一侧的用于完成待测样本和试剂混合的板条加样盘，和设置在所述温育器另一侧的用于存放试剂的试剂冷藏区。
在本发明的另一些具体实施方式中，所述装置还包括设置在所述板条加样盘一侧的空白板条堆栈和加载机构，所述空白板条堆栈和加载机构用于将空白板条推至板条加样盘。
在本发明的一些具体实施方式中，所述装置还包括样本试管载架，所述样本试管载架用于承载样本试管。
在本发明的另一些具体实施方式中，所述装置还包括设置在所述样本试管载架靠近空白板条堆栈和加载机构一侧的稀释板振荡器，所述稀释板振荡器用于对预稀释板进行稀释处理。
在本发明的一些具体实施方式中，所述装置还包括机械臂，所述机械臂上设有加样针；
其中，所述机械臂包括第一机械臂和第二机械臂，所述第一机械臂用于从所述样本试管载架区域吸取样本并分配至板条加样盘的板条内，所述第二机械臂用于从所述试剂冷藏区吸取试剂并分配至板条加样盘的板条内。
在本发明的另一些具体实施方式中，所述装置还包括第一清洗机构和第二清洗机构，所述第一清洗机构用于清洗第一机械臂上的加样针，所述第二清洗机构用于清洗第二机械臂上的加样针。
具体的，样本在进入温育器前，需要经历以下步骤：
1、空白板条通过空白板条堆栈和加载机构被推至板条加样盘位置D0；
2、空白板条被推至板条加样盘后D0位置后，顺时针旋转90度至板条加样盘位置D1，在此位置处，第一机械臂从样本试管载架区域吸取样本并向空白板条进行加样；
3、加样完成后的板条顺时针旋转90度至板条加样盘位置D2，此位置无动作；
4、板条顺时针旋转90度至板条加样盘位置D3，此位置处，由第二机械臂从试剂冷藏区吸取试剂，并分配到该位置处的板条内。
在所有D3位置处的板条完成加样并全部推入到温育器(该推送机构图中未显示)后，板条加样盘从D3位置旋转至D0(此过程应等待D1位置上的板条完成样本分配后进行)，完成板条加样盘的一次循环。板条加样盘满负荷运转时，板条加样盘的D0位置在加载空白板条，D1位置在进行样本分配，D2位置在等待，D3位置在进行试剂分配并在试剂分配后将板条推至温育器。板条加样盘的转动，应等待D0～D3这四个区域内的所有动作都完成，才能进行转动。
加样完成后的板条进入第一温育器温育完成后，推移机构将板条推送至第二温育器，该过程中，还需在含待测样本和试剂的板条中加入标记物特异结合物标记的感光微粒，之后样本进入第二温育器12进行温育，温育后，由推送机构将板条推送至读数单元进行激发光照射并检测发射光量，记录第一次读数。将完成第一次读数后的板条由推回机构推回至第二温育器进行再温育，再温育后，所述板条再由推动机构推送至读数单元进行激发光照射并检测发射光量，记录第二次读数。完成两次读数后，处理单元对两次读数进行处理，当第二次读数和第一次读数的增幅大于标准曲线的最大值时，则判断该免疫测定存在HOOK风险。一种方法是该装置定性的给出HOOK提示，操作人员可对样品进行稀释后再进行测定；第二种方法是该装置直接给出定量的结果，但该结果比线性范围高很多。
与现有技术相比，该装置通过设置读数单元，使得读数单元对温育后的混合液进行两次甚至多次读数，并通过处理单元对读数单元的读数进行处理，进而判断免疫测定是否存在HOOK风险，避免HOOK效应所导致的被检测样本不能被正确区分是由于其浓度超出检测试剂盒的线性范围还是本身浓度就是该值，从而避免实验误诊。
本发明的第五方面提供了一种免疫测定方法，所述方法包括如下步骤：(1)对含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，并将第二次和第一次读数之间的差值增幅记为A，(2)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的差值增幅A’做标准曲线和/或根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数的差值增幅A”做标准；(3)将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线和/或标准进行比较。
需要说明的是，检测待测样本的差值增幅和已知标准物质的差值增幅的反应条件和反应之间均一致。
在本发明的一些实施方式中，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线进行比较。
在本发明的一些实施例中，所述已知标准物质的浓度低于产生HOOK效应的浓度，且所述的已知标准物质为阳性对照。
在本发明的另一些实施例中，所述方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
在本面发明的一些具体实施例中，所述方法包括如下步骤：
(a1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(a2)第一次读数：在步骤(a1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(a3)第二次读数：将步骤(a2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(a4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(a5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；
(a6)如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
在本发明的一些实施方式中，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准进行比较，且所述标准记为临界值；和/或所述已知标准物质为阳性对照。
在本发明的一些实施例中，所述方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于已知标准物质的浓度。
在本发明的一些具体实施例中，所述方法包括如下步骤：
(c1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(c2)第一次读数：在步骤(c1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(c3)第二次读数：将步骤(c2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(c4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(c5)根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数增幅A”做临界值；
(c6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
在本发明的一些实施例中，所述方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数低于所述已知标准物质，则对样品进行稀释后再进行测定。
在本发明的一些具体实施例中，所述方法包括如下步骤：
(d1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(d2)第一次读数：在步骤(d1)的混合液中再加入标记物特异结合物标记 的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(d3)第二次读数：将步骤(d2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(d4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(d5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅A”做临界值；
(d6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定。
在本发明的一些实施例中，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线进行比较；且所述方法还包括步骤(4)：确定样本的浓度。
在本发明的一些具体实施例中，所述方法包括如下步骤：
(b1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(b2)第一次读数：在步骤(b1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(b3)第二次读数：将步骤(b2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(b4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(b5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线；
(b6)通过A值确定待测物质浓度是在标准曲线的上升区间或者是在下降区间，再将待测样本的RLU1代入其对应的标准曲线计算浓度。
根据本发明，所述发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
根据本发明，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
根据本发明，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
本发明第六方面提供了一种用于鉴定免疫测定的系统，所述系统包括：
免疫反应装置，其用于实施化学发光免疫反应，
化学发光免疫反应激发和计数装置，其用于激发和记录化学发光的第一次和第二次读数，并将第二次和第一次读数之间的差值增幅记为A；
处理器。
在一个具体的实施方式中，本发明用于鉴定免疫测定的系统包括免疫反应装置，例如盛放溶液的容器；化学发光免疫反应激发和计数装置，例如光子计数模块和发光二极管；以及处理器，例如电脑，对所述读数进行处理和作图等。这种用于鉴定免疫测定的系统可以例如参考本申请人的实用新型专利CN201532646U，其以引用方式引入本申请。
在本发明的一些实施方式中，所述处理器用于根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A，做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
在本发明的一些具体实施方式中，所述系统的使用方法包括如下步骤：
(1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育 后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；
(6)如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
在本发明的一些实施方式中，所述处理器用于将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
在本发明的一些具体实施方式中，所述系统的使用方法包括如下步骤：
(1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅A做临界值；
(6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
在本发明的一些实施方式中，所述处理器用于将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定。
在本发明的一些具体实施方式中，所述系统的使用方法包括如下步骤：
(1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅A做临界值；
(6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定。
在本发明的一些实施方式中，所述处理器用于根据含待测目标抗原(或抗体)的已知的一系列标准物质的第一次读数和两次读数的增幅A分别做标准曲线，将含待测目标抗原(或抗体)的待测样本的第一次读数和两次读数的增幅A与标准曲线进行比较，来确定样本的浓度。
在本发明的一些具体实施方式中，所述系统的使用方法包括如下步骤：
(1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A做标准曲线；
(6)通过A值确定待测物质浓度是在标准曲线的上升区间或者是在下降区 间，再将待测样本的RLU1代入其对应的标准曲线计算浓度；
所述的校准曲线为根据含待测目标抗原(或抗体)的已知的一系列标准物质的第一次读数与已知的一系列标准物质的浓度所做的曲线。
根据本发明，所述发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
根据本发明，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
根据本发明，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
本发明第七方面提供了一种试剂盒，其包括第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)、标记物特异结合物标记的感光微粒，其特征在于，所述试剂盒的使用方法包括如下步骤：(1)对含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，并将第二次和第一次读数之间的差值增幅记为A，(2)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的差值增幅A’做标准曲线和/或根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数的差值增幅A”做标准；(3)将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线和/或标准进行比较。
在本发明的一些实施方式中，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线进行比较。
在本发明的一些实施例中，所述已知标准物质的浓度低于产生HOOK效应的浓度，且所述的已知标准物质为阳性对照。
在本发明的另一些实施例中，所述试剂盒的使用方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
在本发明的一些具体实施例中，所述试剂盒的使用方法包括如下步骤：
(a1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(a2)第一次读数：在步骤(a1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(a3)第二次读数：将步骤(a2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(a4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(a5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；
(a6)如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
在本发明的一些实施方式中，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准进行比较，且所述标准记为临界值；和/或所述已知标准物质为阳性对照。
在本发明的一些实施例中，所述试剂盒的使用方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于已知标准物质的浓度。
在本发明的一些具体实施例中，所述试剂盒的使用方法包括如下步骤：
(c1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(c2)第一次读数：在步骤(c1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(c3)第二次读数：将步骤(c2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(c4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(c5)根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数增 幅A”做临界值；
(c6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
在本发明的一些实施例中，所述试剂盒的使用方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数低于所述已知标准物质，则对样品进行稀释后再进行测定。
在本发明的一些具体实施例中，所述试剂盒的使用方法包括如下步骤：
(d1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(d2)第一次读数：在步骤(d1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(d3)第二次读数：将步骤(d2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(d4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(d5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅A”做临界值；
(d6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定。
在本发明的一些实施例中，所述试剂盒的使用方法还包括步骤(4)：将含待测目标抗原(或抗体)的待测样本的第一次读数和两次读数的增幅A与标准曲线进行比较，来确定样本的浓度。
在本发明的一些具体实施例中，所述试剂盒的使用方法包括如下步骤：
(b1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
(b2)第一次读数：在步骤(b1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
(b3)第二次读数：将步骤(b2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
(b4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
(b5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线；
(b6)通过A值确定待测物质浓度是在标准曲线的上升区间或者是在下降区间，再将待测样本的RLU1代入其对应的标准曲线计算浓度；
所述的校准曲线为根据含待测目标抗原(或抗体)的已知的一系列标准物质的第一次读数与已知的一系列标准物质的浓度所做的曲线。。
在此，需要特别说明的是，上述方法为非疾病诊断目的的方法，所述方法用于在双抗体夹心免疫法或者双抗原夹心免疫法检测过程中，通过两次读数来拓宽检测范围，并可以准确判断出待检样本是否存在HOOK效应及简便快速地计算出待测样本中待测物的浓度。
优选地，所述抗原是指具有免疫原性的物质。例如蛋白质、多肽。代表性的抗原包括(但不限于)：细胞因子、肿瘤标志物、金属蛋白类、心血管糖尿病相关蛋白等。
所述抗体是指机体产生的能识别特定外来物的免疫球蛋白。
本发明一些实施例中，所述抗原或抗体选自胰岛素(INS)、乙型肝炎病毒表面抗体(HBsAb)、甲胎蛋白(AFP)和促甲状腺素(TSH)等。
可用本发明方法检测的样本没有特别限制，可以是任何含有待测目标抗原(或抗体)的样本，代表性的例子可包括血清样本、尿液样本、唾液样本等。本发明优选的样本是血清样本。
优选的，所述第一抗体和第二抗体指可特异性结合于所述抗原的抗体。
对于同一抗原而言，相应的第一抗体和第二抗体可以是相同的也可以是不同的，并且可同时结合于所述的抗原。
所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
对于同一抗体而言，相应的第一抗原和第二抗原可以是相同的也可以是不同的，并且可同时结合于所述的抗体。
优选的，所述标记物与标记物特异结合物之间能够特异性结合。
更优选的，所述标记物为生物素，所述标记物特异结合物为链霉亲和素。
优选的，所述发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒。发光化合物可以是Dioxene(二氧杂环己烯)或thioxene(二甲基噻吩)的衍生物等，镧系元素化合物可以是Eu(TTA)3/TOPO或Eu(TTA)3/Phen等，该微粒可由市场上购得。发光微粒的表面官能团可以是任何能联接蛋白质的基团，如羧基，醛基，胺基，环氧乙基或卤代烷基等各种已知的可连接蛋白质的官能团。
优选的，所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。当其与发光微粒距离足够近的情况下，单线氧离子传递到发光微粒，与发光微粒中的发光化合物反应，产生紫外光，紫外光再进一步激发镧系元素化合物，产生一定波长的光子。感光化合物可以是酞菁染料等，该微粒也可由市场上购得。
优选的，以600～700nm的红色激发光照射，检测反应溶液的发射光量。发射光的检测波长为520～620nm。
进一步的，红色激光(600～700nm)照射感光微粒，感光微粒释放的单线态氧离子，一部分单线态氧离子被发光微粒接收，从而发射出520～620nm高能级的光。
在检测范围内，待测目标抗原的浓度表现为双抗体夹心复合物的数量，并与光子数成正比；但当待测目标抗原浓度过高时，部分待测抗原分别与单个抗体结合，导致双抗夹心复合物减少，光信号偏低，不能反映待测目标抗原的真实浓度。
同理，在检测范围内，待测目标抗体的浓度表现为双抗原夹心复合物的数量，并与光子数成正比；但当待测目标抗体浓度过高时，部分待测抗体分别与单个抗原结合，导致双抗原夹心复合物减少，光信号偏低，不能反映待测目标抗体的真实浓度。
本发明的方法，通过两次读数，比较两次读数所得信号值增幅之间的关系，从而可以起到拓宽检测范围的作用。两次读数的差异由以下三个方面决定：
第一方面，第一次读数时，感光微粒受红色激光(600～700nm)照射后，释放出单线态氧离子。一部分单线态氧离子传递至发光微粒后，通过一系列的化学反应，发射出520～620nm高能级的光；而一部分单线态氧离子则与未被抗体(或抗原)结合的待测目标抗原(或抗体)反应，使得待测目标抗原(或抗体)的浓度降低。对于低浓度的样本，待测目标抗原(或抗体)浓度下降后，双抗夹心复合物减少，第二次读数信号值会降低；而对于高浓度样本，待测目标抗原(或抗体)浓度降低后，双抗夹心复合物增多，第二次读数信号值反而升高。
第二方面，对于低浓度样本而言，感光微粒在第一次读数过程中受红色激光(600～700nm)照射，释放单线态氧离子后，其能量有所损耗，第二次读数信号会降低。
第三方面，对于HD-HOOK效应而言，第一次读数时，抗原抗体反应尚未达到平衡，在两次读数的间隔时间，反应仍会朝正方向进行，第二次读数信号会增高。
综上所述，本发明在反应未达到平衡时进行第一次读数，感光微粒受激发光照射释放单线态氧，一部分传递到发光微粒，一部分能与未结合的待检测目标抗原或抗体反应，消耗部分待检测目标抗原或抗体，使得反应平衡逆向移动，另一方面感光微粒在激发过一次后，有所损耗，当第二次读数时，待测目标抗原或抗体浓度低的样本的信号值会降低；而浓度高样本的双抗夹心复合物与感光微粒的结合在第一次读数时远未到达平衡，第二次读数时反应会朝正反应方向移动，故而信号会增高，随着待测目标抗原(或抗体)浓度的升高，第二次光激发光的信号值与第一次信号值的增高幅度也增高。信号的增幅与样本浓度正相关，比较两次信号的增幅可以来拓宽检测范围，以在检测过程中，简便快速地计算出其浓度。
与现有技术相比，本发明所述方法基于光激化学发光平台(发光氧通道)的免洗和反应的均一性，能实现对一个反应进行多次信号测量而不中断免疫反应的进行，检测出在不同反应时间的光信号，两次信号的大小比较能区分出HD-HOOK效应样本，所述方法不受检测范围限制，有效地拓宽检测范围100倍以上。同时本发明的方法能够100％正确地鉴别双抗夹心法检测中HD-HOOK效应样本，所述方法能够显著提高双抗体夹心法免疫测定的准确性，并降低双抗体夹心法免疫测定的假阴性率。另外，本发明的方法操作简单，通过两次读数来拓宽检测范围，并在检测过程中，简便快速地计算出待测物浓度。
本发明第八方面提供了一种免疫测定装置，其包括：
读数单元，用于记录化学发光免疫反应并对温育后的混合液进行多次读数；
与所述读数单元连接的处理单元，所述处理单元根据所述读数单元的读数判断免疫测定是否存在HOOK风险。
在本发明的一些实施方式中，所述装置还包括移动机构，用于将温育后的混合液移动至读数单元进行读数。
在本发明的另一些实施方式中，所述装置还包括温育器，用于为化学发光免疫反应提供合适的环境温度。
在本发明的一些实施方式中，所述装置还包括复位机构，用于将完成读数后的混合液复位至所述温育器进行再温育。
在本发明的一些具体实施方式中，所述移动机构为推移机构，所述复位机构为推回机构，所述混合液采用板条盛放。
在本发明的另一些具体实施方式中，所述读数单元用于记录化学发光免疫反应并对温育后的混合液进行两次读数。
在本发明的一些具体实施方式中，所述温育器包括第一温育器和第二温育器，所述推移机构用于将第一温育器内温育后的混合液推送至第二温育器进行温育，且所述推移机构用于将第二温育器内温育后的混合液推送至读数单元进行第一次读数；
所述推回机构用于将完成第一次读数后的混合液推回至第二温育器进行再温育；
所述推移机构还用于将第二温育器内再温育后的混合液推送至读数单元进行第二次读数；
当所述处理单元检测到第二次读数和第一次读数的增幅大于标准曲线的最大值时，则判断免疫测定存在HOOK风险。
在本发明的另一些具体实施方式中，所述推回机构包括：
底板；
设置在所述底板上的导轨；
设置在所述导轨上的移杯机构，所述移杯机构用于承载板条；
驱动装置，用于带动所述移杯机构沿所述导轨移动；
设置在所述底板两端的光电传感器，所述光电传感器用于检测所述移杯机构的位置；
与所述光电传感器连接的位置调节机构，所述位置调节机构能够根据所述光电传感器发出的位置信号对所述移杯机构的位置进行调整。
在本发明的一些具体实施方式中，所述导轨为直轨或变轨。
在本发明的一些具体实施方式中，所述装置还包括设置在所述温育器一侧的用于完成待测样本和试剂混合的板条加样盘，和设置在所述温育器另一侧的用于存放试剂的试剂冷藏区。
在本发明的另一些具体实施方式中，所述装置还包括设置在所述板条加样盘一侧的空白板条堆栈和加载机构，所述空白板条堆栈和加载机构用于将空白板条推至板条加样盘。
在本发明的一些具体实施方式中，所述装置还包括样本试管载架，所述样本试管载架用于承载样本试管。
在本发明的另一些具体实施方式中，所述装置还包括设置在所述样本试管载架靠近空白板条堆栈和加载机构一侧的稀释板振荡器，所述稀释板振荡器用于对预稀释板进行稀释处理。
在本发明的一些具体实施方式中，所述装置还包括机械臂，所述机械臂上设有加样针；
其中，所述机械臂包括第一机械臂和第二机械臂，所述第一机械臂用于从所述样本试管载架区域吸取样本并分配至板条加样盘的板条内，所述第二机械臂用于从所述试剂冷藏区吸取试剂并分配至板条加样盘的板条内。
在本发明的另一些具体实施方式中，所述装置还包括第一清洗机构和第二清洗机构，所述第一清洗机构用于清洗第一机械臂上的加样针，所述第二清洗机构用于清洗第二机械臂上的加样针。
具体的，样本在进入温育器前，需要经历以下步骤：
1、空白板条通过空白板条堆栈和加载机构被推至板条加样盘位置D0；
2、空白板条被推至板条加样盘后D0位置后，顺时针旋转90度至板条加样盘位置D1，在此位置处，第一机械臂从样本试管载架区域吸取样本并向空白板条进行加样；
3、加样完成后的板条顺时针旋转90度至板条加样盘位置D2，此位置无动 作；
4、板条顺时针旋转90度至板条加样盘位置D3，此位置处，由第二机械臂从试剂冷藏区吸取试剂，并分配到该位置处的板条内。
在所有D3位置处的板条完成加样并全部推入到温育器(该推送机构图中未显示)后，板条加样盘从D3位置旋转至D0(此过程应等待D1位置上的板条完成样本分配后进行)，完成板条加样盘的一次循环。板条加样盘满负荷运转时，板条加样盘的D0位置在加载空白板条，D1位置在进行样本分配，D2位置在等待，D3位置在进行试剂分配并在试剂分配后将板条推至温育器。板条加样盘的转动，应等待D0～D3这四个区域内的所有动作都完成，才能进行转动。
加样完成后的板条进入第一温育器温育完成后，推移机构将板条推送至第二温育器，该过程中，还需在含待测样本和试剂的板条中加入标记物特异结合物标记的感光微粒，之后样本进入第二温育器12进行温育，温育后，由推送机构将板条推送至读数单元进行激发光照射并检测发射光量，记录第一次读数。将完成第一次读数后的板条由推回机构推回至第二温育器进行再温育，再温育后，所述板条再由推动机构推送至读数单元进行激发光照射并检测发射光量，记录第二次读数。完成两次读数后，处理单元对两次读数进行处理，当第二次读数和第一次读数的增幅大于标准曲线的最大值时，则判断该免疫测定存在HOOK风险。一种方法是该装置定性的给出HOOK提示，操作人员可对样品进行稀释后再进行测定；第二种方法是该装置直接给出定量的结果，但该结果比线性范围高很多。
与现有技术相比，本发明的优点在于，该装置通过设置读数单元，使得读数单元对温育后的混合液进行两次甚至多次读数，并通过处理单元对读数单元的读数进行处理，进而判断免疫测定是否存在HOOK风险，避免HOOK效应所导致的被检测样本不能被正确区分是由于其浓度超出检测试剂盒的线性范围还是本身浓度就是该值，从而避免实验误诊。
附图说明
以下将结合附图来对本发明进行更详细的描述。其中：
图1显示了本发明所述的鉴别HD-HOOK效应样本的测定装置及免疫测定装置的机箱内部结构示意图一。
图2显示了本发明所述的鉴别HD-HOOK效应样本的测定装置及免疫测定装 置的机箱内部结构示意图二。
图3显示了本发明所述的鉴别HD-HOOK效应样本的测定装置及免疫测定装置的推回机构示意图一。
图4显示了本发明所述的鉴别HD-HOOK效应样本的测定装置及免疫测定装置的推回机构示意图二。
图5显示了本发明所述的鉴别HD-HOOK效应样本的测定装置及免疫测定装置的示意图一。
图6显示了本发明所述的鉴别HD-HOOK效应样本的测定装置及免疫测定装置的示意图二。
图7显示了本发明所述的鉴别HD-HOOK效应样本的测定装置及免疫测定装置一个完整测试流程的时序图。
图8：HCG+β采用常规方法所得信号值与样本浓度关系曲线图。
图9：HCG+β采用本发明方法所得信号值和A分别与样本浓度关系曲线图。
图10：Ferr采用常规方法所得信号值与样本浓度关系曲线图。
图11：Ferr采用本发明方法所得信号值和A分别与样本浓度关系曲线图。
图12：C肽采用常规方法所得信号值与样本浓度关系曲线图。
图13：C肽采用本发明方法所得信号值和A分别与样本浓度关系曲线图。
图14：HBsAb采用常规方法所得信号值与样本浓度关系曲线图。
图15：HBsAb采用本发明方法所得信号值和A分别与样本浓度关系曲线图。
图16：INS采用常规检测方法所得信号值与样本浓度的关系曲线图。
图17：INS采用本发明方法所得第一次读数信号和增幅A与样本浓度关系曲线图。
图18：HBsAb采用常规检测方法所得信号值与样本浓度的关系曲线图。
图19：HBsAb采用本发明方法所得第一次读数信号和增幅A与样本浓度关系曲线图。
图20：AFP采用常规检测方法所得信号值与样本浓度的关系曲线图。
图21：AFP采用本发明方法所得第一次读数信号和增幅A与样本浓度关系曲线图。
图22：TSH采用常规检测方法所得信号值与样本浓度的关系曲线图。
图23：TSH采用本发明方法所得第一次读数信号和增幅A与样本浓度关系 曲线图。
图24：Ferr采用常规检测方法所得信号值与样本浓度的关系曲线图。
图25：Ferr采用本发明方法所得第一次读数信号和增幅A与样本浓度关系曲线图。
图26：C肽采用常规检测方法所得信号值与样本浓度的关系曲线图。
图27：C肽采用本发明方法所得第一次读数信号和增幅A与样本浓度关系曲线图。
图28：HBsAb采用常规检测方法所得信号值与样本浓度的关系曲线图。
图29：HBsAb采用本发明方法所得第一次读数信号和增幅A与样本浓度关系曲线图。
在附图中，相同的部件使用相同的附图标记。附图并未按照实际的比例。
具体实施方式
在进一步描述本发明具体实施方式之前，应理解，本发明的保护范围不局限于下述特定的具体实施方案；还应当理解，本发明实施例中使用的术语是为了描述特定的具体实施方案，而不是为了限制本发明的保护范围。
当实施例给出数值范围时，应理解，除非本发明另有说明，每个数值范围的两个端点以及两个端点之间任何一个数值均可选用。除非另外定义，本发明中使用的所有技术和科学术语与本技术领域技术人员通常理解的意义相同。除实施例中使用的具体方法、设备、材料外，根据本技术领域的技术人员对现有技术的掌握及本发明的记载，还可以使用与本发明实施例中所述的方法、设备、材料相似或等同的现有技术的任何方法、设备和材料来实现本发明。
除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明，具体可参见Sambrook等MOLECULAR CLONING：A LABORATORY MANUAL，Second edition，Cold Spring Harbor Laboratory Press，1989and Third edition，2001；Ausubel等，CURRENT PROTOCOLS IN MOLECULAR BIOLOGY，John Wiley&Sons，New York，1987and periodic updates；the series METHODS IN ENZYMOLOGY，Academic Press，San Diego；Wolffe， CHROMATIN STRUCTURE AND FUNCTION，Third edition，Academic Press，San Diego，1998；METHODS IN ENZYMOLOGY，Vol.304，Chromatin(P.M.Wassarman and A.P.Wolffe，eds.)，Academic Press，San Diego，1999；和METHODS IN MOLECULAR BIOLOGY，Vol.119，Chromatin Protocols(P.B.Becker，ed.)Humana Press，Totowa，1999等。
本发明的发明人经过广泛而深入的研究发现，在不中断反应的前提下通过设立两次读数，并将两次读数的增幅A与临界值作比较，可判断待测样本是否需要稀释后再进行测定；或者建立两次读数的增幅A与样本浓度之间的关系，通过两次读数来拓宽检测范围，以在检测过程中，简便快速地计算出其浓度；或者研究两次读数的增幅与样本是否为HD-HOOK效应样本的之间的关系，可以简便有效地排除双抗夹心免疫测定中HD-HOOK效应所导致的假阴性，及提高双抗夹心免疫测定的准确性。另外，本发明的发明人还提供了一种测定装置，该装置可以实现对温育后的混合液进行两次或两次以上的读数，利用该装置可以实施上述鉴别HD-HOOK效应样本的方法及免疫测定方法。
如图1和图2所示，显示了本发明所述的一种鉴别HD-HOOK效应样本的测定装置及免疫测定装置的机箱内部结构示意图，包括：
温育器1，用于为化学发光免疫反应提供合适的环境温度，所述温育器1包括第一温育器11和第二温育器12。
读数单元2，用于记录化学发光免疫反应并对温育后的混合液进行两次读数，所述读数单元2可以是光电倍增管或者激光激发器；
设置于所述温育器1和所述读数单元2之间的推移机构3，所述推移机构包括第一推移机构31和第二推移机构32，第一推移机构31横穿温育器1，第二推移机构32与第一推移机构31的尾端相连接，所述第二推移机构32位于机箱内部。
所述第一推移机构31和第二推移机构32相配合，用于将第一温育器11内温育后的板条推送至读数单元2进行第一次读数，并用于将第二温育器12内温育后的板条推送至读数单元2进行第二次读数。
所述板条为含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)的混合液。所述混合液中还加入了标记物特异结合物标记的感光微粒。
设置于所述读数单元2和所述第二温育器12之间的推回机构，图3所示推回机构为直轨推回机构4，图4所示推回机构为变轨推回机构4′，两种推回机构均可用于将完成第一次读数后的板条推回至第二温育器12进行再温育。
第一次读数时，对温育后的板条进行激发光照射并检测发射光量；第二次读数时，对再温育后的板条进行激发光照射并检测发射光量。
处理单元(图中未显示)，当第二次读数和第一次读数的增幅大于标准曲线的最大值时，则对板条进行稀释后再进行测定。所述标准曲线为根据含待测目标抗原或抗体的已知的一系列标准物质的两次读数的增幅所做，所述标准物质的浓度低于产生HOOK效应的浓度。其中，所述处理单元可以是电脑，对所述读数进行处理和作图等。
可以理解的，将温育器设置为第一温育器和第二温育器，是为了方便免疫测定装置的机械实现，本发明并不做限制。
同样可以理解的，所述移动机构也可以为机械臂抓取式，所述复位机构也可以为机械臂抓取式，本发明并不做限制。
在本发明的一些具体实施例中，所述推回机构包括：
底板41和设置在所述底板41上的导轨，所述导轨为直轨421，如图3所示。所述导轨也可以为变轨422，如图4所示。
驱动装置，用于带动所述移杯机构43沿所述导轨移动。具体的，所述驱动装置包括步进电机441，所述步进电机441通过电机固定板442固定在导轨2的一端，所述步进电机441的输出端设有同步带轮443，所述导轨2的另一端设有惰轮444，所述同步带轮443和所述惰轮444上套设有同步带445。所述惰轮444的轴心穿设有惰轮轴446，所述惰轮轴446固定在惰轮板447上。
设置在所述导轨上的移杯机构43，所述移杯机构43用于承载板条，所述移杯机构43包括移杯拖链板431和与所述移杯拖链板431连接的移杯连接板432，所述移杯连接板432通过同步带压板448连接所述同步带445。
设置在所述底板41两端的光电传感器45，所述光电传感器45连接传感器挡片46，光电传感器45通过传感器挡片46来检测所述移杯机构43的位置。
与所述光电传感器45连接的位置调节机构，所述位置调节机构能够根据所述光电传感器45发出的位置信号对所述移杯机构43的位置进行调整。具体的，所述位置调节机构包括位置调节板46和控制器(图中未显示)，所以控制器分 别连接所述位置调节板46和所述光电传感器45连接，所述控制器根据接收到的光电传感器45发出的位置信号控制位置调节板46来调整移杯机构43的位置。一般的，该位置调节机构用于微调整，当移杯机构43没有达到指定位置或者到达位置有偏差时，该位置调节机构对其进行微调整。
在本发明的另一些具体实施例中，如图5所示，该装置还包括设置在所述温育器1一侧的能够转动的板条加样盘5，空白板条在所述板条加样盘5完成待测样本和试剂的混合，所述样本含待测目标抗原(或抗体)，所述试剂为与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)。
在本发明的一些具体实施例中，该装置还包括设置在所述温育器1另一侧的用于放置试剂的试剂冷藏区8。
在本发明的另一些具体实施例中，还包括设置在所述板条加样盘5一侧的用于将空白板条推至板条加样盘5的空白板条堆栈和加载机构6。
在本发明的一些具体实施例中，还包括样本试管载架7，所述样本试管载架7用于承载样本试管。
在本发明的另一些具体实施例中，还包括机械臂(图中未显示)，所述机械臂上设有加样针；
其中，所述机械臂包括第一机械臂和第二机械臂，所述第一机械臂用于从所述样本试管载架区域吸取样本，所述第二机械臂用于从所述试剂冷藏区吸取试剂。
在本发明的一些具体实施例中，还包括第一清洗机构9和第二清洗机构10，所述第一清洗机构9用于清洗第一机械臂上的加样针，所述第二清洗机构10用于清洗第二机械臂上的加样针。
若设置为直线导轨421，该模块的长度加长，使得机箱(即为图1和图2所示整体结构)和泵11的位置较紧凑(如图5所示)，从而导致拆装会较为复杂。若设置为变轨422，作为一种实施方式，所述变轨422可以设计为曲线导轨，该模块的宽度加宽，使得机箱和第二洗针机构10的位置较紧凑(如图6所示)，但是不会影响拆装。
具体的，样本在进入温育器1前，需要经历以下步骤：
1、空白板条通过空白板条堆栈和加载机构6被推至板条加样盘5位置D0；
2、空白板条被推至板条加样盘5后，顺时针旋转90度至板条加样盘位置 D1，在此位置处，第一机械臂从样本试管载架区域7吸取样本并向空白板条进行加样；
3、加样完成后的板条顺时针旋转90度至板条加样盘位置D2，此位置无动作；
4、板条顺时针旋转90度至板条加样盘位置D3，此位置处，由第二机械臂从试剂冷藏区8吸取试剂，并分配到该位置处的板条内。
在所有D3位置处的板条完成加样并全部推入到温育器1后，板条加样盘5从D3位置旋转至D0(此过程应等待D1位置上的板条完成样本分配后进行)，完成板条加样盘5的一次循环。板条加样盘满负荷运转时，板条加样盘5的D0位置在加载空白板条，D1位置在进行样本分配，D2位置在等待，D3位置在进行试剂分配并在试剂分配后将板条推至温育器1。板条加样盘5的转动，应等待D0～D3这四个区域内的所有动作都完成，才能进行转动。
加样完成后的板条进入第一温育器11温育完成后，推移机构3将所述板条推送至第二温育器，在该过程中加入标记物特异结合物标记的感光微粒，之后板条进入第二温育器12进行温育，由推移机构3将板条推送至读数单元2进行激发光照射并检测发射光量，记录第一次读数。将完成第一次读数后的板条由直轨推回机构4或变轨推回机构4′推回至第二温育器12进行再温育，再温育后，所述板条再由推移机构3推送至读数单元2进行激发光照射并检测发射光量，记录第二次读数。
完成两次读数后，处理单元2对两次读数进行处理，当第二次读数和第一次读数的增幅大于标准曲线的最大值时，且同时所述待测样本的第一次读数低于所述已知标准物质，一种方法是该装置定性的给出Hook提示，操作人员可对样品进行稀释后再进行测定；第二种方法是该装置直接给出定量的结果，但该结果比线性范围高很多。图7给出了一个完整的测试流程时序图，该图为无稀释流程的测试时序图。图中，a、b、c、d和e分别表示第一批板条、第二批板条、第三批板条、第四批板条和第五批板条。
1)假设每次从空白板条堆栈和加载机构6推入8个空白板条至板条加样盘5，该过程大约需要20秒，在图7中忽略不计；
2)板条加样盘5旋转的时间忽略不计；
3)当空白板条旋转至D1位置时，板条在该D1位置进行样本的分配，如图 7中D1所示位置，假设样本分配使用1吸8分，将一个样本分配到不同的八个板条内，每组加样动作需要30秒，则8个板条共计240秒；
4)板条在D3位置进行试剂分配，如图7中D3所示位置，假设试剂分配使用1吸8分，分配完试剂R1后紧接着分配试剂R2，假设每个板条分配试剂的时间为30秒，则8个板条共计480秒；
5)板条从D3位置推至第一温育器11的时间忽略不计，该过程可以在第二机械臂清洗加样针时完成；
6)图7中F表示第一次温育时间；
7)每个板条分配感光微粒的时间在图7中忽略不计，通用液加载区12如图5和图6中所示；
8)图7中F表示第二次温育时间；
9)图7中G表示读数单元读一个板条的时间(含机械移动和丢弃板条时间)。
上述过程在试剂分配阶段分配了两种试剂R1和R2，可以理解的，也可以分配三种试剂R1、R2和R3，假设R1、R2和R3均是在样本分配完成后添加，例如HBeAb，R3为50μl中和e抗原，运行过程和仅有R1和R2情况类似，只是在试剂分配阶段多了一种试剂，R1、R2和R3的分配顺序是任意的。
同样可以理解的，R1、R2和R3中，有一种试剂在样本分配前添加，例如CA19-9，R3为15μl样品稀释液，在D1位置使用第二机械臂完成一种试剂的分配，然后再由第一机械臂完成样品的分配，其它过程和仅有R1和R2的情况一致。
同样可以理解的，R1、R2和R3中，R3为预稀释液，需要使用预稀释板，例如HCV，10μl样品+100μl稀释液，然后取25μl稀释后的样品参与测试。在空白板条转动至D1后，第二机械臂将稀释液R3分配到预稀释板中，第一机械臂将样本分配到预稀释板中，如果需要可以进行反复吸打，其中，分配稀释液的过程可以进行一吸多分的联合加样，例如做五个项目，一个项目需要预稀释，其它四个项目不需要预稀释，则第一机械臂吸五份样本，一份分配到预稀释板内，其它四份分配到空白板条内。之后，对预稀释板进行震荡处理，参见图5和图6，样本试管载架7靠近空白板条堆栈和加载机构6的一侧设有稀释板振荡器11，震荡处理后，第一机械臂将稀释后的样品分配到板条内。后续过程和仅有R1和R2的情况一致，此处不再赘述。
如本发明所述，术语“第一抗体”和“第二抗体”指可特异性结合于某一抗原(如肿瘤标志物)的抗体。对于同一抗原(如肿瘤标志物)而言，相应的第一抗体和第二抗体可以是不同的也可以是相同的，并且可同时结合于所述的抗原。术语“第一抗原”和“第二抗原”指可特异性结合于某一抗体(如乙肝表面抗体)的抗原。对于同一抗体(如乙肝表面抗体)而言，相应的第一抗原和第二抗原可以是不同的也可以是相同的，并且可同时结合于所述的抗体。
如本发明所述，术语“抗原”是指具有免疫原性的物质，例如蛋白质、多肽。代表性的抗原包括(但不限于)：细胞因子、肿瘤标志物、金属蛋白类、心血管糖尿病相关蛋白等。
如本发明所述，术语“肿瘤标志物”是指在肿瘤的发生和增殖过程中，由肿瘤细胞本身所产生的或者是由机体对肿瘤细胞反应而产生的，反应肿瘤存在和生长的一类物质。本领域代表性的肿瘤标志物包括(但不限于)：甲胎蛋白(AFP)、癌抗原125(CA125)等。
双抗夹心法的基本原理：
双抗体夹心法的基本原理是本领域技术人员所熟知的。常规的做法是将第一抗体固定于固相载体，然后将第一抗体与抗原反应，再与标记的第二抗体反应，最后进行化学发光或酶联显色反应检测信号。
光激化学发光法的基本原理：
光激化学发光法的基本原理是本领域技术人员所熟知的。常规的做法是通过感光微粒和发光微粒在一定范围内结合，产生离子氧能量的传递，发出光信号，从而对待测样本进行检测。其中，感光微粒内部填充有感光化合物，而发光微粒内部填充有发光化合物和镧系元素。在红色激光(600～700nm)的激发下，感光微粒释放出高能态的单线态氧离子(4μS)，其传播距离约为200nm。当感光微粒和发光微粒的距离足够接近时，感光微粒释放的单线态氧离子能到达发光微粒，并通过一系列的化学反应，发射出520～620nm高能级的光，而被仪器检测到。
在本发明的一个优选实施例中，充分利用了第一抗体固定在发光微粒上的特点，同时采用生物素标记第二抗体，链霉亲和素包被感光微粒，将血清样本或抗原标准质控品液与第一抗体包被的发光微粒、生物素标记第二抗体依次或同时加入反应容器中，再加入链霉亲和素标记的感光微粒，从而发生以下反应：
(1)发光微粒上的第一抗体与血清样本或抗原标准质控品液中相应的抗原 结合，形成“抗原－第一抗体－发光微粒”三元复合物；
(2)第二抗体与血清样本或抗原标准质控品液中相应的抗原结合，最终形成“第二抗体－抗原－第一抗体－发光微粒”双抗夹心复合物；
生物素和链霉亲和素特异性结合，使得双抗夹心复合物与感光微粒结合到一起。
此时，感光微粒和发光微粒之间的距离小于200nm，红色激光(600～700nm)照射感光微粒后，释放的单线态氧能够被发光微粒接收。通过一系列化学反应，发射出520～620nm高能级的光，并通过化学发光量的强弱对待测样本进行定性或定量检测。
在本发明的另一个优选实施例中，充分利用了第一抗原固定在发光微粒上的特点，同时采用生物素标记第二抗原，链霉亲和素包被感光微粒，将血清样本或抗原标准质控品液与第一抗原包被的发光微粒、生物素标记第二抗原依次或同时加入反应容器中，再加入链霉亲和素标记的感光微粒，从而发生以下反应：
(1)发光微粒上的第一抗原与血清样本或抗原标准质控品液中相应的抗体积结合，形成“抗体－第一抗原－发光微粒”三元复合物；
(2)第二抗原与血清样本或抗原标准质控品液中相应的抗体结合，最终形成“第二抗原－抗体－第一抗原－发光微粒”双抗夹心复合物；
生物素和链霉亲和素特异性结合，使得双抗夹心复合物与感光微粒结合到一起。
此时，感光微粒和发光微粒之间的距离小于200nm，红色激光(600～700nm)照射感光微粒后，释放的单线态氧能够被发光微粒接收。通过一系列化学反应，发射出520～620nm高能级的光，并通过化学发光量的强弱对待测样本进行定性或定量检测。
以下，进一步说明本发明的相关操作细节。
(1)第一抗体(或抗原)包被的发光微粒，记为试剂1，可购买于博阳生物科技有限公司。
(2)第二抗体(或抗原)可用各种本领域已知的标记物及其特异结合物系统进行标记。优选地是通过生物素-亲和素系统标记第二抗体(或抗原)。生物素标记的第二抗体(或抗原)，记为试剂2，可购买于博阳生物科技有限公司。
(3)链霉亲和素包被的感光微粒，记为通用液，可购买于博阳生物科技有 限公司。
(4)校准品：
用待测抗原(或抗体)配置一定浓度范围内(峰值校准品浓度等于HD-HOOK效应浓度)的已知标准物质溶液。将校准品、试剂1、试剂2混匀，温育反应后加入LiCA通用液，继续温育反应一段时间后第一次读数(RLU1)，再温育一段时间后进行第二次读数(RLU2)，计算A＝(RLU2/RLU1-1)×100％，根据标准物质的RLU1和两次读数的增幅A分别与标准物质浓度做校准曲线和标准曲线；标准物质的RLU1与浓度的校准曲线表现为在非HD-HOOK效应阶段，RLU1随浓度的升高而升高，记为RLU1的上升区间，浓度升高到HD-HOOK效应阶段后，RLU1随浓度的升高而降低，记为RLU1的下降区间；标准物质的两次度数的增幅与浓度的标准曲线表现为增幅随浓度的升高而升高，不受HD-HOOK效应影响。
已知标准物质溶液的浓度范围根据需要可跨越HD-HOOK效应浓度或低于HD-HOOK效应浓度。
(5)样品的检测：
可用本发明方法检测的样品没有特别限制，可以是任何含有抗原(或抗体)的样品，代表性的例子可包括血清样本、尿液样本、唾液样本等。优选的样品是血清样品。
(6)样品浓度计算：
将待测样本两次读数增幅A值与校准品的A比较，若待测样本A大于校准品的A，则此样本浓度大于校准品的浓度；若同时此样本RLU1小于此校准品的RLU1，则表明此样本的RLU1低是由于HD-HOOK效应导致的，需要稀释检测；
或者将待测样本两次读数增幅A值代入校准品A与校准品浓度的标准曲线中，判断出待测样本浓度是处于RLU1的上升区间还是下降区间，再将待测样本的RLU1代入所在区间的校准品RLU1与校准品浓度的校准曲线中计算待测样本浓度；
或者将测样本两次读数增幅A值与HD-HOOK效应分界值R0比较，如果A小于R0，则此样本不是HD-HOOK效应样本，将待测样本的RLU1代入校准品RLU1与校准品浓度的校准曲线中计算待测样本浓度；如果A大于等于R0，则鉴定此样本为HD-HOOK效应样本，需要稀释检测。
实施例1：检测人血清样本中人绒毛膜促性腺激素及β亚单位(HCG+β)
采用博阳生物科技(上海)有限公司生产的人绒毛膜促性腺激素及β亚单位(HCG+β)检测试剂盒(光激化学发光法)来检测血清样本中人绒毛膜促性腺激素及β亚单位(HCG+β)的含量。所述试剂盒包括校准品1-校准品6(亦即，已知的一系列标准物质)、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
另需用到的组分：LiCA通用液(链霉亲和素标记的感光微粒)，该产品为博阳生物科技公司生产光激化学发光分析系统的辅助试剂。与仪器及相应的光激化学发光法检测试剂盒配套使用，用于抗原、抗体的检测。
对由Roche检测(超过检测限样本进行稀释检测)得18例HCG+β浓度的病人血清样本，分别进行常规方法及本发明方法检测。
常规检测方法：将待测样本、校准品，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)分别加入反应杯后，37℃温育15min，加入通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，计算得样本浓度，结果如表1所示。
采用本发明两次读数法：将待测样本、校准品，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)，37℃温育15min，加入通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，结果如表1所示。
表1：常规检测及本发明检测的结果
Figure PCTCN2017112145-appb-000001
Figure PCTCN2017112145-appb-000002
注：HCG+β常规检测的检测范围为0-10000mIU/ml，超出检测上限样本显示浓度为>10000mIU/ml。
Roche检测结果作为真实浓度，由表1和图8可知，常规检测中，浓度上升到54531mIU/ml信号值随浓度升高而增高，浓度继续升高，信号值随HCG+β浓 度升高而降低，即浓度大于54531mIU/ml则HD-HOOK。在常规检测中，检测范围为0-10000mIU/ml，超出检测上限样本显示浓度为>10000mIU/ml。当HD-HOOK效应样本浓度持续升高，信号持续下降，就会出现将超高浓度样本报告成偏低浓度情况，如样本18。故而在常规检测中就不能分辨待测样本的检测结果是真实浓度还是超高值样本受HD-HOOK效应影响而报告的偏低浓度。
本发明方法通过两次读数来鉴别HOOK效应导致的报告浓度偏低样本。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度的指标之一。由表1和图9可知，信号值随浓度续升高到54531mIU/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。故直接比较待测样本的A值与校准品的A值，即可判断待测样本浓度与校准品浓度的大小关系。样本10～18的增幅A均大于校准品6的增幅(11.1％)，且A值持续增高，表明样本10～18的HCG+β浓度均大于10000mIU/ml，且浓度持续升高，这与Roche的浓度结果相符，样本18信号值低于校准品6，常规方法检测浓度为8713.02mIU/ml，通过本发明方法能鉴别其为HD-HOOK效应样本，需进行稀释检测。
实施例2：检测样本中铁蛋白(Ferr)
采用博阳生物科技(上海)有限公司生产的铁蛋白(Ferr)检测试剂盒(化学发光法)来检测样本中铁蛋白(购自Fitzgerald，Catalog No：30-AF10)的含量。所述试剂盒包括校准品1-校准品6(亦即，已知的一系列标准物质)、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作标校准曲线计算待测物浓度。
另需用到的组分：LiCA通用液(链霉亲和素标记的感光微粒)，该产品为博阳生物科技公司生产光激化学发光分析系统的辅助试剂。与仪器及相应的光激化学发光法检测试剂盒配套使用，用于抗原、抗体的检测。
将高浓度的铁蛋白抗原进行梯度稀释，分别采用常规检测方法和本发明检测 方法测定含不同浓度铁蛋白的样本的浓度值。
常规检测方法：将校准品1-校准品6、待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结果如表2所示。
采用本发明两次读数法：将校准品1-校准品6、待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，结果如表2所示，
表2：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000003
Figure PCTCN2017112145-appb-000004
注：铁蛋白常规检测的检测范围为0-2000ng/ml，超出检测上限样本显示浓度为>2000ng/ml。
在常规检测中，由表2和图10可知，浓度上升到51000ng/ml信号值随浓度升高而增高，浓度继续升高，信号值随Ferr浓度升高而降低，常规检测范围为0-2000ng/ml，超出检测上限样本显示浓度为>2000ng/ml。当HD-HOOK效应样本浓度持续升高，当浓度升高到2550000ng/ml，信号下降到校准品6的信号以下，就会出现将超高浓度样本报告成偏低浓度情况，如样本15。故而在常规检测中就不能分辨待测样本的检测结果是真实浓度还是超高值样本受HD-HOOK效应影响而报告的偏低浓度。
本发明方法通过两次读数来鉴别HOOK效应导致的报告浓度偏低样本。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度的指标之一。由表2和图11可知，信号值随浓度续升高到51000ng/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。故直接比较待测样本的A值与校准品的A值，即可判断待测样本浓度与校准品浓度的大小关系。样本4～15的增幅A均大于校准品6的增幅(-5.5％)，表明样本4～15的Ferr浓度均大于2000ng/ml。这与实际浓度相符，样本15信号值低于校准品6，常规方法检测浓度为1860.97ng/ml，通过本发明方法能鉴别其为浓度超过检测范围样本，需进行稀释检测。
实施例3：检测样本中C肽(CP)
采用博阳生物科技(上海)有限公司生产的C肽(CP)检测试剂盒(化学发光法)来检测样本中C肽(购自Fitzgerald，Catalog No：30-AC96)的含量。所 述试剂盒包括校准品1-校准品6(亦即，已知的一系列标准物质)、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
另需用到的组分：LiCA通用液(链霉亲和素标记的感光微粒)，该产品为博阳生物科技公司生产光激化学发光分析系统的辅助试剂。与仪器及相应的光激化学发光法检测试剂盒配套使用，用于抗原、抗体的检测。
将高浓度的C肽抗原进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度C肽的样本的浓度值。
常规检测方法：将校准品1-校准品6、待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结果如表3所示。
采用本发明两次读数法：将校准品1-校准品6、待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，结果如表3所示，
表3：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000005
Figure PCTCN2017112145-appb-000006
注：C肽常规检测的检测范围为0-30ng/ml，超出检测上限样本显示浓度为>30ng/ml。
在常规检测中，由表3和图12可知，浓度上升到10000ng/ml信号值随浓度升高而增高，浓度继续升高，信号值随C肽浓度升高而降低，常规检测范围为0-30ng/ml，超出检测上限样本显示浓度为>30ng/ml。当浓度升高到 33500000ng/ml，信号下降到校准品6的信号以下，就会出现将超高浓度样本报告成偏低浓度情况，如样本16、17。故而在常规检测中就不能分辨待测样本的检测结果是真实浓度还是超高值样本受HD-HOOK效应影响而报告的偏低浓度。
本发明方法通过两次读数来鉴别HOOK效应导致的报告浓度偏低样本。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度的指标之一。由表3和图13可知，信号值随浓度续升高到10000ng/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。故直接比较待测样本的A值与校准品的A值，即可判断待测样本浓度与校准品浓度的大小关系。样本5～17的增幅A均大于校准品6的增幅(-4.9％)，表明样本5～17的C肽浓度均大于30ng/ml，超过检测上限。这与实际浓度相符，样本16、17信号值低于校准品6，常规方法检测浓度分别为8.15ng/ml、0.76ng/ml，通过本发明方法能鉴别其为超过检测上限样本，需进行稀释检测。
实施例4：检测人血清样本中乙型肝炎病毒表面抗原(HBsAg)
采用博阳生物科技(上海)有限公司生产的乙型肝炎病毒表面抗原(HBsAg)检测试剂盒(光激化学发光法)检测样本中HBsAg浓度，所述试剂盒包括校准品1-校准品6(亦即，已知的一系列标准物质)、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作标准曲线计算待测物浓度。
另需用到的组分：LiCA通用液(链霉亲和素标记的感光微粒)，该产品为博阳生物科技公司生产光激化学发光分析系统的辅助试剂。与仪器及相应的光激化学发光法检测试剂盒配套使用，用于抗原、抗体的检测。
先用本发明方法检测校准品1-校准品6、待测的血清样本1-15：将待测物，试剂1(抗体包被的发光微粒)和试剂2(生物素标记的抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增 幅A＝(RLU2/RLU1-1)×100％，结果如表4所示。
表4：本发明方法检测结果
Figure PCTCN2017112145-appb-000007
本发明方法得到15个血清样本的浓度如表4所示，先通过与校准品6的增幅比较区分出超过检测上限(336.56IU/mL)的样本，即A值大于27％则判断为HOOK效应样本，推荐稀释后检测；而A小于27％则为检测范围内样本，可直接用校准曲线计算出样本浓度。
通过对样本进行梯度稀释后检测浓度变化以验证以上结论的可靠性，将样本1-样本15均进行2被稀释和4倍稀释，同时用常规检测方法检测未稀释的原倍样本、2被稀释样本和4倍稀释样本，通过观察稀释后浓度的变化判断该样本是否有HOOK效应，即若稀释后样本浓度反而升高即为HOOK效应样本。非HOOK效应样本在稀释后浓度会降低。结果如表5所示：
表5：稀释验证结果
Figure PCTCN2017112145-appb-000008
从表5可知，血清样本1、2、3、5、6、9、12、13、14、15稀释后检测浓 度升高，即证明为HD-HOOK效应样本，浓度大于336.56IU/mL。血清样本4、7、8、10、11稀释后浓度降低，证明不是HD-HOOK效应样本，与本发明方法的结果完全相同。
实施例5：检测人血清样本中CA125
采用博阳生物科技(上海)有限公司生产的糖类抗原125(CA125)检测试剂盒(光激化学发光法)检测样本中CA125浓度，所述试剂盒包括校准品1-校准品6亦即，已知的一系列标准物质)、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
另需用到的组分：LiCA通用液(链霉亲和素标记的感光微粒)，该产品为博阳生物科技公司生产光激化学发光分析系统的辅助试剂。与仪器及相应的光激化学发光法检测试剂盒配套使用，用于抗原、抗体的检测。
先用本发明方法检测校准品1-校准品6、待测的血清样本1-18：将待测物，试剂1(抗体包被的发光微粒)和试剂2(生物素标记的抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，结果如表6。
表6：本发明方法检测结果
Figure PCTCN2017112145-appb-000009
Figure PCTCN2017112145-appb-000010
本发明方法得到18个血清样本的浓度如表6所示，先通过与校准品6的增幅比较区分出超过检测上限的样本，即A值大于7.4％则判断为超过检测上限的样本，推荐稀释后检测；而A小于7.4％则为非HOOK效应样本，可直接用校准曲线计算出样本浓度。
通过对样本进行梯度稀释后检测浓度变化以验证以上结论的可靠性，将样本1-样本18均进行2被稀释和4倍稀释，同时用常规检测方法检测未稀释的原倍样本、2被稀释样本和4倍稀释样本，通过观察稀释后浓度的变化判断该样本是否有HOOK效应，即若稀释后样本浓度反而升高即为HOOK效应样本。非HOOK 效应样本在稀释后浓度会降低。结果如7所示：
表7：稀释验证结果
Figure PCTCN2017112145-appb-000011
从表7可知，血清样本16、17、18稀释后检测浓度升高，即证明为HD-HOOK效应样本，血清样本1-样本15稀释后浓度降低，证明不是HD-HOOK效应样本。
与本发明方法的结果完全相同。不稀释样本的情况下，常规方法检测原倍样本，则会因为HD-HOOK效应将血清样本16、17、18误判为偏低浓度样本。
实施例6：检测样本中乙型肝炎病毒表面抗体(HBsAb)
采用博阳生物科技(上海)有限公司生产的乙型肝炎病毒表面抗体检测试剂盒HBsAbHBsAb(光激化学发光法)来检测样本中乙型肝炎病毒表面抗体(购自北京中科京达生物技术有限公司，Clone No：M2201)的含量。所述试剂盒包括校准品1-校准品6(亦即，已知的一系列标准物质)、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作标准曲线计算待测物浓度。
另需用到的组分：LiCA通用液(链霉亲和素标记的感光微粒)，该产品为博阳生物科技公司生产光激化学发光分析系统的辅助试剂。与仪器及相应的光激化学发光法检测试剂盒配套使用，用于抗原、抗体的检测。
将高浓度的HBsAb进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度HBsAb的样本的浓度值。
常规检测方法：将校准品1-校准品6、待测样本1-14，试剂1(HBsAg包被的发光微粒)和试剂2(生物素标记的HBsAg)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结果如表8所示。
采用本发明两次读数法：将校准品1-校准品6、待测样本1-14，试剂1(HBsAg包被的发光微粒)和试剂2(生物素标记的HBsAg)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，结果如表8所示。
表8：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000012
Figure PCTCN2017112145-appb-000013
注：HBsAb常规检测的检测范围为0-1000mIU/ml，超出检测上限样本显示浓度为>1000mIU/ml。
在常规检测中，由表8和图14可知，浓度上升到10000mIU/ml信号值随浓度升高而增高，浓度继续升高，信号值随HBsAb浓度升高而降低，常规检测范围为0-1000mIU/ml，超出检测上限样本显示浓度为>1000mIU/ml。当浓度升高到 335000mIU/ml及以上，信号下降到校准品6的信号以下，就会出现将超高浓度样本报告成偏低浓度情况，如样本12、13、14。故而在常规检测中就不能分辨待测样本的检测结果是真实浓度还是超高值样本受HD-HOOK效应影响而报告的偏低浓度。
本发明方法通过两次读数来鉴别HOOK效应导致的报告浓度偏低样本。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度的指标之一。由表8和图15可知，信号值随浓度续升高到10000mIU/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。故直接比较待测样本的A值与校准品的A值，即可判断待测样本浓度与校准品浓度的大小关系。样本8～14的增幅A均大于校准品6的增幅(35.9％)，表明样本8～14的HBsAb浓度均大于1000mIU/ml，超过检测上限。这与实际浓度相符，样本12、13、14。信号值低于校准品6，常规方法检测浓度分别为802.57mIU/ml、352.22mIU/ml、147.9mIU/ml，通过本发明方法能鉴别其为超过检测上限样本，需进行稀释检测。
实施例7：本发明方法在定性试剂盒Anti-HCV中应用
采用博阳生物科技(上海)有限公司生产的丙型肝炎病毒抗体检测试剂盒(化学发光法)来检测样本中Anti-HCV的含量。所述试剂盒包括参考品、阴性对照、阳性对照、试剂1(发光HCV抗原，亦即，HCV抗原包被的发光微粒)和试剂2(生物素标记HCV抗原，亦即生物素标记的HCV抗原)。
参考品、阴性对照、阳性对照：参考品是用作参考以判断待测样本阴阳性的已知浓度标准品；阴性对照、阳性对照是用作判断试验有效性的已知浓度标准品。将高浓度的Anti-HCV进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度Anti-HCV样本的信号值。
常规检测方法：将梯度稀释的一系列Anti-HCV样本，试剂1(发光HCV抗原，亦即，HCV抗原包被的发光微粒)和试剂2(生物素标记HCV抗原，亦即生物素标记的HCV抗原)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结 果如表9所示。
采用本发明两次读数法：将梯度稀释的一系列Anti-HCV样本，试剂1(发光HCV抗原，亦即，HCV抗原包被的发光微粒)和试剂2(生物素标记HCV抗原，亦即生物素标记的HCV抗原)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，结果如表9所示。
表9：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000014
Figure PCTCN2017112145-appb-000015
如表9所示，抗原稀释倍数降低到10000倍后，信号值随浓度升高而降低，出现HOOK效应，浓度继续升高到一定值时(如样本12)，RLU下降到参考值cut off以下，常规检测方法下就会误判其结果为阴性，本发明方法则可先观察两次信号的增幅A＝(RLU2/RLU1-1)×100％，比较待测样本的A值与阳性对照的A值(-25％)，判断出待测样本与阳性参照之间的大小关系，如上表所示，样本12的A值(47％)远高于阳性对照的A值(-25％)，表明次样本浓度高于阳性对照，是阳性样本，其信号的不丰富是因为HOOK效应，应该进行稀释验证。
实施例8：检测样本中胰岛素(INS)
采用博阳生物科技(上海)有限公司生产的胰岛素(INS)检测试剂盒(化学发光法)来检测样本中胰岛素(购自Fitzgerald，Catalog No：30R-2704)的含量。
将高浓度的胰岛素抗原进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度胰岛素的样本的信号值。
常规检测方法：将已知浓度的待测物样本，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结果如表10所示。
采用本发明两次读数法：将知浓度的待测物样本，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，检测结果如表10所示：
表10：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000016
Figure PCTCN2017112145-appb-000017
由表10和图16可知，浓度从3μIU/ml到10000μIU/ml信号值随浓度升高而增高，浓度继续升高，信号值随胰岛素浓度升高而降低，即浓度大于10,000μIU/ml则HD-HOOK，在常规检测中，抗原浓度高于此检测范围的样本报告浓度将会偏低(报告浓度均小于10,000μIU/ml)。
本发明方法通过两次读数来拓宽检测范围。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度区间的指标之一。由表10和图17可知，信号值随浓度续升高到10,000μIU/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。
当胰岛素校准品浓度覆盖到从3μIU/ml到1,000,000μIU/ml范围，用本发明方法分别作出RLU1和A校准曲线和标准曲线(如图17)，随浓度升高，A持续上升，RLU1分为3μIU/ml到10,000μIU/ml的上升区间和10,000μIU/ml到1,000,000μIU/ml的下降区间。用本发明方法检测得到待测样本的RLU1、RLU2和A。先通过A值确定待测物质浓度是在3μIU/ml到10,000μIU/ml的上升区间或者是10,000μIU/ml到1,000,000μIU/ml的下降区间，再将待测物质的RLU1代入 其对应的校准曲线计算确切浓度。
由表10可知，胰岛素浓度为10,000μIU/ml时，有信号峰值，其对应A为20％，若待测物A<20％，则待测样本不是HD-HOOK样本，将其RLU1代入浓度小于10,000μIU/ml的校准曲线计算浓度；若A≥20％，则待测样本是HD-HOOK样本，将其RLU1代入浓度大于10,000μIU/ml的校准曲线计算浓度，从而将检测上限从10,000μIU/ml拓宽到了1,000,000μIU/ml。
实施例9：检测样本中乙型肝炎病毒表面抗体(HBsAb)
采用博阳生物科技(上海)有限公司生产的乙型肝炎病毒表面抗体检测试剂盒(光激化学发光法)来检测样本中乙型肝炎病毒表面抗体(购自北京中科京达生物技术有限公司，Clone No：M2201)的浓度。
将高浓度的HBsAb进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度HBsAb的样本的信号值。
常规检测方法：梯度稀释的HBsAb样本，试剂1(HBsAg包被的发光微粒)和试剂2(生物素标记的HBsAg)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读取RLU。
采用两次读数法：梯度稀释的HBsAb样本，试剂1(HBsAg包被的发光微粒)和试剂2(生物素标记的HBsAg)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，检测结果如表11所示：
表11：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000018
Figure PCTCN2017112145-appb-000019
由表11和图18可知，浓度从1mIU/ml到10000mIU/ml信号值随浓度升高而增高，浓度继续升高，信号值随HBsAb浓度升高而降低，即浓度大于10,000mIU/ml则HD-HOOK，在常规检测中，抗原浓度高于此检测范围的样本报告浓度将会偏低(报告浓度均小于10,000mIU/ml)。
本发明方法通过两次读数来拓宽检测范围。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度区间的指标之一。由表11和图19可知，信号值随浓度续升高到10,000mIU/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。
当HBsAb校准品浓度覆盖到从1mIU/ml到3,350,000mIU/ml范围，用本发明方法分别作出RLU1和A的校准曲线和标准曲线(如图19)，随浓度升高，A持续上升，RLU1分为1mIU/ml到10,000mIU/ml的上升区间和10,000mIU/ml到3,350,000mIU/ml的下降区间。用本发明方法检测得到待测样本的RLU1、RLU2和A。先通过A值确定待测物质浓度是在1mIU/ml到10,000mIU/ml的上升区间或者是10,000mIU/ml到3,350,000mIU/ml的下降区间，再将待测物质的RLU1代入其对应的校准曲线计算确切浓度。
由表11可知，HBsAb浓度为10,000mIU/ml时，有信号峰值，其对应A为37.5％。若待测物A<37.5％，则待测样本不是HD-HOOK样本，将其RLU1代入 浓度小于10,000mIU/ml的校准曲线计算浓度；若A≥37.5％,则待测样本是HD-HOOK样本，将其RLU1代入浓度大于10,000mIU/ml的校准曲线计算浓度，从而将检测上限从10,000mIU/ml拓宽到了3,350,000mIU/ml。
实施例10：检测样本中胎儿甲种球蛋白(AFP)
采用博阳生物科技(上海)有限公司生产的胎儿甲种球蛋白检测试剂盒(光激化学发光法)来检测样本中AFP(购自Fitzgerald，Catalog No：30-1370)的含量。
将高浓度的AFP抗原进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度AFP的样本的信号值。所述常规检测方法和本发明方法参照实施例8。检测结果如下：
表12：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000020
由表12和图20可知，浓度从5ng/ml到10000ng/ml信号值随浓度升高而增高，浓度继续升高，信号值随AFP浓度升高而降低，即浓度大于10,000ng/ml则 HD-HOOK，在常规检测中，抗原浓度高于此检测范围的样本报告浓度将会偏低(报告浓度均小于10,000ng/ml)。
本发明方法通过两次读数来拓宽检测范围。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度区间的指标之一。由表12和图21可知，信号值随浓度续升高到10,000ng/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。
当AFP校准品浓度覆盖到从5ng/ml到1,000,000ng/ml范围，用本发明方法分别作出RLU1和A的校准曲线和标准曲线(如图21)，随浓度升高，A持续上升，RLU1分为5ng/ml到10,000ng/ml的上升区间和10,000ng/ml到1,000,000ng/ml的下降区间。用本发明方法检测得到待测样本的RLU1、RLU2和A。先通过A值确定待测物质浓度是在5ng/ml到10,000ng/ml的上升区间或者是10,000ng/ml到1,000,000ng/ml的下降区间，再将待测物质的RLU1代入其对应的校准曲线计算确切浓度。
由表12可知，AFP浓度为10,000ng/ml时，有信号峰值，其对应A为18％。若待测物A<18％，则待测样本不是HD-HOOK样本，将其RLU1代入浓度小于10,000ng/ml的校准曲线计算浓度；若A≥18％,则待测样本是HD-HOOK样本，将其RLU1代入浓度大于10,000ng/ml的校准曲线计算浓度，从而将检测上限从10,000ng/ml拓宽到了1,000,000ng/ml。
实施例11：检测样本中促甲状腺素(TSH)
采用博阳生物科技(上海)有限公司生产的促甲状腺素检测试剂盒(光激化学发光法)来检测样本中促甲状腺素(购自Fitzgerald，Catalog No：30R-AT009)的含量。
将高浓度的TSH抗原进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度TSH的样本的信号值。所述常规检测方法和本发明方法参照实施例8。检测结果如下：
表13：常规检测与本发明检测结果
Figure PCTCN2017112145-appb-000021
由表13和图22可知，浓度从1μIU/ml到10000μIU/ml信号值随浓度升高而增高，浓度继续升高，信号值随TSH浓度升高而降低，即浓度大于10,000μIU/ml则HD-HOOK，在常规检测中，抗原浓度高于此检测范围的样本报告浓度将会偏低(报告浓度均小于10,000μIU/ml)。
本发明方法通过两次读数来拓宽检测范围。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度区间的指标之一。由表13和图23可知，信号值随浓度续升高到10,000μIU/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。
当TSH校准品浓度覆盖到从1μIU/ml到1,000,000μIU/ml范围，用本发明方法分别作出RLU1和A的校准曲线和标准曲线(如图23)，随浓度升高，A持续上升，RLU1分为1μIU/ml到10,000μIU/ml的上升区间和10,000μIU/ml到1,000,000μIU/ml的下降区间。用本发明方法检测得到待测样本的RLU1、RLU2 和A。先通过A值确定待测物质浓度是在3μIU/ml到10,000μIU/ml的上升区间或者是10,000μIU/ml到1,000,000μIU/ml的下降区间，再将待测物质的RLU1代入其对应的校准曲线计算确切浓度。
由表13可知，TSH浓度为10,000μIU/ml时，有信号峰值，其对应A为17.0％，若待测物A<17.0％，则待测样本不是HD-HOOK样本，将其RLU1代入浓度小于10,000μIU/ml的校准曲线计算浓度；若A≥17.0％，则待测样本是HD-HOOK样本，将其RLU1代入浓度大于10,000μIU/ml的校准曲线计算浓度，从而将检测上限从10,000μIU/ml拓宽到了1,000,000μIU/ml。
实施例12：检测人血清样本中乙型肝炎病毒表面抗原(HBsAg)
采用依据本发明的一种免疫测定方法所涉及的试剂盒，检测样本中HBsAg浓度，所述试剂盒包括校准品1-校准品6、峰值校准品、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
峰值校准品的选取：将已知的HOOK样本梯度稀释，常规检测其信号值，选取信号值最高的样本作为峰值校准品，即小于此浓度样本不会发生HOOK效应，而高于此浓度就有HOOK效应。其A值记为R0作为判断待测物是否HOOK的临界值。
另需用到的组分：LiCA通用液(链霉亲和素标记的感光微粒)，该产品为博阳生物科技公司生产光激化学发光分析系统的辅助试剂。与仪器及相应的光激化学发光法检测试剂盒配套使用，用于抗原、抗体的检测。
先用本发明方法检测校准品1-校准品6、峰值校准品和待测的血清样本1-15：将待测物，试剂1(抗体包被的发光微粒)和试剂2(生物素标记的抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，检测结果如表14所示：
表14：本发明检测方法检测结果
Figure PCTCN2017112145-appb-000022
本发明方法得到15个血清样本的浓度如表14所示，先通过与峰值校准品的增幅R0比较区分出HOOK效应的样本，即A值大于31％则判断为HOOK效应样本，推荐稀释后检测；而A小于31％则为非HOOK效应样本，可直接用校准曲线计算出样本浓度。
通过对样本进行梯度稀释后检测浓度变化以验证以上结论的可靠性，将样本1-样本15均进行2被稀释和4倍稀释，同时用常规检测方法检测未稀释的原倍样本、2被稀释样本和4倍稀释样本，通过观察稀释后浓度的变化判断该样本是否有HOOK效应，即若稀释后样本浓度反而升高即为HOOK效应样本。非HOOK效应样本在稀释后浓度会降低。结果如表15所示：
表15：稀释验证结果
Figure PCTCN2017112145-appb-000023
从表15可知，血清样本1、2、3、5、6、9、12、13、14、15稀释后检测浓度升高，即证明为HD-HOOK效应样本，血清样本4、7、8、10、11稀释后浓度降低，证明不是HD-HOOK效应样本。与本发明方法的结果完全相同。
实施例13：检测人血清样本中CA125
采用依据本发明的一种免疫测定方法所涉及的试剂盒，检测样本中CA125浓度，所述试剂盒包括校准品1-校准品6、峰值校准品、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
峰值校准品的选取：将已知的HOOK样本梯度稀释，常规检测其信号值，选取信号值最高的样本作为峰值校准品，即小于此浓度样本不会发生HOOK效应，而高于此浓度就有HOOK效应。其A值记为R0作为判断待测物是否HOOK的临界值。
先用本发明方法检测校准品1-校准品6、峰值校准品和待测的血清样本1-18：将待测物，试剂1(抗体包被的发光微粒)和试剂2(生物素标记的抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，检测结果如表16所示：
表16：本发明检测方法检测结果
Figure PCTCN2017112145-appb-000024
Figure PCTCN2017112145-appb-000025
本发明方法得到18个血清样本的浓度如表16所示，先通过与峰值校准品的增幅R0比较区分出HOOK效应的样本，即A值大于18.7％则判断为HOOK效应样本，推荐稀释后检测；而A小于18.7％则为非HOOK效应样本，可直接用校准曲线计算出样本浓度。
通过对样本进行梯度稀释后检测浓度变化以验证以上结论的可靠性，将样本1-样本18均进行2被稀释和4倍稀释，同时用常规检测方法检测未稀释的原倍样本、2被稀释样本和4倍稀释样本，通过观察稀释后浓度的变化判断该样本是否有HOOK效应，即若稀释后样本浓度反而升高即为HOOK效应样本。非HOOK效应样本在稀释后浓度会降低。结果如表17所示：
表17：稀释验证结果
Figure PCTCN2017112145-appb-000026
Figure PCTCN2017112145-appb-000027
从表17可知，血清样本16、17、18稀释后检测浓度升高，即证明为HD-HOOK效应样本，血清样本1-样本15稀释后浓度降低，证明不是HD-HOOK效应样本。与本发明方法的结果完全相同。
实施例14：检测样本中铁蛋白(Ferr)
采用依据本发明的一种免疫测定方法所涉及的试剂盒来检测样本中铁蛋白(购自Fitzgerald，Catalog No：30-AF10)的含量。所述试剂盒包括校准品1-校准品6、峰值校准品、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
峰值校准品的选取：将已知的HOOK样本梯度稀释，常规检测其信号值，选取信号值最高的样本作为峰值校准品，在本实验中即为样本9，小于此浓度样本 不会发生HOOK效应，而高于此浓度就有HOOK效应。其A值记为R0作为判断待测物是否HOOK的临界值。
将高浓度的铁蛋白抗原进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度铁蛋白的样本的浓度值。
常规检测方法：将校准品1-校准品6、峰值校准品和待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结果如下表所示。
采用本发明两次读数法：将校准品1-校准品6、峰值校准品和待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，检测结果如表18所示：
表18：常规检测和本发明检测结果
Figure PCTCN2017112145-appb-000028
Figure PCTCN2017112145-appb-000029
注：铁蛋白常规检测的检测范围为0-2000ng/ml，超出检测上限样本显示浓度为>2000ng/ml。
由表18和图24可知，常规检测高浓度抗原梯度稀释的样本，浓度上升到51000ng/ml信号值随浓度升高而增高，浓度继续升高，信号值随Ferr浓度升高而降低，即浓度大于51000ng/ml则HD-HOOK，51000ng/ml的样本9即为峰值校准品，R0为13.9％。
在常规检测中，检测范围为0-2000ng/ml，超出检测上限样本显示浓度为>2000ng/ml。当HD-HOOK效应样本浓度持续升高，信号持续下降，就会出现将超高浓度样本报告成偏低浓度情况，如样本15。故而在常规检测中就不能分辨待测样本的检测结果是真实浓度还是超高值样本受HD-HOOK效应影响而报告的偏低浓度。
本发明方法通过两次读数来鉴别HOOK样本。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度的指标之一。由表18和图25可知，信号值随浓度续升高到51000ng/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。故直接比较待测样本的A值与校准品的A值，即可判断待测样本浓度与校准品浓度的大小关系。样本10～15的增幅A均大于峰值校准品的增幅R0 (13.9％)，表明样本10～15的Ferr浓度均大于51000ng/ml，为HD-HOOK样本。这与实际浓度相符，样本15信号值低于校准品6，常规方法检测浓度为1860.97ng/ml，通过本发明方法能鉴别其为HD-HOOK效应样本，需进行稀释检测。
实施例15：检测样本中C肽(CP)
采用依据本发明的一种免疫测定方法所涉及的试剂盒来检测样本中C肽(购自Fitzgerald，Catalog No：30-AC96)的含量。所述试剂盒包括校准品1-校准品6、峰值校准品、试剂1(发光抗体，亦即，抗体包被的发光微粒)、试剂2(生物素标记抗体，亦即生物素标记的抗体)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
峰值校准品的选取：将已知的HOOK样本梯度稀释，常规检测其信号值，选取信号值最高的样本作为峰值校准品，在本实验中即为样本9，小于此浓度样本不会发生HOOK效应，而高于此浓度就有HOOK效应。其A值记为R0作为判断待测物是否HOOK的临界值。
将高浓度的C肽抗原进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度C肽的样本的浓度值。
常规检测方法：将校准品1-校准品6、峰值校准品和待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结果如表19所示。
采用本发明两次读数法：将校准品1-校准品6、峰值校准品和待测样本1-15，试剂1(发光抗体，亦即，鼠单克隆抗体包被的发光微粒)和试剂2(生物素标记抗体，亦即生物素标记的鼠单克隆抗体)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，检
测结果如表19所示：
表19：常规检测和本发明检测结果
Figure PCTCN2017112145-appb-000030
注：C肽常规检测的检测范围为0-30ng/ml，超出检测上限样本显示浓度为>30ng/ml。
由表19和图26可知，常规检测高浓度抗原梯度稀释的样本，浓度上升到10000ng/ml信号值随浓度升高而增高，浓度继续升高，信号值随C肽浓度升高而降低，即浓度大于10000ng/ml则HD-HOOK，10000ng/ml的样本9即为峰值校准品，R0为23.3％。
在常规检测中，检测范围为0-30ng/ml，超出检测上限样本显示浓度为>30ng/ml。当HD-HOOK效应样本浓度持续升高，信号持续下降，就会出现将超高浓度样本报告成偏低浓度情况，如样本16、17。故而在常规检测中就不能分辨待测样本的检测结果是真实浓度还是超高值样本受HD-HOOK效应影响而报告的偏低浓度。
实施例16：检测样本中乙型肝炎病毒表面抗体(HbsAb)
采用依据本发明的一种免疫测定方法所涉及的试剂盒来检测样本中乙型肝炎病毒表面抗体(购自北京中科京达生物技术有限公司，Clone No：M2201)的浓度。所述试剂盒包括校准品1-校准品6、峰值校准品、试剂1(发光抗原，亦即，抗原包被的发光微粒)、试剂2(生物素标记抗原，亦即生物素标记的抗原)。
校准品1-校准品6：常规试剂盒中的已知浓度样本，浓度远小于HOOK样本，作校准曲线计算待测物浓度。
峰值校准品的选取：将已知的HOOK样本梯度稀释，常规检测其信号值，选取信号值最高的样本作为峰值校准品，在本实验中即为样本9，小于此浓度样本不会发生HOOK效应，而高于此浓度就有HOOK效应。其A值记为R0作为判断待测物是否HOOK的临界值。
将高浓度的HBsAb进行梯度稀释，分别采用常规检测方法和本发明检测方法测定含不同浓度HBsAb的样本的浓度值。
常规检测方法：将校准品1-校准品6、峰值校准品和待测样本1-14，试剂1(HBsAg包被的发光微粒)和试剂2(生物素标记的HBsAg)加入反应杯后，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育10min，光子计数器读数，读取RLU，结果如表20所示。
采用本发明两次读数法：将校准品1-校准品6、峰值校准品和待测样本1-14，试剂1(HBsAg包被的发光微粒)和试剂2(生物素标记的HBsAg)，37℃温育15min，加入LiCA通用液(链霉亲和素标记的感光微粒)，37℃温育3min，读数RLU1，37℃继续温育7min，读数RLU2，并计算第二次信号值的增幅A＝(RLU2/RLU1-1)×100％，检测结果如表20所示：
表20：常规检测和本发明检测结果
Figure PCTCN2017112145-appb-000031
Figure PCTCN2017112145-appb-000032
注：HBsAb常规检测的检测范围为0-1000mIU/ml，超出检测上限样本显示浓度为>1000mIU/ml。
由表20和图28可知，常规检测高浓度HBsAb梯度稀释的样本，浓度上升到10000mIU/ml信号值随浓度升高而增高，浓度继续升高，信号值随HBsAb浓度升高而降低，即浓度大于10000mIU/ml则HD-HOOK，10000mIU/ml的样本9即为峰值校准品，R0为37.5％。
在常规检测中，检测范围为0-1000mIU/ml，超出检测上限样本显示浓度为>1000mIU/ml。当HD-HOOK效应样本浓度持续升高，信号持续下降，就会出现将超高浓度样本报告成偏低浓度情况，如样本12、13、14。故而在常规检测中就不能分辨待测样本的检测结果是真实浓度还是超高值样本受HD-HOOK效应影响而报告的偏低浓度。
本发明方法通过两次读数来鉴别HOOK样本。每个待测样本先后检测到信号值结果RLU1、RLU2，将第二次读数的增幅A＝(RLU2/RLU1-1)×100％作为判断样本浓度的指标之一。由表20和图29可知，信号值随浓度续升高到10000mIU/ml，之后信号值开始随浓度升高而下降，但是增幅A却是随浓度持续上升的。故直接比较待测样本的A值与校准品的A值，即可判断待测样本浓度与校准品浓度的大小关系。样本10～14的增幅A均大于峰值校准品的增幅R0(37.5％)，表明样本10～14的HBsAb浓度均大于10000mIU/ml，为HD-HOOK样本。这与实际浓度相符，样本12、13、14信号值低于校准品6，常规方法检测浓度分别为802.57mIU/ml、352.22mIU/ml、147.9mIU/ml，通过本发明方法能鉴别其为HD-HOOK效应样本，需进行稀释检测。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (92)

  1. 一种鉴别HD-HOOK效应样本的方法，其特征在于，所述方法包括如下步骤：对校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0，对比待测样本的第二次和第一次读数之间的增幅A’是否大于R0，如果大于R0则样本具有HD-HOOK效应，如果小于R0则不具有HD-HOOK效应。
  2. 根据权利要求1所述的方法，其特征在于，所述方法包括如下步骤：
    (1)将校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (5)将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0；
    (6)将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则鉴定此样本为HD-HOOK效应样本。
  3. 根据权利要求1或2所述的方法，其特征在于，将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则待测样本为HD-HOOK效应样本，需要稀释；如果A’小于R0，则直接用校准曲线计算出样本浓度；
    所述的校准曲线为根据校准品的第一次读数与校准品的浓度所做的曲线。
  4. 根据权利要求2所述的方法，其特征在于，发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
  5. 根据权利要求2所述的方法，其特征在于，步骤(2)和(3)中，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
  6. 根据权利要求2所述的方法，其特征在于，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
  7. 一种用于鉴定免疫测定中的HD-HOOK效应的系统，所述系统包括：
    免疫反应装置，其用于实施化学发光免疫反应，
    化学发光免疫反应激发和计数装置，其用于激发和记录化学发光的第一次和第二次读数，
    处理器，其用于根据待测样本的第二次和第一次读数之间的差值增幅A’来确定HD-HOOK效应样本的存在。
  8. 根据权利要求7所述的系统，所述系统包括：
    免疫反应装置，其用于实施化学发光免疫反应，
    化学发光免疫反应激发和计数装置，其用于激发和记录化学发光的第一次和第二次读数，
    处理器，其用于对比待测样本的第二次和第一次读数之间的差值增幅A’是否大于峰值校准品的第二次和第一次读数之间差值的增幅R0，如果大于R0则样本具有HD-HOOK效应，如果小于R0则不具有HD-HOOK效应，
    其中化学发光的第二次读数是针对同一免疫反应间隔一段时间后再次激发和读数得到的。
  9. 根据权利要求7所述的系统，其特征在于，所述系统的使用方法包括如下步骤：
    (1)将校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (5)将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0；
    (6)将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则鉴定此样本为HD-HOOK效应样本。
  10. 根据权利要求9所述的系统，其特征在于，将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则待测样本为HD-HOOK效应样本，需要稀释；如果A’小于R0，则直接用校准曲线计算出样本浓度；
    所述的校准曲线为根据校准品的第一次读数与校准品的浓度所做的曲线。
  11. 根据权利要求9所述的系统，其特征在于，发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
  12. 根据权利要求9所述的系统，其特征在于，步骤(2)和(3)中，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
  13. 根据权利要求9所述的系统，其特征在于，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
  14. 一种试剂盒，包括校准品、峰值校准品、第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)、标记物特异结合物标记的感光微粒，其特征在于，所述试剂盒的使用方法包括如下步骤：对校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，根据待测样本的第二次和第一次读数之间的差值增幅A’来确定HD-HOOK效应样本的存在。
  15. 根据权利要求14所述的试剂盒，其特征在于，所述试剂盒的使用方法包括如下步骤：对校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，对比待测样本的第二次和第一次读数之间的差值增幅A’是否大于峰值校准品的第二次和第一次读数之间差值的增幅R0，如果大于R0则样本具有HD-HOOK效应，如果小于R0则不具有HD-HOOK效应。
  16. 根据权利要求14所述的试剂盒，其特征在于，所述试剂盒的使用方法包括如下步骤：
    (1)将校准品、峰值校准品、含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (5)将峰值校准品的第二次和第一次读数之间差值的增幅A记为R0；
    (6)将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则鉴定此样本为HD-HOOK效应样本。
  17. 根据权利要求14所述的试剂盒，其特征在于，将待测样本的两次读数增幅A’值与R0比较，如果A’大于等于R0，则待测样本为HD-HOOK效应样本，需要稀释；如果A’小于R0，则直接用校准曲线计算出样本浓度；
    所述的校准曲线为根据校准品的第一次读数与校准品的浓度所做的曲线。
  18. 根据权利要求16所述的试剂盒，其特征在于，发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
  19. 根据权利要求16所述的试剂盒，其特征在于，步骤(2)和(3)中，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
  20. 根据权利要求16所述的试剂盒，其特征在于，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
  21. 一种鉴别HD-HOOK效应样本的测定装置，其特征在于，包括：
    读数单元，用于记录化学发光免疫反应并对温育后的混合液进行多次读数；
    与所述读数单元连接的处理单元，所述处理单元根据所述读数单元的读数判断免疫测定是否存在HD-HOOK风险。
  22. 根据权利要求21所述的免疫测定装置，其特征在于，还包括移动机构，用于将温育后的混合液移动至读数单元进行读数。
  23. 根据权利要求21或22所述的免疫测定装置，其特征在于，还包括温育器，用于为化学发光免疫反应提供合适的环境温度。
  24. 根据权利要求23所述的免疫测定装置，其特征在于，还包括复位机构，用于将完成读数后的混合液复位至所述温育器进行再温育。
  25. 根据权利要求24所述的免疫测定装置，其特征在于，所述移动机构为推移机构，所述复位机构为推回机构，所述混合液采用板条盛放。
  26. 根据权利要求21-25中任意一项所述的免疫测定装置，其特征在于，所述读数单元用于记录化学发光反应并对温育后的混合液进行两次读数。
  27. 根据权利要求25所述的免疫测定装置，其特征在于，
    所述温育器包括第一温育器和第二温育器，所述推移机构用于将第一温育器内温育后的混合液推送至第二温育器进行温育，且所述推移机构用于将第二温育器内温育后的混合液推送至读数单元进行第一次读数；
    所述推回机构用于将完成第一次读数后的混合液推回至第二温育器进行再温育；
    所述推移机构还用于将第二温育器内再温育后的混合液推送至读数单元进行第二次读数；
    当所述处理单元检测到第二次读数和第一次读数的增幅A大于标准曲线的最大值时，则判断免疫测定存在HOOK风险。
  28. 根据权利要求25所述的免疫测定装置，其特征在于，所述推回机构包括：
    底板；
    设置在所述底板上的导轨；
    设置在所述导轨上的移杯机构，所述移杯机构用于承载板条；
    驱动装置，用于带动所述移杯机构沿所述导轨移动；
    设置在所述底板两端的光电传感器，所述光电传感器用于检测所述移杯机构的位置；
    与所述光电传感器连接的位置调节机构，所述位置调节机构能够根据所述光电传感器发出的位置信号对所述移杯机构的位置进行调整。
  29. 根据权利要求28所述的免疫测定装置，其特征在于，所述导轨为直轨或 变轨。
  30. 根据权利要求23-25中任意一项或权利要求27-29中任意一项所述的免疫测定装置，其特征在于，还包括设置在所述温育器一侧的用于完成待测样本和试剂混合的板条加样盘，和设置在所述温育器另一侧的用于存放试剂的试剂冷藏区。
  31. 根据权利要求30所述的免疫测定装置，其特征在于，还包括设置在所述板条加样盘一侧的空白板条堆栈和加载机构，所述空白板条堆栈和加载机构用于将空白板条推至板条加样盘。
  32. 根据权利要求31所述的免疫测定装置，其特征在于，还包括样本试管载架，所述样本试管载架用于承载样本试管。
  33. 根据权利要求32所述的免疫测定装置，其特征在于，还包括设置在所述样本试管载架靠近空白板条堆栈和加载机构一侧的稀释板振荡器，所述稀释板振荡器用于对预稀释板进行稀释处理。
  34. 根据权利要求32所述的免疫测定装置，其特征在于，还包括机械臂，所述机械臂上设有加样针；
    其中，所述机械臂包括第一机械臂和第二机械臂，所述第一机械臂用于从所述样本试管载架区域吸取样本并分配至板条加样盘的板条内，所述第二机械臂用于从所述试剂冷藏区吸取试剂并分配至板条加样盘的板条内。
  35. 根据权利要求34所述的免疫测定装置，其特征在于，还包括第一清洗机构和第二清洗机构，所述第一清洗机构用于清洗第一机械臂上的加样针，所述第二清洗机构用于清洗第二机械臂上的加样针。
  36. 一种免疫测定方法，所述方法包括如下步骤：(1)对含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，并将第二次和第一次读数之间的差值增幅记为A，(2)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的差值增幅A’做标准曲线或根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数的差值增幅A”做标准；(3)将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线和/或标准进行比较。
  37. 根据权利要求36所述的免疫测定方法，其特征在于，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线 进行比较。
  38. 根据权利要求37所述的免疫测定方法，其特征在于，所述已知的一系列标准物质的浓度低于产生HOOK效应的浓度，和/或已知标准物质为阳性对照。
  39. 根据权利要求38所述的免疫测定方法，其特征在于，所述方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
  40. 根据权利要求39所述的免疫测定方法，其特征在于，所述方法包括如下步骤：
    (a1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (a2)第一次读数：在步骤(a1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (a3)第二次读数：将步骤(a2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (a4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (a5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；
    (a6)如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
  41. 根据权利要求36所述的免疫测定方法，其特征在于，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准进行比较，且所述标准记为临界值；
    和/或所述已知标准物质为阳性对照。
  42. 根据权利要求41所述的免疫测定方法，其特征在于，所述方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于已知标准物质的浓度。
  43. 根据权利要求42所述的免疫测定方法，其特征在于，所述方法包括如下步骤：
    (c1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (c2)第一次读数：在步骤(c1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (c3)第二次读数：将步骤(c2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (c4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (c5)根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数增幅A”做临界值；
    (c6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
  44. 根据权利要求41所述的免疫测定方法，其特征在于，所述方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数低于所述已知标准物质，则对样品进行稀释后再进行测定。
  45. 根据权利要求44所述的免疫测定方法，其特征在于，所述方法包括如下步骤：
    (d1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (d2)第一次读数：在步骤(d1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (d3)第二次读数：将步骤(d2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (d4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (d5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅 A”做临界值；
    (d6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定。
  46. 根据权利要求37所述的免疫测定方法，其特征在于，所述方法还包括步骤(4)：确定样本的浓度。
  47. 根据权利要求46所述的免疫测定方法，其特征在于，所述方法包括如下步骤：
    (b1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (b2)第一次读数：在步骤(b1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (b3)第二次读数：将步骤(b2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (b4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (b5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线；
    (b6)通过A值确定待测物质浓度是在标准曲线的上升区间或者是在下降区间，再将待测样本的RLU1代入其对应的校准曲线计算浓度；
    所述的校准曲线为根据含待测目标抗原(或抗体)的已知的一系列标准物质的第一次读数与已知的一系列标准物质的浓度所做的曲线。
  48. 根据权利要求36-47中任意一项所述的免疫测定方法，其特征在于，所述发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
  49. 根据权利要求40、43、45和47中任意一项所述的方法，其特征在于，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长 为520～620nm。
  50. 根据权利要求40、43、45和47中任意一项所述的免疫测定方法，其特征在于，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
  51. 一种用于鉴定免疫测定的系统，所述系统包括：
    免疫反应装置，其用于实施化学发光免疫反应，
    化学发光免疫反应激发和计数装置，其用于激发和记录化学发光的第一次和第二次读数，并将第二次和第一次读数之间的差值增幅记为A；
    处理器。
  52. 根据权利要求51所述的系统，其特征在于，所述处理器用于根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A，做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
  53. 根据权利要求52所述的系统，其特征在于，所述系统的使用方法包括如下步骤：
    (1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；
    (6)如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
  54. 根据权利要求51所述的系统，其特征在于，所述处理器用于将含待测目 标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
  55. 根据权利要求54所述的系统，其特征在于，所述系统的使用方法包括如下步骤：
    (1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅A做临界值；
    (6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
  56. 根据权利要求51所述的系统，其特征在于，所述处理器用于将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定；
    和/或所述的已知标准物质为阳性对照。
  57. 根据权利要求56所述的系统，其特征在于，所述系统的使用方法包括如下步骤：
    (1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅A做临界值；
    (6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定。
  58. 根据权利要求51所述的系统，其特征在于，所述处理器用于根据含待测目标抗原(或抗体)的已知的一系列标准物质的第一次读数和两次读数的增幅A分别做校准曲线和标准曲线，将含待测目标抗原(或抗体)的待测样本的第一次读数和两次读数的增幅A分别与校准曲线和标准曲线进行比较，来确定样本的浓度。
  59. 根据权利要求58所述的系统，其特征在于，所述系统的使用方法包括如下步骤：
    (1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (2)第一次读数：在步骤(1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (3)第二次读数：将步骤(2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A做标准曲线；
    (6)通过A值确定待测物质浓度是在标准曲线的上升区间或者是在下降区间，再将待测样本的RLU1代入其对应的校准曲线计算浓度。
  60. 根据权利要求51-59任意一项所述的系统，其特征在于，所述发光微粒 是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
  61. 根据权利要求53、55、57和59任意一项所述的系统，其特征在于，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
  62. 根据权利要求53、55、57和59任意一项所述的系统，其特征在于，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
  63. 一种试剂盒，其包括包括第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)、标记物特异结合物标记的感光微粒，其特征在于，所述试剂盒的使用方法包括如下步骤：(1)对含待测目标抗原(或抗体)的待测样本进行化学发光免疫反应，激发和记录化学发光的第一次和第二次读数，并将第二次和第一次读数之间的差值增幅记为A，(2)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的差值增幅A’做标准曲线或根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数的差值增幅A”做标准；(3)将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线和/或标准进行比较。
  64. 根据权利要求63所述的试剂盒，其特征在于，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准曲线进行比较。
  65. 根据权利要求64所述的试剂盒，其特征在于，所述已知的一系列标准物质的浓度低于产生HOOK效应的浓度，和/或已知标准物质为阳性对照。
  66. 根据权利要求65所述的试剂盒，其特征在于，所述试剂盒的使用方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
  67. 根据权利要求66所述的试剂盒，其特征在于，所述试剂盒的使用方法包括如下步骤：
    (a1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (a2)第一次读数：在步骤(a1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (a3)第二次读数：将步骤(a2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (a4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (a5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线，其中标准物质的浓度低于产生HOOK效应的浓度；
    (a6)如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述标准曲线的最大值，则对样品进行稀释后再进行测定。
  68. 根据权利要求63所述的试剂盒，其特征在于，将含待测目标抗原(或抗体)的待测样本的第二次和第一次读数之间的差值增幅A与所述标准进行比较，且所述标准记为临界值。
  69. 根据权利要求68所述的试剂盒，其特征在于，所述试剂盒的使用方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于已知标准物质的浓度；
    和/或所述的已知标准物质为阳性对照。
  70. 根据权利要求69所述的试剂盒，其特征在于，所述试剂盒的使用方法包括如下步骤：
    (c1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (c2)第一次读数：在步骤(c1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (c3)第二次读数：将步骤(c2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (c4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (c5)根据含待测目标抗原(或抗体)的已知的一个标准物质的两次读数增 幅A”做临界值；
    (c6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，则所述待测样本的浓度高于所述已知标准物质的浓度。
  71. 根据权利要求68所述的试剂盒，其特征在于，所述试剂盒的使用方法还包括步骤(4)：如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数低于所述已知标准物质，则对样品进行稀释后再进行测定。
  72. 根据权利要求71所述的试剂盒，其特征在于，所述试剂盒的使用方法包括如下步骤：
    (d1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (d2)第一次读数：在步骤(d1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (d3)第二次读数：将步骤(d2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (d4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (d5)根据含待测目标抗原(或抗体)的一个已知标准物质的两次读数增幅A”做临界值；
    (d6)将含待测目标抗原(或抗体)的待测样本的两次读数的增幅A与临界值作比较，如果含待测目标抗原(或抗体)的待测样本的两次读数的增幅A大于所述临界值，且同时所述待测样本的第一次读数所得信号值低于所述已知标准物质，则对样品进行稀释后再进行测定。
  73. 根据权利要求64所述的试剂盒，其特征在于，所述试剂盒的使用方法还包括步骤(4)：确定样本的浓度。
  74. 根据权利要求73所述的试剂盒，其特征在于，所述试剂盒的使用方法包括如下步骤：
    (b1)将含待测目标抗原(或抗体)的待测样本与第一抗体(或抗原)包被 的发光微粒、标记物标记的第二抗体(或抗原)混合，温育得混合液；
    (b2)第一次读数：在步骤(b1)的混合液中再加入标记物特异结合物标记的感光微粒，温育后进行激发光照射并检测发射光量，光子计数器读数，计为RLU1；
    (b3)第二次读数：将步骤(b2)中进行第一次读数后的反应溶液进一步温育后，再进行激发光照射并检测发射光量，光子计数器读数，计为RLU2；
    (b4)计算样本第二次读数所得信号值相对于第一次读数所得信号值的增幅A，A＝(RLU2/RLU1-1)×100％；
    (b5)根据含待测目标抗原(或抗体)的已知的一系列标准物质的两次读数的增幅A’做标准曲线；
    (b6)通过A值确定待测物质浓度是在标准曲线的上升区间或者是在下降区间，再将待测样本的RLU1代入其对应的校准曲线计算浓度；
    所述标准曲线为根据含待测目标抗原(或抗体)的已知的一系列标准物质的第一次读数与已知的一系列标准物质的浓度所做的曲线。
  75. 根据权利要求63-74中任意一项所述的试剂盒，其特征在于，所述发光微粒是指填充有发光化合物和镧系元素化合物的高分子微粒；所述感光微粒是填充有感光化合物的高分子微粒，在红色激光激发下，可以产生单线态氧离子。
  76. 根据权利要求67、70、72和74中任意一项所述的试剂盒，其特征在于，以600～700nm的红色激发光照射，检测反应溶液的发射光量；发射光的检测波长为520～620nm。
  77. 根据权利要求67、70、72和74中任意一项所述的试剂盒，其特征在于，所述抗原是指具有免疫原性的物质；所述抗体是指机体产生的能识别特定外来物的免疫球蛋白；所述第一抗体和第二抗体指可特异性结合于所述目标抗原的抗体；所述第一抗原和第二抗原指可特异性结合于所述目标抗体的抗原。
  78. 一种免疫测定装置，其特征在于，包括：
    读数单元，用于记录化学发光免疫反应并对温育后的混合液进行多次读数；
    与所述读数单元连接的处理单元，所述处理单元根据所述读数单元的读数判断免疫测定是否存在HOOK风险。
  79. 根据权利要求78所述的免疫测定装置，其特征在于，还包括移动机构，用于将温育后的混合液移动至读数单元进行读数。
  80. 根据权利要求78或79所述的免疫测定装置，其特征在于，还包括温育器，用于为化学发光免疫反应提供合适的环境温度。
  81. 根据权利要求80所述的免疫测定装置，其特征在于，还包括复位机构，用于将完成读数后的混合液复位至所述温育器进行再温育。
  82. 根据权利要求81所述的免疫测定装置，其特征在于，所述移动机构为推移机构，所述复位机构为推回机构，所述混合液采用板条盛放。
  83. 根据权利要求78-82中任意一项所述的免疫测定装置，其特征在于，所述读数单元用于记录化学发光反应并对温育后的混合液进行两次读数。
  84. 根据权利要求82所述的免疫测定装置，其特征在于，
    所述温育器包括第一温育器和第二温育器，所述推移机构用于将第一温育器内温育后的混合液推送至第二温育器进行温育，且所述推移机构用于将第二温育器内温育后的混合液推送至读数单元进行第一次读数；
    所述推回机构用于将完成第一次读数后的混合液推回至第二温育器进行再温育；
    所述推移机构还用于将第二温育器内再温育后的混合液推送至读数单元进行第二次读数；
    当所述处理单元检测到第二次读数和第一次读数的增幅A大于标准曲线的最大值时，则判断免疫测定存在HOOK风险。
  85. 根据权利要求82所述的免疫测定装置，其特征在于，所述推回机构包括：
    底板；
    设置在所述底板上的导轨；
    设置在所述导轨上的移杯机构，所述移杯机构用于承载板条；
    驱动装置，用于带动所述移杯机构沿所述导轨移动；
    设置在所述底板两端的光电传感器，所述光电传感器用于检测所述移杯机构的位置；
    与所述光电传感器连接的位置调节机构，所述位置调节机构能够根据所述光电传感器发出的位置信号对所述移杯机构的位置进行调整。
  86. 根据权利要求85所述的免疫测定装置，其特征在于，所述导轨为直轨或变轨。
  87. 根据权利要求80-82中任意一项或权利要求84-86中任意一项所述的免疫 测定装置，其特征在于，还包括设置在所述温育器一侧的用于完成待测样本和试剂混合的板条加样盘，和设置在所述温育器另一侧的用于存放试剂的试剂冷藏区。
  88. 根据权利要求87所述的免疫测定装置，其特征在于，还包括设置在所述板条加样盘一侧的空白板条堆栈和加载机构，所述空白板条堆栈和加载机构用于将空白板条推至板条加样盘。
  89. 根据权利要求88所述的免疫测定装置，其特征在于，还包括样本试管载架，所述样本试管载架用于承载样本试管。
  90. 根据权利要求89所述的免疫测定装置，其特征在于，还包括设置在所述样本试管载架靠近空白板条堆栈和加载机构一侧的稀释板振荡器，所述稀释板振荡器用于对预稀释板进行稀释处理。
  91. 根据权利要求89所述的免疫测定装置，其特征在于，还包括机械臂，所述机械臂上设有加样针；
    其中，所述机械臂包括第一机械臂和第二机械臂，所述第一机械臂用于从所述样本试管载架区域吸取样本并分配至板条加样盘的板条内，所述第二机械臂用于从所述试剂冷藏区吸取试剂并分配至板条加样盘的板条内。
  92. 根据权利要求91所述的免疫测定装置，其特征在于，还包括第一清洗机构和第二清洗机构，所述第一清洗机构用于清洗第一机械臂上的加样针，所述第二清洗机构用于清洗第二机械臂上的加样针。
PCT/CN2017/112145 2016-11-22 2017-11-21 鉴定hd-hook效应样本和免疫测定的方法、系统、试剂盒及装置 WO2018095314A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US16/462,968 US20190353664A1 (en) 2016-11-22 2017-11-21 Method, system, reagent kit, and device for determining hd-hook-effect sample and immunoassay
EP17874359.7A EP3546937A4 (en) 2016-11-22 2017-11-21 METHOD, SYSTEM, REAGENT KIT AND SYSTEM FOR CHECKING A HIGH DOSE HOOK SAMPLE AND FOR IMMUNOASSAY
KR1020197014563A KR102220361B1 (ko) 2016-11-22 2017-11-21 Hd-hook 효과 시료 식별 및 면역 측정 방법, 시스템, 키트 및 장치
JP2019547751A JP6980800B2 (ja) 2016-11-22 2017-11-21 Ｈｄ−ｈｏｏｋ効果サンプルと免疫測定を同定する方法、システム、キット及び装置
ZA2018/05983A ZA201805983B (en) 2016-11-22 2018-09-06 Method, system, reagent kit and system for verifying hd-hook effect sample

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
CN201611026623.1 2016-11-22
CN201611034237.7A CN108132344B (zh) 2016-11-22 2016-11-22 免疫测定方法、用于鉴定免疫测定的系统和试剂盒
CN201611034237.7 2016-11-22
CN201611026623.1A CN108152505B (zh) 2016-11-22 2016-11-22 免疫测定方法、用于鉴定免疫测定的系统和试剂盒
CN201611034252.1A CN108204959B (zh) 2016-11-22 2016-11-22 鉴别hd-hook效应样本的方法和鉴定免疫测定中的hd-hook效应的系统
CN201611034252.1 2016-11-22
CN201710695530.6A CN109406498B (zh) 2017-08-15 2017-08-15 一种免疫测定装置
CN201710695530.6 2017-08-15

Publications (1)

Publication Number Publication Date
WO2018095314A1 true WO2018095314A1 (zh) 2018-05-31

Family

ID=62195759

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/112145 WO2018095314A1 (zh) 2016-11-22 2017-11-21 鉴定hd-hook效应样本和免疫测定的方法、系统、试剂盒及装置

Country Status (6)

Country Link
US (1) US20190353664A1 (zh)
EP (1) EP3546937A4 (zh)
JP (1) JP6980800B2 (zh)
KR (1) KR102220361B1 (zh)
WO (1) WO2018095314A1 (zh)
ZA (1) ZA201805983B (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019223692A1 (zh) * 2018-05-21 2019-11-28 博阳生物科技（上海）有限公司 一种化学发光分析测定方法及使用该方法的系统、试剂盒

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210208080A1 (en) * 2018-05-21 2021-07-08 Beyond Diagnostics (Shanghai) Co., Ltd Chemiluminescence analytical method and system and kit using same
USD919830S1 (en) * 2019-05-29 2021-05-18 Chemclin Diagnostics Co., Ltd. Chemiluminescent immunoassay system
CN113125419B (zh) * 2019-12-31 2023-06-23 科美博阳诊断技术(上海)有限公司 一种供体试剂及其应用
CN114624451A (zh) * 2022-05-12 2022-06-14 深圳市帝迈生物技术有限公司 识别钩状效应的方法和识别装置
CN115267163B (zh) * 2022-06-23 2023-03-28 北京铂茵生物科技有限公司 一种化学发光免疫分析试剂盒装载、混匀及制冷系统

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2203836A (en) * 1987-04-24 1988-10-26 Dr Ch Ng Soo Ling Determination of analyte concentration
CN101055272A (zh) * 2006-04-13 2007-10-17 奥林巴斯生命及材料科学欧洲有限公司 免疫测定方法
US20080213797A1 (en) * 2007-03-01 2008-09-04 Abbott Laboratories Immunoassays exhibiting a reduction in prozone phenomena
CN101326440A (zh) * 2005-04-29 2008-12-17 金伯利-克拉克环球有限公司 在钩状效应区域内具有检测能力的测定装置
CN201532646U (zh) 2009-10-21 2010-07-21 上海博阳医疗仪器有限公司 光子计数装置
CN102341706A (zh) * 2009-08-07 2012-02-01 爱科来株式会社 前带现象检测方法、分析方法、前带现象检测装置和分析装置
EP2790019A1 (de) * 2013-04-10 2014-10-15 Siemens Healthcare Diagnostics Products GmbH High Dose Hook Erkennung
CN104991056A (zh) * 2015-08-05 2015-10-21 武汉林勉生物技术有限公司 一种血清学检测与定量分析的方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3633497A1 (de) * 1986-10-02 1988-04-14 Hoechst Ag Immunometrisches bestimmungsverfahren
JP2946850B2 (ja) * 1991-06-30 1999-09-06 株式会社島津製作所 抗原抗体反応におけるプロゾーン判定方法及び分析方法
WO1998025143A1 (en) * 1996-12-03 1998-06-11 Erkki Soini Biospecific, two photon excitation, fluorescence detection and device
DE10064827A1 (de) * 2000-12-22 2002-06-27 Dade Behring Marburg Gmbh Nachweisverfahren
US7381370B2 (en) * 2003-07-18 2008-06-03 Dade Behring Inc. Automated multi-detector analyzer
WO2009126336A1 (en) * 2008-04-11 2009-10-15 Becton, Dickinson And Company Methods of controlling the sensitivity and dynamic range of a homogeneous assay
GB0809995D0 (en) * 2008-05-31 2008-07-09 Spd Swiss Prec Diagnostics Gmb Assay device

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2203836A (en) * 1987-04-24 1988-10-26 Dr Ch Ng Soo Ling Determination of analyte concentration
CN101326440A (zh) * 2005-04-29 2008-12-17 金伯利-克拉克环球有限公司 在钩状效应区域内具有检测能力的测定装置
CN101055272A (zh) * 2006-04-13 2007-10-17 奥林巴斯生命及材料科学欧洲有限公司 免疫测定方法
US20080213797A1 (en) * 2007-03-01 2008-09-04 Abbott Laboratories Immunoassays exhibiting a reduction in prozone phenomena
CN102341706A (zh) * 2009-08-07 2012-02-01 爱科来株式会社 前带现象检测方法、分析方法、前带现象检测装置和分析装置
CN201532646U (zh) 2009-10-21 2010-07-21 上海博阳医疗仪器有限公司 光子计数装置
EP2790019A1 (de) * 2013-04-10 2014-10-15 Siemens Healthcare Diagnostics Products GmbH High Dose Hook Erkennung
CN104991056A (zh) * 2015-08-05 2015-10-21 武汉林勉生物技术有限公司 一种血清学检测与定量分析的方法

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
"METHODS IN ENZYMOLOGY", ACADEMIC PRESS
"METHODS IN ENZYMOLOGY", vol. 304, 1999, ACADEMIC PRESS, article "Chromatin"
"METHODS IN MOLECULAR BIOLOGY", vol. 119, 1999, HUMANA PRESS, article "Chromatin Protocols"
AUSUBEL ET AL.: "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY", 1987, JOHN WILEY & SONS
MA, HONGWEI ET AL.: "Development of Serum Cardiac Troponin I Light Induced Chemiluminescent Immunoassay", LABORATORY MEDICINE, vol. 22, no. 4, 31 December 2007 (2007-12-31), pages 398 - 401, XP009515536 *
S. AMARASIRI FERNANDO ET AL., STUDIES OF THE 'HOOK' EFFECT IN THE ONE-STEP SANDWICH IMMUNOASSAY JOURNAL OF IMMUNOLOGICAL METHODS, vol. 151, 31 December 1992 (1992-12-31), pages 47 - 66, XP023657574 *
SAMBROOK ET AL.: "MOLECULAR CLONING: A LABORATORY MANUAL", 1989, COLD SPRING HARBOR LABORATORY PRESS
WOLFFE: "CHROMATIN STRUCTURE AND FUNCTION", 1998, ACADEMIC PRESS

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019223692A1 (zh) * 2018-05-21 2019-11-28 博阳生物科技（上海）有限公司 一种化学发光分析测定方法及使用该方法的系统、试剂盒

Also Published As

Publication number Publication date
US20190353664A1 (en) 2019-11-21
KR20190077421A (ko) 2019-07-03
JP2020507780A (ja) 2020-03-12
EP3546937A1 (en) 2019-10-02
ZA201805983B (en) 2019-05-29
KR102220361B1 (ko) 2021-02-25
EP3546937A4 (en) 2020-07-01
JP6980800B2 (ja) 2021-12-15

Similar Documents

Publication Publication Date Title
WO2018095314A1 (zh) 鉴定hd-hook效应样本和免疫测定的方法、系统、试剂盒及装置
EP2995952B1 (en) Reagent kit, measurement kit, and method of measuring test substance
CN109470874B (zh) 免疫测定方法、用于鉴定免疫测定的系统和试剂盒
CN108152505B (zh) 免疫测定方法、用于鉴定免疫测定的系统和试剂盒
CN106918708A (zh) 一种用于检测胰岛素的竞争法胶乳增强免疫透射比浊试剂盒
US8741218B2 (en) Automatic analyzer
CN115389760A (zh) 用于免疫分析试条的检测试剂
CN108204959B (zh) 鉴别hd-hook效应样本的方法和鉴定免疫测定中的hd-hook效应的系统
CN209127196U (zh) 一种tsh化学发光免疫检测试剂盒
WO2021134299A1 (zh) 选择性检测不同待测物质的免疫分析仪、方法及试剂盒
CN207976394U (zh) 时间分辨荧光免疫层析法检测amh的试纸条和试剂盒
JP2997747B2 (ja) 試料中の物質の測定法
JP2023059089A (ja) 検体中の検出対象を検出又は定量する方法、および検体中の検出対象を検出又は定量するための試薬
JP2021511525A (ja) ウイルス感染と細菌感染を区別するためのマルチプレックスラテラルフローアッセイ
CN109406498A (zh) 一种免疫测定装置
JP2021511524A (ja) ウイルス感染と細菌感染を区別するためのマルチプレックスラテラルフローアッセイ
White Development of a novel diagnostic system based on the use of the compact disc as a platform for biological analysis
JP2000187034A (ja) 免疫測定法
JP2000146968A (ja) 酵素免疫測定法
JPH0980053A (ja) 改良された免疫測定法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17874359

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 20197014563

Country of ref document: KR

Kind code of ref document: A

Ref document number: 2019547751

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017874359

Country of ref document: EP

Effective date: 20190624