WO2021134304A1 - Kit, méthode et analyseur de dosage immunologique de criblage d'une infection à torch - Google Patents

Kit, méthode et analyseur de dosage immunologique de criblage d'une infection à torch Download PDF

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Publication number
WO2021134304A1
WO2021134304A1 PCT/CN2019/130129 CN2019130129W WO2021134304A1 WO 2021134304 A1 WO2021134304 A1 WO 2021134304A1 CN 2019130129 W CN2019130129 W CN 2019130129W WO 2021134304 A1 WO2021134304 A1 WO 2021134304A1
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antigen
sample
reagent
antigens
coated
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PCT/CN2019/130129
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English (en)
Chinese (zh)
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于丽娜
李可
何建文
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深圳迈瑞生物医疗电子股份有限公司
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Priority to PCT/CN2019/130129 priority Critical patent/WO2021134304A1/fr
Priority to CN201980101478.4A priority patent/CN114585920A/zh
Publication of WO2021134304A1 publication Critical patent/WO2021134304A1/fr

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    • 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
    • G01N33/53—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

Definitions

  • the present invention relates to the field of immunoassays, in particular to the analysis method of Torch screening.
  • TORCH refers to the abbreviation of a group of pathogenic microorganisms in English.
  • T stands for Toxoplasma gondii or Toxoplasma gondii;
  • O stands for other pathogenic microorganisms, such as herpes zoster virus, parvovirus B19, Coxsackie virus, etc.;
  • R stands for rubella virus;
  • C stands for cytomegalovirus;
  • H stands for herpes simplex virus I Type and Type II.
  • This group of pathogens can be transmitted to the fetus through the placenta, causing perinatal infections, and there is a risk of miscarriage, stillbirth, premature delivery, congenital malformations, and intellectual disability. Therefore, antibody testing for TORCH infection has gradually become a routine pregnancy test project.
  • a negative IgG antibody of this group of pathogens indicates that the subject has not been infected or infected but has not produced antibodies; a negative IgM antibody of this group of pathogens indicates that the subject has no active infection, but does not exclude potential infections . If the IgG antibody of a certain pathogen is positive, it means that the subject has been infected by this pathogen in the past or has been vaccinated; if the IgM antibody of a certain pathogen is positive, it indicates that the subject has an active infection of this virus in the near future. Acute infection, higher risk.
  • the present invention proposes a new mixed detection mode.
  • the present invention provides a kit including:
  • a capture mixture which comprises at least two antigens selected from the group consisting of Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen; and
  • the kit of the present invention coats at least two antigens required for Torch detection on a solid-phase carrier, and also includes a labeled anti-human IgM antibody, so that it can simultaneously detect the antigens in Torch detection. Multiple IgM for mixed detection.
  • using the kit of the present invention can finally obtain a mixed detection result, which can simultaneously evaluate the infection risk of multiple pathogenic microorganisms in TORCH, thereby improving the efficiency of screening. That is to say, when the subject is tested using the kit provided in the first aspect of the present invention, a mixed test result is obtained. If the mixed test result is negative, it means that the subject has no activity of at least two pathogenic microorganisms.
  • the mixed test result is positive, it indicates that the subject has an active infection of at least one pathogenic microorganism in the near future, and it is recommended to further screen for the infection of each pathogenic microorganism separately. This can greatly improve the efficiency of preliminary screening.
  • the at least two antigens are coated on the same solid phase carrier.
  • the at least two antigens are respectively coated on different solid carriers.
  • the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed so that, in the same reaction system, each coating is used in the solid phase.
  • the antigen on the carrier and the anti-human IgM antibody single internal reference for example, the internal reference defined in the ISO 1183-1:2004 standard
  • the corresponding luminescence threshold is basically the same.
  • the luminescence threshold is determined by an ROC curve.
  • the capture mixture comprises Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus type 1/2 antigen coated on a solid phase carrier.
  • the capture mixture comprises Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, and parvovirus B19 antigen coated on a solid phase carrier.
  • the capture mixture comprises parvovirus B19 antigen, Coxsackie virus antigen, and herpes zoster virus antigen coated on a solid phase carrier.
  • the capture mixture comprises Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, and Coxsackie virus antigen coated on a solid phase carrier. And herpes zoster virus antigen.
  • the kit of the present invention may further include instructions, which record that when the corresponding pathogen is detected using at least two antigens on the capture mixture and the labeled anti-human IgM antibody, the combined detection value and luminescence The case where the ratio of the threshold value is greater than or equal to 1.1 is determined as a positive result.
  • the instructions further state that when a positive result is determined, it is recommended that the pathogen corresponding to each of the at least two antigens be further examined.
  • the present invention provides an immunoassay method, including the following steps:
  • the sample to be tested is coated with at least two of the toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen, and herpes zoster virus antigen.
  • a solid-phase carrier of three antigens for a period of time, so that the at least two antigens coated on the solid-phase carrier can bind to the IgM antibodies corresponding to the at least two antigens in the sample to be tested;
  • a luminescent substrate is added to the washed complex to detect the mixed detection value of IgM antibodies corresponding to the at least two antigens in the sample to be tested.
  • the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed so that, in the same reaction system, each coating on the solid-phase carrier is used.
  • the corresponding luminescence threshold is basically the same.
  • the at least two antigens coated on the solid-phase carrier are added to the sample to be tested, mixed and incubated, or added to the sample to be tested in a pre-mixed form Mix and incubate.
  • the at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus type 1/2 antigen.
  • the at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, and parvovirus B19 antigen.
  • the at least two antigens are parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen.
  • the at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen and herpes zoster virus antigen .
  • the method when it is judged as a positive result, the method further includes the step of individually detecting the pathogen corresponding to each of the at least two antigens.
  • the present invention provides a sample analyzer that can selectively detect different types of IgM antibodies in blood samples, including:
  • the sample device has a sample storage component and a sample dispensing component, the sample storage component is used to store the sample to be tested, and the sample dispensing component is used to suck the sample to be tested and discharge it into the reaction cup to be added;
  • the reagent device has a reagent storage component and a reagent dispensing component.
  • the reagent storage component is used to store a reagent kit.
  • the reagent kit includes a solid-phase reagent and a labeling reagent.
  • the solid-phase reagent includes a coating with Toxoplasma gondii antigen and rubella.
  • a solid phase component of at least two antigens among viral antigens, cytomegalovirus antigens, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen and herpes zoster virus antigen, and the labeling reagent comprises A labeled anti-human IgM antibody, the reagent dispensing part is used for sucking the solid phase components and labeling reagents in the kit stored on the reagent storage part and discharging them into the reaction cup where the reagent is to be added;
  • the luminescent substrate dispensing device is connected to the container storing the luminescent substrate and is used to inject the luminescent substrate into the reaction cup where the luminescent substrate is to be added;
  • the reaction device has a plurality of placement positions for placing the reaction cup and is used for incubating the reaction solution in the reaction cup;
  • the optical measurement component is used to perform optical measurement on the reaction solution after the incubation to obtain the detection result of the sample to be tested;
  • the control device is electrically connected to the sample device, the reagent device, the luminescent substrate dispensing device and the light measuring component, and is configured to:
  • test instruction includes the type of IgM antibody to be tested
  • the reagent dispensing component is controlled to further add a labeling reagent to the reaction cup, so that the labeling reagent is mixed with the mixture in the reaction cup and incubated for a period of time, so that the labeling reagent can be combined with the solid phase component Binding to the IgM antibody to be tested;
  • the detection result is obtained according to the ratio of the luminescence value and the luminescence threshold value measured in the light measuring component.
  • the at least two antigens coated on the solid phase component are present in the kit in the form of separate aliquots, or in a pre-mixed form in the kit.
  • the kit is the kit provided in the first aspect of the present invention.
  • the use of the solid phase reagent and the labeling reagent of the present invention in preparing a kit for Torch detection is provided.
  • the present invention realizes the evaluation of multiple infection risks based on the obtained one detection result, thereby greatly improving the efficiency of Torch detection and evaluation, shortening the average detection time, and thereby reducing The cost of testing is conducive to the promotion of Torch screening.
  • the embodiments of the present invention can selectively screen the items involved in the Torch detection as required, which further improves the scope and flexibility of the detection.
  • Figure 1 shows a schematic diagram of an immunoassay system according to an embodiment of the present invention
  • Fig. 2 shows a schematic structural diagram of a control device according to an embodiment of the present invention.
  • the present invention provides a multiple IgM antibody evaluation method based on capture mixture, which can realize multiple infection risks based on only one mixed detection result.
  • the evaluation has improved the efficiency of screening. For example, when the result of the method of the present invention is negative, it indicates that all the pathogen IgM items of the mixed test are negative and the risk is low; when the result is positive, it indicates that at least one of the pathogen IgM items of the mixed test is positive, and it is recommended to proceed further an examination. Accordingly, the screening efficiency of Torch detection is improved and the detection time is shortened, thereby solving the problems of high cost of Torch detection and limited promotion.
  • solid support refers to a solid surface to which antigens or antibodies can be attached.
  • solid-phase carrier used in the present invention
  • commercial solid-phase carriers and any solid-phase carrier that can be used in immunoassays can be used in the present invention.
  • Exemplary solid phase carriers can be magnetic beads (such as carboxyl magnetic beads), enzyme-labeled plates, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates, or micro Chip, but the present invention is not limited to this.
  • the term “capture mixture” means that it contains at least two antigens coated on a solid-phase carrier, and the at least two antigens coated on the solid-phase carrier are present in the mixed form.
  • the kit In the embodiment of the present invention, the term “capture mixture” means that it contains at least two antigens coated on a solid-phase carrier, and the at least two antigens coated on the solid-phase carrier are present in the mixed form. In the kit.
  • At least two antigens can be coated on a solid-phase carrier in the following manner: on the one hand, each antigen can be separately coated on a different solid-phase carrier, and then each antigen coated The solid phase carrier is mixed.
  • each antigen can be separately coated on a different solid-phase carrier, and then each antigen coated
  • the solid phase carrier is mixed.
  • the parvovirus B19 antigen, the Coxsackie virus antigen, and the herpes zoster virus antigen can be separately coated on a solid phase carrier, and then mixed together.
  • At least two antigens can also be divided into one or more groups, each group contains one or more antigens, each group of antigens are respectively coated with a different solid phase carrier, and then the coating It is mixed with a solid-phase carrier with antigen, for example, herpes simplex virus type 1/2 antigen is usually coated on the same solid-phase carrier during the coating process.
  • a solid-phase carrier with antigen for example, herpes simplex virus type 1/2 antigen is usually coated on the same solid-phase carrier during the coating process.
  • markers that can be used in the embodiments of the present invention are well known to those skilled in the art, such as alkaline phosphatase (ALP), peroxidase, microperoxidase, horseradish peroxidase, ⁇ -half Enzymes such as lactosidase, glucose oxidase and glucose 6-phosphate dehydrogenase; fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, fluorescein, rhodamine, europium and green fluorescent protein, etc.
  • ALP alkaline phosphatase
  • peroxidase microperoxidase
  • horseradish peroxidase ⁇ -half Enzymes
  • lactosidase glucose oxidase and glucose 6-phosphate dehydrogenase
  • fluorescein isothiocyanate tetramethylrhodamine isothiocyanate
  • fluorescein, rhodamine europium
  • Fluorescent substances such as luminol, isoluminol, phenanthridinium and acridinium esters; coenzymes such as NAD; biotin; radioactive substances such as 35 S, 14 C, 32 P, 131 I and 125 I , but the present invention is not limited to this.
  • a suitable luminescent substrate can be selected according to the type of label used to generate a detectable signal.
  • alkaline phosphatase 3-(2-helicaladamantane)-4-methoxy-4-(3-phosphooxy)-phenyl-1,2-dioxide can be used.
  • Cycloethane is used as a luminescent substrate.
  • the substrate will be decomposed by alkaline phosphatase to remove a phosphate group and generate an unstable intermediate product.
  • the intermediate product generates methyl meta-oxybenzoate anion through intramolecular electron transfer.
  • chemiluminescence is generated.
  • the number of photons generated in the reaction is measured by a photomultiplier tube, and the amount of photons generated is proportional to the content of the detected substance in the sample.
  • the embodiments of the present invention are applicable to various methods such as ELISA, chemiluminescence, electrochemiluminescence, POCT, immunochromatography, up-conversion luminescence, down-conversion luminescence, etc.
  • the antigen used is a recombinant antigen or a natural antigen.
  • ROC receiver operating characteristic
  • ROC curve refers to the curve obtained by dividing the diagnostic test result into several critical points, with the sensitivity corresponding to each critical point as the ordinate and the specificity as the abscissa.
  • ROC curve is an effective tool for comprehensive and accurate evaluation of diagnostic tests.
  • Another function of the ROC curve is to determine the optimal threshold for detection.
  • ROC curve method to determine the critical point In most cases, select the point on the curve as close to the upper left as possible to determine the critical point as the best.
  • Adopting the detection method of the embodiment of the present invention allows a personalized Torch screening scheme to be formulated for the individual to be tested, thereby providing a more flexible screening method.
  • a solid phase carrier coated with Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus type 1/2 antigen can be used;
  • Phase carrier For example, when the individual to be tested has previously received one or more of the Torch tests, a solid carrier coated with the antigens corresponding to the remaining items can be used, which can flexibly provide the test subject Personalized item selection, which broadens the scope of application during testing.
  • the antigen of the embodiment of the present invention may exist in the form of, for example, a multimer, a recombinant antigen, an antigen fragment, or an antigen peptide.
  • the antibodies of the embodiments of the present invention may exist in the form of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, and antigen-binding fragments of antibodies, for example.
  • mixed detection value can be used interchangeably, and refer to the detection result obtained by using the kit, method and system of the present invention.
  • the detection result is a luminescence value.
  • the ratio of the mixed detection value to the luminescence threshold (COI value) is used to determine whether the result is positive or negative. For example, when the ratio is greater than or equal to 1.1, the judgment result is positive, indicating that the IgM antibody test result corresponding to at least one pathogen is positive. When the ratio is between 0.9 and 1.1, the judgment result is a gray area, which is neither positive or negative; when the ratio is less than 0.9, the judgment result is negative, indicating that the IgM antibodies for the item are all negative.
  • the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed so that each coating on the solid-phase carrier is used in the same reaction system.
  • the antigen on the carrier and the anti-human IgM antibody individually detect the internal reference, the corresponding luminescence threshold is basically the same.
  • internal reference refers to the standard and basis for determining the composition of the reaction system, which has the definition as defined in the international standard ISO 18113-1:2009 and can be obtained according to the standard.
  • Internal reference materials are samples used by medical device manufacturers to verify product performance. They are the most important criteria and basis for product selection, preparation, identification, and determination of raw material quality standards, product production process determination, reaction system composition, reaction conditions, etc. .
  • the identification sample of the internal reference product of the enterprise is a sample that judges the value or quantity of a specific disease, state, or the boundary between the existence and nonexistence of the measurement.
  • substantially the same means that the relative deviation is within ⁇ 10%, such as within ⁇ 5%, ⁇ 2%, or ⁇ 1%.
  • concentration of the labeled anti-human IgM antibody is not particularly limited in the present invention, and an exemplary concentration may be about 0.5 ⁇ g/mL to about 5 ⁇ g/mL.
  • the concentration of the solid phase carrier of the type 2 antigen, the solid phase carrier coated with the parvovirus B19 antigen, and the solid phase carrier coated with the Coxsackie virus antigen may be, for example, 0.1 to 0.3 mg/mL.
  • the concentration of the solid phase carrier coated with the herpes zoster virus antigen may be, for example, 0.1 to 0.4 mg/mL.
  • the concentration of the labeled anti-human IgM antibody is about 1.0 ⁇ g/mL, and the luminescence threshold is adjusted to 30,000, then the solid phase carrier (such as magnetic microspheres) coated with the Toxoplasma antigen, Solid-phase carrier coated with rubella virus antigen, solid-phase carrier coated with cytomegalovirus antigen, solid-phase carrier coated with herpes simplex virus type 1/2 antigen, solid-phase carrier coated with parvovirus B19 antigen
  • the concentration of the solid-phase carrier coated with the herpes zoster virus antigen and the solid-phase carrier coated with the Coxsackie virus antigen are respectively: 0.15mg/mL, 0.15mg/mL, 0.15mg/mL, 0.12 mg/mL, 0.2mg/mL, 0.15mg/mL, 0.12mg/mL.
  • the labeled anti-human IgM antibody can be derived from mice, rabbits, goats, sheep, and chickens, but the present invention is not limited thereto.
  • the embodiment of the present invention provides an immunoassay instrument that can selectively detect the type of IgM antibody in a blood sample.
  • the immunoassay analyzer includes a sample device 10, a reagent device 20, a reaction device 30, a light measuring component 40, and a control device 50.
  • the immune analyzer may also include a display part (not shown).
  • the sample device 10 is used to carry the sample to be tested, aspirate the sample and provide it to the reaction device 30.
  • the sample device 10 includes a sample storage part 11 and a sample dispensing part 12.
  • the sample storage component 11 is used to store samples to be tested.
  • the sample storage component 11 may include a sample distribution module (SDM, Sample Delivery Module) and a front-end track.
  • the sample storage component 11 may also be a sample tray.
  • the sample tray includes a plurality of sample positions such as sample tubes. By rotating the tray structure of the sample tray, the sample can be scheduled to the corresponding position, for example, for The position where the sample dispensing part 12 sucks the sample.
  • the sample dispensing component 12 is used to aspirate the sample and discharge it into the reaction cup to be added.
  • the sample dispensing component 12 may include, for example, a sample needle.
  • the sample needle performs a two-dimensional or three-dimensional movement in space through a two-dimensional or three-dimensional drive mechanism, so that the sample needle can move to aspirate the sample carried by the sample storage component 11, and Move to the reaction cup to be added, and discharge the sample into the reaction cup.
  • the reagent device 20 is used to carry reagents, and the reagents are sucked and supplied to the reaction device 30.
  • the reagent device 20 includes a reagent storage part 13 and a reagent dispensing part 14.
  • the reagent storage part 13 is used to store a reagent cartridge.
  • the reagent storage component 13 may be a reagent tray.
  • the reagent tray is arranged in a disc-shaped structure and has multiple positions for carrying reagent containers.
  • the reagent storage component 13 can rotate and drive the reagent container it carries to rotate. It is used to rotate the reagent container to a specific position, for example, a position where the reagent is sucked by the reagent dispensing part 14.
  • the number of reagent storage parts 13 may be one or more.
  • the reagent dispensing part 14 is used for sucking the reagents in the reagent box and discharging them into the reaction cup to be added with the reagents.
  • the reagent dispensing part 14 may include a reagent needle, and the reagent needle can move in a two-dimensional or three-dimensional space through a two-dimensional or three-dimensional drive mechanism, so that the reagent needle can move to absorb the reagent storage part 13 The loaded reagent, and move to the reaction cup where the reagent is to be added, and discharge the reagent into the reaction cup.
  • the reagent storage part 13 is used to store a reagent kit, which includes a solid-phase reagent and a labeling reagent
  • the solid-phase reagent includes the coating with Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, and herpes simplex virus 1/ A solid phase component of at least two antigens of type 2 antigen, parvovirus B19 antigen, Coxsackie virus antigen, and herpes zoster virus antigen
  • the labeling reagent includes an anti-human IgM antibody with a label.
  • the reaction device 30 has at least one placement position for placing the reaction cup and incubating the reaction solution in the reaction cup.
  • the reaction device 30 may be a reaction disc, which is arranged in a disc-shaped structure and has one or more placement positions for placing reaction cups. The reaction disc can rotate and drive the reaction cup in its placement position to rotate for The reaction cup is arranged in the reaction tray and the reaction solution in the incubation reaction cup is incubated.
  • the optical measurement component 40 is used to perform optical measurement on the reaction solution after the incubation to obtain reaction data of the sample.
  • the light measuring component 40 detects the luminous intensity of the reaction solution to be measured, and calculates the concentration of the component to be measured in the sample through a calibration curve.
  • the optical sensing component 40 is separately arranged outside the reaction device 30.
  • the immunoassay analyzer also includes a luminescent substrate dispensing device (not shown).
  • the luminescent substrate dispensing device is connected with the container storing the luminescent substrate and is used for injecting the luminescent substrate into the reaction cup to be added with the luminescent substrate.
  • the control device 50 includes at least: a processing component 51, a RAM 52, a ROM 53, a communication interface 54, a memory 56 and an I/O interface 55.
  • the processing component 51, RAM 52, ROM 53, communication interface 54, memory 56 and the I/O interface 55 communicate through the bus 57.
  • the processing component may be a CPU, GPU, or other chips with computing capabilities.
  • the memory 56 is loaded with various computer programs such as an operating system and application programs for the processor component 51 to execute, and data required to execute the computer programs. In addition, during the sample detection process, any data that needs to be stored locally can be stored in the memory 56.
  • the I/O interface 55 is composed of a serial interface such as USB, IEEE1394 or RS-232C, a parallel interface such as SCSI, IDE or IEEE1284, and an analog signal interface composed of a D/A converter and an A/D converter.
  • An input device composed of a keyboard, a mouse, a touch screen or other control buttons is connected to the I/O interface 55, and the user can use the input device to directly input data to the control device 50.
  • the I/O interface 55 can also be connected to a display with display function, such as: LCD screen, touch screen, LED display screen, etc., and the control device 50 can output the processed data as image display data to the display for display, for example : Analyze data, instrument operating parameters, etc.
  • the communication interface 54 is an interface that can be any currently known communication protocol.
  • the communication interface 54 communicates with the outside world through the network.
  • the control device 50 can transmit data to any device connected through the network through the communication interface 54 in a certain communication protocol.
  • control device 50 is configured to receive a test instruction, the test instruction includes the type of the IgM antibody to be tested, and the following steps are executed in response to the test instruction:
  • the control reagent dispensing component 14 adds the solid phase component corresponding to the type of IgM antibody to be tested from the reagent kit stored in the reagent storage component 13 into the reaction cup on the reaction device 30, so that the to-be-tested The sample and the solid phase component are mixed in the reaction cup and incubated for a period of time, so that the antigen coated on the solid phase component can bind to the test IgM antibody in the test sample;
  • the control reagent dispensing component 14 further adds the labeled reagent in the kit to the reaction cup, so that the labeled reagent is mixed with the mixture in the reaction cup and incubated for a period of time, so that the labeled reagent can be combined with the solid Binding of the IgM antibody to be tested bound on the phase component;
  • the detection result is obtained according to the ratio of the luminescence value measured in the light measuring component 40 to the luminescence threshold value.
  • the immune analyzer provided by the embodiment of the present invention can selectively detect any one or more of the Torch detection items in one detection, which improves the flexibility of Torch detection and meets the needs of users in different scenarios.
  • the solid phase reagent in the solid phase reagent, at least two antigens coated on the solid phase component are present in the kit in the form of separate aliquots.
  • the solid phase reagent includes a solid phase component coated with a Toxoplasma antigen and a solid phase component coated with a rubella virus antigen, which are separated from each other.
  • the user can use the immune analyzer to perform preliminary screening of multiple items in the Torch test item in one test, or use the immune analyzer to screen each item of the Torch test item one by one. The user needs to input the items that need to be detected.
  • the immunoassay analyzer can add the solid phase components corresponding to the items to be tested and the labeling reagents to the sample to be tested according to the user's instructions.
  • control device 50 is configured to control the reagent dispensing part 14 to separate different solid-phase components in the solid-phase reagent. Add to the reaction cup.
  • kits in the solid phase reagent, at least two antigens coated on the solid phase component are present in the kit in a pre-mixed form.
  • the kit is, for example, the above-mentioned kit according to the present invention.
  • Toxoplasma anti-antigen rubella virus antigen
  • cytomegalovirus antigen cytomegalovirus antigen
  • herpes simplex virus type 1 antigen herpes simplex virus type 2 antigen are from Meridian Life Science;
  • Alkaline phosphatase comes from Roche Pharmaceuticals
  • the magnetic beads come from Thermo Fisher;
  • Human IgM antibodies specifically bind antibodies from Jackson ImmunoResearch.
  • the antigen is pre-treated, and the protective components in the buffer matrix are removed by dialysis.
  • the coating is carried out at a ratio of 0.5-40ug (preferably 1-30ug, more preferably 10-20ug) of antigen added per mg of magnetic beads.
  • the carboxyl groups on the surface of the magnetic beads are coupled with the amino groups of the antigen under the catalysis of EDC/NHS.
  • 20 mg of magnetic microspheres modified with carboxyl groups on the surface were taken, dispersed in 10 mM MES buffer by ultrasonic, 80 mg EDC and 120 mg NHS were added, and after ultrasonic mixing, they were placed on a shaker at 37°C for 15 minutes.
  • the mouse, rabbit, goat, sheep, chicken and other antibodies that specifically bind to human IgM antibodies are labeled with signal markers.
  • the signal marker is alkaline phosphatase.
  • the concentration of the signal marker after dilution is in the range of about 0.5 ⁇ g/mL to about 5 ⁇ g/mL. In the embodiment of the present invention, the concentration of the signal marker after dilution is about 1 ⁇ g/mL, and the labeling reagent is prepared.
  • the sample and solid-phase coating are added to the reaction tube, and incubated at 37°C for 10 minutes, so that the solid-phase coating can bind to the corresponding IgM antibody in the sample.
  • the substance bound to the solid phase will be placed in a magnetic field and be attracted, the substance bound to the solid phase of the magnetic beads is retained, and the unbound substance is washed and removed.
  • the second step add the labeling reagent to the reaction tube, mix it, and incubate at 37°C for 10 minutes to combine with the conjugate formed in the first step to form a complex. After the incubation in the reaction tube is completed, the complex is attracted by the magnetic field, and other unbound substances are washed and removed.
  • the third step is to add AMPPD to the reaction tube to generate chemiluminescence.
  • the number of photons produced by the reaction is measured by a photomultiplier tube to obtain the chemiluminescence signal value of the sample.
  • the negative coincidence rate refers to the ratio of the number of samples judged to be negative using the test method of the embodiment of the present invention to the negative samples actually participating in the evaluation
  • the positive coincidence rate refers to the test using the embodiment of the present invention
  • the method obtains the proportion of the number of positive samples judged to be positive to the positive samples actually participating in the evaluation; the true negative and positive results of the samples come from the diagnosis results of the hospital.
  • Embodiment 1 Determining the detection threshold
  • solid phase coating preparation method was used to prepare magnetic microspheres coated with Toxoplasma antigen, magnetic microspheres coated with rubella virus antigen, magnetic microspheres coated with cytomegalovirus antigen, and magnetic microspheres coated with cytomegalovirus antigen.
  • Herpes simplex virus type 1/2 antigen HSV-1 and HSV-2 mixed at 1:1
  • magnetic microspheres magnetic microspheres coated with parvovirus B19 antigen
  • magnetic microspheres coated with herpes zoster virus antigen Balls magnetic microspheres coated with Coxsackie virus antigens
  • labeling reagents prepared by the above-mentioned "preparation of labeling reagents" method, and testing the positive and negative coincidence rates of each pathogen sample at different luminescence thresholds according to the "detection steps" Compliance rate, the results are shown in Table 1 below.
  • Magnetic microspheres coated with Toxoplasma antigen, magnetic microspheres coated with rubella virus antigen, magnetic microspheres coated with cytomegalovirus antigen, and coated magnetic beads according to "Preparation of magnetic bead coating".
  • Magnetic microspheres with herpes simplex virus type 1 antigen, herpes simplex virus type 2 antigen (1:1), magnetic microspheres coated with parvovirus B19 antigen, magnetic microspheres and coatings coated with herpes zoster virus antigen Concentration of magnetic beads with magnetic microspheres with Coxsackie virus antigen.
  • magnetic microspheres coated with toxoplasma antigen, magnetic microspheres coated with rubella virus antigen, magnetic microspheres coated with cytomegalovirus antigen, and herpes simplex virus type 1/2 The magnetic bead concentrations of the antigen magnetic microspheres, the magnetic microspheres coated with parvovirus B19 antigen, the magnetic microspheres coated with the herpes zoster virus antigen, and the magnetic microspheres coated with the Coxsackie virus antigen are respectively: 0.15mg/mL, 0.15mg/mL, 0.15mg/mL, 0.12mg/mL, 0.2mg/mL, 0.15mg/mL, 0.12mg/mL.
  • each magnetic bead coating determined in Table 3 According to the concentration of each magnetic bead coating determined in Table 3, the corresponding magnetic bead coatings were mixed according to the items in Experiments 1 to 4, and the solid phase reagents in Experiments 1 to 4 were prepared respectively.
  • COI Cutoff index
  • the threshold value is the judgment that the test result is positive Or negative cutoff value.
  • the COI value of the test result of the sample needs to be compared with a reference value (reference value 1.10). If it is greater than or equal to 1.10, it means one or more test substances in the sample. Species are positive; if it is less than 0.90, it means that the test substances in the sample are all negative.
  • the COI is between 0.90-1.10, and the result is a gray zone (indeterminate).

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Abstract

L'invention concerne un kit de criblage d'une infection à TORCH, faisant appel à : un mélange de capture lors duquel sont mélangés au moins deux antigènes revêtus sur un support en phase solide et sélectionnés parmi les antigènes suivants : un antigène de toxoplasme, un antigène du virus de la rubéole, un antigène du cytomégalovirus, un antigène du virus de l'herpès simplex de type 1/2, un antigène du parvovirus B19, un antigène du virus Coxsackie, et un antigène du virus du zona ; ainsi qu'un anticorps IgM anti-humain avec un marqueur. Le kit permet de tester des anticorps IgM provoqués par une variété de microorganismes pathogènes différents dans un échantillon à tester, et évalue un risque conformément à un résultat de test mixte. Par ailleurs, la présente invention concerne en outre une méthode d'analyse de dosage immunologique et un analyseur de dosage immunologique de criblage d'une infection à TORCH.
PCT/CN2019/130129 2019-12-30 2019-12-30 Kit, méthode et analyseur de dosage immunologique de criblage d'une infection à torch WO2021134304A1 (fr)

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PCT/CN2019/130129 WO2021134304A1 (fr) 2019-12-30 2019-12-30 Kit, méthode et analyseur de dosage immunologique de criblage d'une infection à torch
CN201980101478.4A CN114585920A (zh) 2019-12-30 2019-12-30 用于筛查torch感染的试剂盒、方法及免疫分析仪

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CN117230161A (zh) * 2023-11-10 2023-12-15 新羿制造科技（北京）有限公司 检测torch病原体的数字pcr试剂盒

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CN117230161A (zh) * 2023-11-10 2023-12-15 新羿制造科技（北京）有限公司 检测torch病原体的数字pcr试剂盒

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