WO2021134307A1 - 用于检测传染性疾病的试剂盒、方法及免疫分析仪 - Google Patents

用于检测传染性疾病的试剂盒、方法及免疫分析仪 Download PDF

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WO2021134307A1
WO2021134307A1 PCT/CN2019/130132 CN2019130132W WO2021134307A1 WO 2021134307 A1 WO2021134307 A1 WO 2021134307A1 CN 2019130132 W CN2019130132 W CN 2019130132W WO 2021134307 A1 WO2021134307 A1 WO 2021134307A1
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reagent
solid
coated
antibody
component
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PCT/CN2019/130132
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English (en)
French (fr)
Inventor
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 CN201980101493.9A priority Critical patent/CN114599977A/zh
Priority to PCT/CN2019/130132 priority patent/WO2021134307A1/zh
Publication of WO2021134307A1 publication Critical patent/WO2021134307A1/zh

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    • 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
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
    • 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
    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses

Definitions

  • the invention relates to the field of immunoassays, in particular to a screening method for pathogenic microorganisms of infectious diseases in the blood.
  • the detection method for blood screening is the separate detection of different infectious diseases. It is costly and time-consuming to conduct a comprehensive examination, which brings difficulties to the popularization of blood screening.
  • the present invention provides a kit including:
  • Capture mixing reagent which is mixed with the anti-HBs antigen antibody coated on the solid phase carrier, the HCV antigen coated on the solid phase carrier, the anti-HIV-1 p24 antibody coated on the solid phase carrier, and the anti-HIV-1 p24 antibody coated on the solid phase carrier. HIV-1/-2 combined antigen on a solid carrier; and
  • the labeling reagent includes a labeled anti-HBs antigen antibody, a labeled anti-human antibody, and a labeled anti-HIV-1 p24 antibody.
  • the above kit can be used for mixed detection of HBs antigen, anti-HCV antibody, HIV-1 p24 antigen and HIV-1/-2 antibody in the sample to be tested.
  • the HBs antigen when the HBs antigen is present in the sample, it can be formed with the anti-HBs antigen antibody coated on the solid phase carrier and the labeled anti-HBs antigen antibody.
  • Complex in the case of anti-HCV antibodies in the sample, it can form a complex with the HCV antigen coated on the solid carrier and the labeled anti-human antibody; there is HIV-1 p24 antigen in the sample Under circumstances, it can form a complex with the anti-HIV-1 p24 antibody coated on the solid carrier and the labeled anti-HIV-1 p24 antibody; there are HIV-1 antibodies and/or HIV-2 in the sample In the case of an antibody, it can form a complex with HIV-1/-2 combined antigen coated on a solid-phase carrier and a labeled anti-human antibody.
  • the kit provided in the first aspect of the present invention when a subject is tested using the kit provided in the first aspect of the present invention, whether the subject is infected with HBV, HCV, and HIV can be evaluated at the same time, and a mixed test result can be obtained. If the mixed test result is Negative, it means that the subject is not infected with any of HBV, HCV and HIV; if the mixed test is positive, it means that the subject is infected with at least one of HBV, HCV and HIV, and it is recommended not to use the subject Provide blood, and recommend that the subject be further screened separately for HBV, HCV and HIV infection. This can greatly improve the efficiency of preliminary screening.
  • the kit of the present invention includes:
  • Capture mixing reagent mixed with anti-HBs antigen antibody coated on a solid phase carrier, HCV antigen coated on a solid phase carrier, anti-HIV-1 p24 antibody coated on a solid phase carrier, and a solid phase carrier. HIV-1/-2 combined antigen on phase carrier and TP antigen coated on solid phase carrier;
  • Labeling reagents include labeled anti-HBs antigen antibodies, labeled anti-human antibodies, and labeled anti-HIV-1 p24 antibodies.
  • the above kit can be used for mixed detection of HBs antigen, anti-HCV antibody, HIV-1 p24 antigen, HIV-1/-2 antibody and anti-TP antibody in the sample to be tested.
  • the kit of the present invention includes:
  • Capture mixed reagent mixed with anti-HBs antigen antibody coated on a solid phase carrier, HCV antigen coated on a solid phase carrier, anti-HIV-1 p24 antibody coated on a solid phase carrier, and coated on a solid phase HIV-1/-2 combined antigen on the carrier and HBc antigen coated on the solid carrier;
  • Labeling reagents include labeled anti-HBs antigen antibodies, labeled anti-human antibodies, and labeled anti-HIV-1 p24 antibodies.
  • the above kit can be used for mixed detection of HBs antigen, anti-HCV antibody, HIV-1 p24 antigen, HIV-1/-2 antibody and anti-HBc antibody in the sample to be tested.
  • the various antibodies or antigens coated on the solid-phase carrier are coated on the same solid-phase carrier.
  • the various antibodies or antigens coated on the solid-phase carrier are respectively coated on different solid-phase carriers.
  • the concentration of each component in the capture mixed reagent and the labeling reagent is designed so that each of the capture mixed reagents is used to coat the solid phase in the same reaction system.
  • the corresponding luminescence threshold is basically the same.
  • the luminescence threshold is determined by an ROC curve.
  • the labeled anti-HBs antigen antibody, the labeled anti-human antibody, and the labeled anti-HIV-1 p24 antibody are present in the reagent in the form of a pre-mixed labeling reagent. In the box.
  • the kit of the present invention may further include an instruction manual, which states that when the capture mixed reagent and the labeling reagent are used to detect the corresponding pathogen, the ratio of the mixed detection value to the luminescence threshold value is greater than or equal to 1.1 as a positive result. .
  • the specification further states that when it is judged as a positive result, it is recommended that each of the pathogens corresponding to each component on the coated solid-phase carrier be further examined separately.
  • the present invention provides an immunoassay method, including the following steps:
  • the sample to be tested is mixed with the solid phase reagent and incubated for a period of time, so that the components in the solid phase reagent coated on the solid phase carrier can bind to the corresponding substance to be tested in the sample to be tested, and the solid phase
  • the reagents include anti-HBs antigen antibody coated on a solid phase carrier, HCV antigen coated on a solid phase carrier, anti-HIV-1p24 antibody coated on a solid phase carrier, and HIV coated on a solid phase carrier -1/-2 combined antigen;
  • a luminescent substrate is added to the washed complex to detect the mixed detection value of the test substance in the test sample.
  • the solid phase reagent may further comprise one or more of TP antigen coated on a solid phase carrier and HBc antigen coated on a solid phase carrier.
  • the solid-phase reagent comprises an anti-HBs antigen antibody coated on a solid-phase carrier, an HCV antigen coated on a solid-phase carrier, and an anti-HIV-antibody coated on a solid-phase carrier.
  • the solid-phase reagent comprises an anti-HBs antigen antibody coated on a solid-phase carrier, an HCV antigen coated on a solid-phase carrier, and an anti-HIV-antibody coated on a solid-phase carrier.
  • the concentration of each component in the solid phase reagent and the labeling reagent is designed such that each of the solid phase reagents is coated on the solid phase carrier under the same reaction system
  • the corresponding luminescence thresholds are basically the same.
  • the components of the solid-phase reagent coated on the solid-phase carrier are added to the sample to be tested, mixed and incubated, or added to the sample in a pre-mixed form. Mix and incubate the test sample.
  • each component with a label in the labeling reagent is added to the mixture of the sample to be tested and the solid phase reagent to be mixed and incubated, or added in a pre-mixed form Mix and incubate the sample to be tested and the mixture of the solid phase reagent.
  • the method further includes the step of individually detecting each of the pathogens corresponding to the components on the coated solid phase carrier.
  • the present invention provides an immunoassay instrument that can selectively detect different infectious pathogenic microorganisms 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 labeled reagent.
  • the solid-phase reagent includes a solid-phase reagent coated on a solid-phase carrier.
  • the labeling reagent includes a labeling component for labeling an antibody against HBs antigen, a labeling component for labeling an anti-human antibody, and a labeling component for labeling an anti-HIV-1 p24 antibody, and the reagent dispensing component is used for suction
  • the solid phase reagent and the labeling reagent in the reagent kit stored on the reagent storage component are discharged into the reaction cup to be added reagent;
  • 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 including the type of the substance to be tested
  • each solid phase component in the solid phase reagent is present in the kit in the form of separate packaging, or is present in the kit in a pre-mixed form.
  • each labeling component in the labeling reagent is present in the kit in the form of separate aliquots, or in the form of pre-mixing in the kit.
  • the kit is the kit described according to the first aspect of the present invention.
  • the use of the solid phase reagent and labeling reagent of the present invention in preparing a kit for blood screening is provided.
  • the above-mentioned scheme is adopted to implement mixed detection of multiple infectious pathogens in the sample to be tested. Based on the obtained test result, multiple infection risks are evaluated, which greatly improves the efficiency of blood screening and evaluation, shortens the average detection time, and reduces This reduces the cost of testing and is conducive to the promotion of blood screening.
  • the detection mode provided by the embodiment of the present invention can set the targeted infectious pathogen according to the target population, which further improves the scope and flexibility of screening.
  • 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 blood screening evaluation method based on capturing mixed reagents and labeling reagents. Considering that the positive result of any infectious disease of the blood product will cause the blood to be unusable, the method provided by the present invention can judge the negative or positive of the infectious disease of the blood product based on a mixed test result. In the present invention, when the test result is negative, it indicates that all the infectious pathogens tested in the mixed test are negative; when the result is positive, it indicates that at least one of the infectious pathogens tested in the mixed test is positive. The blood product is not recommended or recommended to be positive The individual from which the sample originated is further examined. Accordingly, this mixed detection method for infectious diseases can effectively improve the efficiency of blood screening and reduce the cost of blood screening.
  • 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 mixed reagent” means that it contains at least two first ligands coated on a solid support, and the at least two first ligands coated on the solid support are The mixed form is included in the kit.
  • the term “labeled mixed reagent” means that it contains at least two second ligands with a label, and the at least two first ligands with a label are present in the kit in a mixed form.
  • the solid-phase carrier can be coated in the following manner: On the one hand, each substance to be coated can be separately coated on a different solid-phase carrier, and then each coated The solid carrier coating is mixed.
  • the anti-HBs antigen antibody, HCV antigen, anti-HIV-1 p24 antibody, and HIV-1/-2 combined antigen can be separately coated on a solid phase carrier, and then mixed together.
  • the substances to be coated can also be divided into one or more groups, each group contains one or more substances to be coated, and each group of substances is respectively coated with a different solid phase carrier. Then, the coating material can be mixed.
  • antibodies to HBs antigen, HCV antigen, anti-HIV-1 p24 antibody and HIV-1/-2 combined antigen can be formed into a group, and the group can be coated with the same solid phase carrier. Be.
  • 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.
  • HBs antigen (HBsAg), also known as hepatitis B virus surface antigen, is the coat protein of hepatitis B virus;
  • Anti-HBs antigen antibody (Anti-HBsAg) is an antibody that specifically binds to HBsAg.
  • HCV antigen (HCV Ag) is an antigen of hepatitis C virus
  • Anti-HCV antibody (Anti-HCV) is an antibody that specifically binds to HCV antigen.
  • HIV-1 p24 antigen (p24 antigen) is a highly conserved protein produced by the expression of HIV-1 gag gene; “anti-HIV-1 p24 antibody” (Anti-p24) is related to HIV- 1 An antibody that specifically binds to the p24 antigen.
  • HIV is divided into type 1 and type 2.
  • the HIV-1 antigen is directed against the type 1 HIV virus
  • the HIV-2 antigen is directed against the type 2 HIV virus.
  • HIV-1/-2 combined antigen refers to the combination of HIV-1 and HIV-2 in a certain ratio (5:1-1:5) to prepare the "combined antigen", and then the magnetic beads are packaged. Be.
  • TP antigen As used herein, "TP antigen” (TP Ag) is a syphilis antigen; "anti-TP antibody” (Anti-TP) is an antibody that specifically binds to TP antigen.
  • HBc antigen (HBcAg) is the hepatitis B virus core antigen
  • Anti-HBc antigen antibody (Anti-HBc) is an antibody that specifically binds to the HBc antigen.
  • 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 test result of at least one biomarker is positive. When the ratio is between 0.9 and 1.1, the judgment result is a gray zone, which is neither positive or negative; when the ratio is less than 0.9, the judgment result is negative, indicating that the target biomarker test results are all negative.
  • the concentration of the anti-HBs antigen antibody coated on the solid phase carrier and the concentration of the labeled anti-HBs antigen antibody are designed so that they are used alone in the preset reaction system
  • the corresponding luminescence threshold is basically the preset luminescence threshold
  • the concentration of the HCV antigen coated on the solid phase carrier and the concentration of the labeled anti-human antibody are designed to make it in place
  • the corresponding luminescence threshold is basically equal to the preset luminescence threshold
  • the concentration of HIV-1/-2 combined antigen coated on the solid-phase carrier is equal to the total
  • the concentration of the labeled anti-human antibody is designed so that when it is used alone to detect the internal reference, the corresponding luminescence threshold is basically equal to the preset luminescence threshold; the one coated on the solid carrier
  • the concentration of the anti-HIV-1 p24 antibody and the concentration of the labeled anti-HIV-1 p24 antibody are designed so that the
  • the concentration of the TP antigen or HBc coated on the solid-phase carrier and the concentration of the labeled anti-human antibody are designed so that it performs the internal reference separately under the preset reaction system.
  • the corresponding light-emitting threshold is substantially equal to the preset light-emitting threshold.
  • internal reference refers to the standard and basis determined by the system composition, which has the definition as 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 company's internal reference product is a sample that judges the value or quantity of a specific disease, state, or the boundary between the existence and absence of the measurement.
  • substantially the same means that the relative deviation is within ⁇ 10%, such as within ⁇ 5%, ⁇ 2%, or ⁇ 1%.
  • the concentration of the labeled anti-HBs antigen antibody may be, for example, 0.1-5ug/mL.
  • the concentration of the labeled anti-HIV-1 p24 antibody may be, for example, 0.1-5 ug/mL.
  • the concentration of the labeled anti-human antibody may be, for example, about 0.1 ⁇ g/mL to about 5 ⁇ g/mL.
  • the concentration of the solid-phase carrier coated with the antibody against the HBs antigen and the solid-phase carrier of the HIV p24 antibody may be, for example, 0.1 mg/mL to 2 mg/mL.
  • the concentration of the solid phase carrier coated with HCV antigen and the solid phase carrier coated with TP antigen may be, for example, 0.1 mg/mL to 2 mg/mL.
  • the labeled anti-HBs antigen antibody, labeled anti-human antibody, and labeled anti-HIV-1 p24 antibody can be derived from mice, rabbits, goats, sheep, and chickens, but The present invention is not limited to this.
  • the antibody in the labeling reagent can be connected to the label by a method conventional in the art (for example, chemical bonding).
  • the embodiment of the present invention provides an immunoassay instrument that can selectively detect different infectious pathogenic microorganisms in blood samples.
  • the immunoassay analyzer includes a sample device 10, a reagent device 20, a reaction device 30, an optical measuring part 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 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 component 13 is used to store a reagent kit, which includes a solid-phase reagent and a labeling reagent
  • the solid-phase reagent includes a solid-phase component of an antibody against HBs antigen coated on a solid-phase carrier, and a coating A solid phase component with HCV antigen, a solid phase component coated with anti-HIV-1 p24 antibody, and a solid phase component coated with HIV-1/-2 combined antigen
  • the labeling reagent includes a labeled anti-HBs antigen The labeling component of the antibody, the labeling component of the anti-human antibody and the labeling component of the anti-HIV-1 p24 antibody.
  • 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 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 substance 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 the substance 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 sample to be tested Mixing with the added solid phase component in the reaction cup and incubating for a period of time, so that the substance coated on the added solid phase component can be combined with the test substance in the test sample;
  • the control reagent dispensing component 14 further adds the labeled component corresponding to the type of the substance to be tested in the reagent box into the reaction cup, so that the added labeled component is mixed with the mixture in the reaction cup and incubated for a period of time Time so that the added labeling component can bind to the test substance bound to the added solid 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 different infectious pathogenic microorganisms in the sample to be tested in one test, improve the flexibility of the test, and meet the needs of users in different scenarios.
  • each solid phase component in the solid phase reagent is present in the kit in the form of separate packaging.
  • the user can use the immune analyzer to conduct preliminary screening of multiple infectious pathogenic microorganisms in one test, or use the immune analyzer to screen a single infectious pathogenic microorganism, and only need user input The items that need to be tested are sufficient.
  • the immune analyzer can add the solid phase component corresponding to the item to be detected and the corresponding labeled component to the sample to be tested according to the user's instruction.
  • each solid phase component in the solid phase reagent is present in the kit in a pre-mixed form.
  • the kit is, for example, the above-mentioned kit according to the present invention.
  • the immunoassay analyzer adds the mixed solid-phase reagent and corresponding labeled components to the sample to be tested according to the needs of the user to achieve different detection requirements .
  • each labeling component in the labeling reagent is present in the kit in the form of separate aliquots, or in a pre-mixed form in the kit.
  • Anti-HBs antigen antibody, HCV antigen, anti-p24 antibody, HIV-1/-2 specific combined antigen and TP antigen are from Meridian Life Science;
  • Alkaline phosphatase comes from Roche Pharmaceuticals
  • Antibodies that specifically bind to human antibodies are from Jackson ImmunoResearch.
  • the antigen or antibody is pretreated, and the protective components in the buffer matrix are removed by dialysis.
  • the coating is carried out at a ratio of 1-40 ⁇ g of antigen/antibody (preferably 5-30 ug, more preferably 10-20 ug) 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.
  • the anti-HBs antigen antibody, the antibody specifically binding to the human antibody, and the anti-p24 antibody are labeled with signal markers.
  • the signal marker is alkaline phosphatase. Choose 50mM MES pH 6.0 buffer to dilute the anti-hepatitis B surface antigen antibody signal markers, the antibody signal markers that specifically bind to human antibodies, and the anti-p24 antibody signal markers to prepare labeled components.
  • the sample and the 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 test substance 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 labeling reagent is added to the reaction tube and incubated, mixed, and incubated at 37°C for 10 minutes to bind to the conjugate formed in the first step. 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.
  • Example 1 Determining the luminescence threshold of blood screen detection
  • the magnetic microspheres coated with Anti-HBsAg obtained by "Preparation of solid phase coating” are used as solid phase reagents and Anti-HBsAg with alkaline phosphatase obtained by "Preparation of labeled components" As a labeling reagent, it tests the positive and negative coincidence rates of HBsAg under different luminescence thresholds according to the "detection steps". In the same way, the positive and negative coincidence rates of Anti-HCV, HIV-1 p24 antigen and HIV-1/-2 antibody were tested respectively. The results are summarized in Table 1.
  • Example 2 Determination of the concentration of each solid-phase coating and determination of the concentration of each labeled component
  • the corresponding labeling components are all antibodies with alkaline phosphatase that specifically bind to human antibodies ( For example, mouse antibody), the concentration of the labeling component is fixed to about 1 ⁇ g/mL, the concentration of the magnetic bead coating is adjusted, and the luminescence threshold is adjusted to 50,000, allowing ⁇ 5% deviation.
  • Table 3 The results are shown in Table 3 below.
  • the anti-hepatitis B surface antigen (HBsAg) antibody, HCV antigen, anti-p24 antibody, HIV-1/-2 specific combined antigen and TP antigen are respectively coated on Magnetic microspheres.
  • HBsAg antibody, HCV antigen, anti-p24 antibody, HIV-1/-2 specific combined antigen and TP antigen are respectively coated on Magnetic microspheres.
  • each magnetic bead coating choose 50mM Tris pH 7.4 buffer to dilute each magnetic bead coating to obtain 0.2mg/mL magnetic microspheres coated with anti-HBsAg antibody, 0.2mg/mL magnetic microspheres coated with HCV antigen, 0.2mg/mL magnetic microspheres coated with anti-HBsAg antibody, 0.2mg/mL magnetic microspheres coated with HCV antigen, 0.2mg/mL mg/mL magnetic microspheres coated with anti-p24 antibody, 0.25mg/mL magnetic microspheres coated with HIV-1/-2 specific combined antigen for dilution and 0.2mg/mL coated with TP antigen Magnetic microspheres.
  • the magnetic bead coatings were mixed according to the items in Experiments 1-2, and the solid-phase reagents in Experiments 1-2 were prepared respectively.
  • the labeling reagent For the labeling reagent, prepare 0.8 ⁇ g/ml of anti-HBsAg antibody with alkaline phosphatase, 1 ⁇ g/mL of alkaline phosphatase-specific antibody binding to human antibodies, and 1.2 ⁇ g/mL anti-HIV-1 p24 antibody with alkaline phosphatase. Next, the marking components were mixed according to the items in Experiments 1-2, and the labeling reagents in Experiments 1-2 were prepared respectively.
  • Example 4 Use the reagent combination in experiments 1 to 2 for testing
  • 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 of the 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 (undefined).
  • the positive coincidence rate of Experiment 1 ie simultaneous detection of HbsAg, Anti-HCV, HIV-1 p24 antigen and HIV-1/-2 antibody
  • the negative coincidence rate was 99.50%
  • the overall coincidence rate 99.79%
  • Experiment 2 that is, simultaneous detection of HbsAg, Anti-HCV, HIV-1 p24 antigen, HIV-1/-2 antibody and Anti-TP
  • the positive coincidence rate was 100%
  • the negative coincidence rate was 99.30%
  • the overall coincidence rate was 99.75 %. This can meet the needs for rapid and effective screening of infectious diseases.

Abstract

一种血液筛查试剂盒，包括捕获混合试剂，其混合有包被在固相载体上的抗HBs抗原的抗体、HCV抗原、抗HIV-1 p24抗体和HIV-1/-2联合抗原；和标记试剂，其包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体。该试剂盒能够对血液中多种病原微生物对应的不同待测物质进行混合检测，并根据一个混合检测结果来评估风险。此外，还涉及免疫分析方法及免疫分析仪。

Description

用于检测传染性疾病的试剂盒、方法及免疫分析仪 技术领域
本发明涉及免疫分析领域，具体涉及血液中感染性疾病的病原微生物的筛查方式。
背景技术
外伤性出血、产后大出血、严重烧伤、各种血液病以及实施了外科手术的伤员，需要靠输血来救治。由于血液难以人工制造或是用其它的物质所代替，需要通过献血来获取。而来自不健康的供血者的血液会传播乙型肝炎、丙型肝炎、艾滋病和梅毒等多种严重的传染病。
为了保障临床用血的安全和血液制品的质量，同时防止供受血之间的交叉感染和采血及血液制品工作者的健康，必须防止上述疾病的感染者进入供血队伍，防止上述疾病病原体阳性血浆直接输注病人或用于血液制品生产。
因此有必要进行血液筛查以确保血液质量，防止输血造成的疾病的感染。当前，血液筛查时的检测方法是对不同传染性疾病的分开检测。在进行全面检查成本较高、耗时长，为血液筛查的普及带来了困难。
据此，在血液筛查领域，存在着对低成本、高效率的检测传染病病原体感染的强烈需求。
发明内容
为了改善当前血液筛查成本高、耗时长的问题。基于血液筛查的特点，经研究，本发明人提出了一种新的混合检测模式，从而完成本发明。
在第一方面，本发明提供了一种试剂盒，包括：
捕获混合试剂，其混合有包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体和包被在固相载体上的HIV-1/-2联合抗原；和
标记试剂，其包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体。
使用上述试剂盒可以对待测样本中的HBs抗原、抗HCV抗体、HIV-1 p24抗原和HIV-1/-2抗体进行混合检测。
需要说明的是，根据本发明的试剂盒，在样本中存在HBs抗原的情况下，其可以与包被在固相载体上的抗HBs抗原的抗体和带有标记物的抗HBs抗原的抗体形成复合体；在样本中存在抗HCV抗体的情况下，其可以与包被在固相载体上的HCV抗原和带有标记物的抗人抗体形成复合体；在样本中存在HIV-1 p24抗原的情况下，其可以与包被在固相载体上的抗HIV-1 p24抗体和带有标记物的抗HIV-1 p24抗体形成复合体；在样本中存在HIV-1抗体和/或HIV-2抗体的情况下，其可以与包被在固相载体上的HIV-1/-2联合抗原和带有标记物的抗人抗体形成复合体。也就是说，当使用本发明第一方面提供的试剂盒对受试者进行检测，能够同时对受试者是否感染HBV、HCV和HIV进行评估，得出一个混合检测结果，若该混合检测结果呈阴性，则表明受试者没有感染HBV、HCV和HIV的任一种；若该混合检测呈阳性，表明受试者感染了HBV、HCV和HIV中至少一种，建议不使用该受试者提供的血液，并可建议该受试者进一步单独针对HBV、HCV和HIV感染进行筛查。由此能够大大提高初步筛查的效率。
在一些实施方式中，所述捕获混合试剂可进一步混合有包被在固相载体上的TP抗原和包被在固相载体上的HBc抗原中的一种或更多种。
在一个具体的实施方式中，本发明的试剂盒包括：
捕获混合试剂，混合有包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的TP抗原；
标记试剂，包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体。
使用上述试剂盒可以对待测样本中的HBs抗原、抗HCV抗体、HIV-1 p24抗原、HIV-1/-2抗体和抗TP抗体进行混合检测。
在一个具体的实施方式中，本发明的试剂盒包括：
捕获混合试剂，混合有包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的HBc抗原；
标记试剂，包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体。
使用上述试剂盒可以对待测样本中的HBs抗原、抗HCV抗体、HIV-1 p24抗原、HIV-1/-2抗体和抗HBc抗体进行混合检测。
在一些实施方式中，固相载体上所包被的各种抗体或抗原包被在同一固相载体。
在一些实施方式中，固相载体上所包被的各种抗体或抗原分别包被在不同的固相载体上。
在具体的实施方式中，所述捕获混合试剂和所述标记试剂中各组分的浓度被设计为使得，在同一个反应体系下采用所述捕获混合试剂中的每一种包被在固相载体上的组分和所述标记试剂中对应带标记物的组分单独对内部参考品(例如根据ISO 18113-1:2009标准定义的内部参考品)进行检测时，所对应的发光阈值基本上相同。
在示例性的实施方式中，所述发光阈值通过ROC曲线确定。
在一些实施方式中，带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体以预先混合的标记试剂的形式存在于所述试剂盒中。
本发明的试剂盒可进一步包括说明书，其记载了当使用所述捕获混合试剂和所述标记试剂检测所对应的病原体时，将混合检测值与发光阈值的比大于等于1.1的情况确定为阳性结果。
在具体的实施方式中，所述说明书进一步记载了当判断为阳性结果时，建议对包被固相载体上的各个组分所对应病原体的每一种单独作进一步检查。
在第二方面，本发明提供了一种免疫分析方法，包括以下步骤：
将待测样本与固相试剂混合并孵育一段时间，使得所述固相试剂中包被在固相载体上的组分能与所述待测样本中对应的待测物质结合，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1p24抗体和包被在固相载体上的HIV-1/-2联合抗原；
对所述待测样本和所述固相试剂的混合物进行清洗，除去未结合的物质；
在所述经清洗的混合物中加入标记试剂并孵育，使得所述标记试剂能与所述固相载体上结合的所述待测物质结合，形成复合物，所述标记试剂包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体；
对所述复合物进行清洗，除去未结合的物质；
在经清洗的复合物中加入发光底物，以检测所述待测样本中所述待测物质的混合检测值。
在一些实施方式中，所述固相试剂可进一步包含包被在固相载体上的TP抗原和包 被在固相载体上的HBc抗原中的一种或更多种。
在一个具体的实施方式中，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的TP抗原；并且所述标记试剂包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1p24抗体。
在一个具体的实施方式中，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的HBc抗原；并且所述标记试剂包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1p24抗体。
在一些实施方式中，所述固相试剂和所述标记试剂中各组分的浓度被设计为使得，在同一个反应体系下所述固相试剂中的每一种包被在固相载体上的组分和所述标记试剂中对应带标记物的组分单独对内部参考品进行检测时，所对应的发光阈值基本上相同。
在一些实施方式中，所述固相试剂中包被在固相载体上的各个组分被分别加入到所述待测样本中进行混合并孵育，或者以预先混合的形式被加入到所述待测样本中进行混合并孵育。
在一些实施方式中，所述标记试剂中带有标记物的各个组分被分别加入到所述待测样本与所述固相试剂的混合物中进行混合并孵育，或者以预先混合的形式被加入到所述待测样本与所述固相试剂的混合物中进行混合并孵育。
在具体的实施方式中，当判断为阳性结果时，所述方法进一步包括对包被固相载体上的组分所对应病原体的每一种进行单独检测的步骤。
在第三方面，本发明提供了可选择性检测血液样本中不同传染性病原微生物的免疫分析仪，包括：
样本装置，具有样本存储部件和样本分注部件，所述样本存储部件用于存储待测样本，所述样本分注部件用于吸取所述待测样本并排放到待加样的反应杯中；
试剂装置，具有试剂存储部件和试剂分注部件，所述试剂存储部件用于存储试剂盒，所述试剂盒包括固相试剂和标记试剂，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体的固相组分、包被有HCV抗原的固相组分、包被有抗HIV-1 p24抗体的固 相组分和包被有HIV-1/-2联合抗原的固相组分，所述标记试剂包含标记抗HBs抗原的抗体的标记组分、标记抗人抗体的标记组分和标记抗HIV-1 p24抗体的标记组分，所述试剂分注部件用于吸取所述试剂存储部件上存储的试剂盒中的固相试剂和标记试剂并排放到待加试剂的反应杯中；
发光底物分注装置，与存储发光底物的容器连接并用于将发光底物注入到待加发光底物的反应杯中；
反应装置，具有多个用于放置所述反应杯的放置位并用于孵育所述反应杯中的反应液；
光测部件，用于对孵育完成的反应液进行光测定，以得到待测样本的检测结果；
控制装置，与所述样本装置、所述试剂装置、所述发光底物分注装置和所述光测部件电连接，配置用于：
接收测试指令，所述测试指令包括待测物质的类型；
响应于所述测试指令：
控制所述样本分注部件将所述样本存储部件中的待测样本加入到所述反应装置上的反应杯中；
控制所述试剂分注部件将所述待测物质的类型所对应的固相组分加入到所述反应装置上的反应杯中，以使所述待测样本与所加入的固相组分在所述反应杯中混合并孵育一段时间，使得加入的固相组分上包被的物质能与所述待测样本中的所述待测物质结合；
控制所述试剂分注部件进一步将所述待测物质的类型所对应的标记组分加入所述反应杯，以使所加入的标记组分与所述反应杯中的混合物混合并孵育一段时间，使得所加入的标记组分能与所加入的固相组分上结合的所述待测物质结合；
控制所述发光底物分注装置将发光底物加入所述反应杯中；以及
根据所述光测部件中所测得发光值与发光阈值的比值获得检测结果。
在一些实施方式中，所述固相试剂中的各个固相组分以单独分装的形式存在于所述试剂盒中，或者以预先混合的形式存在于所述试剂盒中。
在一些实施方式中，所述标记试剂中的各个标记组分以单独分装的形式存在于所述试剂盒中，或者以预先混合的形式存在于所述试剂盒中。
在一些实施方式中，所述试剂盒为根据本发明第一方面所描述的试剂盒。
在第四方面，提供了本发明的固相试剂和标记试剂在制备用于血液筛查的试剂盒中 的用途。
采用上述方案对待测样本中的多项感染病原体实施混合检测，基于所得到一个检测结果实现了对多项感染风险的评估，从而大大提高了血液筛查评估效率、缩短了平均检测时长，从而降低了检测成本，有利于血液筛查的推广。另一方面，本发明实施方式提供的检测模式能够根据目标人群设定所针对的感染病原体，进一步提升了筛查时的适用范围和灵活性。
附图说明
图1示出了本发明实施方式的免疫分析系统的示意图；
图2示出了本发明实施方式的控制装置的结构示意图。
具体实施方式
下文将结合具体实施方式和实施例，具体阐述本发明，本发明的优点和各种效果将由此更加清楚地呈现。本领域技术人员应理解，这些具体实施方式和实施例是用于说明本发明，而非限制本发明。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
如前文所述，针对当前血液筛查项目多、成本高的问题，本发明提供了一种基于捕获混合试剂和标记试剂的血液筛查评估方式。考虑到血液制品任意一项传染性疾病结果的阳性，都会造成该血液无法使用，本发明提供的方式基于一个混合检测结果，就能对血液制品传染疾病的阴阳性进行判断。在本发明中，当检测结果为阴性时，表明混合检测的所有感染病原体为阴性；结果为阳性时，表明混合检测的感染病原体中至少有一项为阳性，该血液制品不建议使用或者建议该阳性样本所源自的个体做进一步检查。据此，这种对传染性疾病进行混合检测的方式可以有效的提高血液筛查的效率、降低血液筛查的成本。
在本发明实施方式中，术语“固相载体”、“固相支持物”、“固体支持物”和“固体载体”可以互换使用，其是指可以附着抗原或抗体的固体表面。对用于本发明的固相载体没有特别的限制，商品化的固相载体及任何可用于免疫分析的固相载体均可用于本发明。示例性的固相载体可以是磁珠(如羧基磁珠)、酶标板、塑料板、塑料管、乳胶珠、琼脂 糖珠、玻璃、硝酸纤维素膜、尼龙膜、二氧化硅板或微芯片，但本发明不限于此。
在本发明实施方式中，术语“捕获混合试剂”表示，其包含包被在固相载体上的至少两种第一配体，并且包被在固相载体上的至少两种第一配体以混合的形式存在于试剂盒中。术语“标记混合试剂”表示，其包含带有标记物的至少两种第二配体，并且带有标记物的至少两种第一配体以混合的形式存在于试剂盒中。
在本发明实施方式中，可以通过以下方式在固相载体上进行包被：一方面，可以将每种待包被的物质单独包被在不同的固相载体上，随后将经包被的各个固相载体包被物混合。例如，可以分别将抗HBs抗原的抗体、HCV抗原、抗HIV-1 p24抗体和HIV-1/-2联合抗原分别包被在固相载体上，随后混合在一起。另一方面，也可以将待包被的物质分为一个或更多个组，每个组中含有一种或更多种待包被的物质，将每组物质分别与不同的固相载体包被，随后将包被物混合，例如，可以将HBs抗原的抗体、HCV抗原、抗HIV-1 p24抗体和HIV-1/-2联合抗原构成一个组，将该组与同一固相载体进行包被。
可用于本发明实施方式中的标记物是本领域技术人员所熟知的，例如可以是碱性磷酸酶(ALP)、氧化物酶、微过氧化物酶、辣根过氧化物酶、β-半乳糖苷酶、葡萄糖氧化酶以及葡萄醣6-磷酸脱氢酶等的酶；异硫氰酸荧光素、四甲基罗丹明异硫氰酸酯、荧光素、罗丹明、铕以及绿色荧光蛋白等的荧光物质；鲁米诺、异鲁米诺、菲啶鎓以及吖啶酯等的化学发光物质；NAD等辅酶；生物素； 35S、 14C、 32P、 131I以及 125I等的放射性物质，但本发明不限于此。
本领域技术人员能够依据所使用的标记物的种类选择合适的发光底物，从而产生可检测的信号。例如，当使用碱性磷酸酶作为标记物时，可采用3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1,2-二氧环乙烷作为发光底物，该底物会被碱性磷酸酶所分解，脱去一个磷酸基，生成不稳定的中间产物，该中间产物通过分子内电子转移产生间氧苯甲酸甲酯阴离子，处于激发态的间氧苯甲酸甲酯阴离子从激发态返回基态时，产生化学发光。再通过光电倍增管对反应中所产生的光子数进行测量，所产生光子的量与样本内检测物的含量成正比。
本发明实施方式适用于ELISA、化学发光、电化学发光、POCT、免疫层析法、上转换发光、下转换发光等多种方法，所采用的抗原为重组抗原或天然抗原。
在本发明的范围中，术语ROC(receiver operating characteristic)曲线是指将诊断试验结果划分为若干临界点，以每个临界点对应的灵敏度为纵坐标，特异度为横坐标，作图得到的曲线。ROC曲线是一种全面、准确评价诊断试验的有效工具。ROC曲线的另 一个作用是确定检测的最佳阈值。ROC曲线法确定临界点多数情况下，选择曲线上尽量靠近左上方的点，确定临界点为最佳。在应用中，根据ROC曲线，结合各切点的灵敏度和特异度结果，选择曲线上尽量靠近左上方约登指数(Youden index)最大的切点为最佳临界点，从而使试验的灵敏度和特异度均较高，同时误诊率和漏诊率较小。
采用本发明实施方式的检测方式，允许针对不同人群选择不同试剂组分，据此，在另一方面，本发明提供了一种更具针对性且更灵活的筛查手段。例如，当需要对待测个体进行常规的筛查时，可以使用针对HBs抗原、抗HCV抗体、HIV-1 p24抗原和HIV-1/-2抗体的组分；又如，当需要进行更全面检测时，可以使用针对HBs抗原、抗HCV抗体、HIV-1 p24抗原、HIV-1/-2抗体和抗TP抗体的组分；再如，当待测个体为欧美人群时，可以使用针对HBs抗原、抗HCV抗体、HIV-1 p24抗原、HIV-1/-2抗体和抗HBc抗体的组分，由此能够可以灵活地针对不同的待测者选择合适的筛查项目，从而拓宽了检测时适用范围。
如本文所使用的，“HBs抗原”(HBsAg)又称乙型肝炎病毒表面抗原，是乙肝病毒的外壳蛋白；“抗HBs抗原抗体”(Anti-HBsAg)是与HBsAg特异性结合的抗体。
如本文所使用的，“HCV抗原”(HCV Ag)是丙型肝炎病毒抗原；“抗HCV抗体”(Anti-HCV)是与HCV抗原特异性结合的抗体。
如本文所使用的，“HIV-1 p24抗原”(p24抗原)是HIV-1 gag基因表达产生的一种高度保守的蛋白；“抗HIV-1 p24抗体”(Anti-p24)是与HIV-1 p24抗原特异性结合的抗体。
如本文所使用的，HIV分为1型和2型。HIV-1抗原是针对1型HIV病毒，HIV-2抗原是针对2型HIV病毒。“HIV-1/-2联合抗原”指的是，将HIV-1和HIV-2按照一定的比例(5:1-1:5)混合，制备得“联合抗原”，进而进行磁珠的包被。
如本文所使用的，“TP抗原”(TP Ag)是梅毒抗原；“抗TP抗体”(Anti-TP)是与TP抗原特异性结合的抗体。
如本文所使用的，“HBc抗原”(HBcAg)是乙型肝炎病毒核心抗原；“抗HBc抗原抗体”(Anti-HBc)是与HBc抗原特异性结合的抗体。
在用于制备固相试剂时，本发明实施方式的抗原例如可以多聚体、重组抗原、抗原片段或抗原肽的形式存在。
在用于制备固相试剂和/或标记试剂时。本发明实施方式的抗体例如可以单克隆抗体、多克隆抗体、重组抗体、嵌合抗体、人源化抗体、抗体的抗原结合片段的形式存在。
在本发明实施方式中，“混合检测值”、“混合检测结果”和“一个检测结果”可以互换使用，是指采用本发明的试剂盒、方法和系统所得到检测结果。例如，当使用碱性磷酸酶作为标记物时，检测结果为发光值。
在本发明实施方式中，通过混合检测值与发光阈值的比值(COI值)确定结果为阳性或阴性。例如，当比值大于等于1.1时，判断结果为阳性，表明有至少一种生物标志物的检测结果为阳性。当比值介于0.9～1.1时，判断结果为灰区，既不确定为阳性或阴性；当比值小于0.9时，判断结果为阴性，表明所针对生物标志物检测结果均为阴性。
在本发明实施方式中，所述捕获混合试剂和所述标记试剂中各组分的浓度被设计为使得，在同一个反应体系下采用所述捕获混合试剂中的每一种包被在固相载体上的组分和所述标记试剂中相应带标记物的组分单独对内部参考品进行检测时，所对应的发光阈值基本上相同。例如，在确定的反应体系下，采用某一种待测物质对应的固相包被物和标记组分单独对ISO 18113-1:2009中定义的内部参考品进行检测，保持标记组分的浓度为一恒定值，调节固相包被物的浓度，调配发光阈值至预设值；或者，保持固相包被物的浓度为一恒定值，调节标记组分的浓度，调配发光阈值至预设值。
在本发明实施方式中，包被在固相载体上的抗HBs抗原的抗体的浓度与带有标记物的抗HBs抗原的抗体的浓度被设计为，使得其在预设反应体系下单独用于检测内部参考品时，所对应的发光阈值基本上为预设发光阈值；包被在固相载体上的HCV抗原的浓度与带有标记物的抗人抗体的浓度被设计为，使得其在所述预设反应体系下单独用于检测内部参考品时，所对应的发光阈值基本上等于所述预设发光阈值；包被在固相载体上的HIV-1/-2联合抗原的浓度与所述带有标记物的抗人抗体的浓度被设计为，使得其单独用于检测内部参考品进行时，所对应的发光阈值基本上等于所述预设发光阈值；包被在固相载体上的抗HIV-1 p24抗体的浓度与带有标记物的抗HIV-1 p24的抗体的浓度被设计为使得其在所述预设反应体系下单独用于检测内部参考品时，所对应的发光阈值基本上为所述预设发光阈值。进一步地，包被在固相载体上的TP抗原或HBc的浓度与所述带有标记物的抗人抗体的浓度被设计为，使得其在所述预设反应体系下单独对内部参考品进行检测时，所对应的发光阈值基本上等于所述预设发光阈值。
在本发明实施方式中，“内部参考品”是指应体系组成确定的标准和依据，其具有如国际标准ISO 18113-1:2009中的定义并可依据该标准而获得。内部参考品是医疗器械生产者用于验证产品性能的样本，是产品原材料选择、制备、鉴定以及确定原材料质量标准，产品生产工艺确定，反应体系组成、反应条件等的最重要的确定标准和依据。对于 定性项目，企业内部参考品的鉴别样本，是判断特定疾病、状态或被测量存在和不存在的界限的数值或量值的样本。
在本发明实施方式中，“基本上相同”是指相对偏差在±10％以内，如±5％以内、±2％以内或±1％以内。
在本发明实施方式中，带有标记物的抗HBs抗原的抗体的浓度可以例如为0.1-5ug/mL。
在本发明实施方式中，带有标记物的抗HIV-1 p24抗体的浓度可以例如为0.1-5ug/mL。
在本发明实施方式中，带有标记物的抗人抗体的浓度可以例如为约0.1μg/mL至约5μg/mL。
在本发明实施方式中，包被有抗HBs抗原的抗体的固相载体和HIV p24抗体的固相载体的浓度可以例如为0.1mg/mL到2mg/mL。
在本发明实施方式中，包被有HCV抗原的固相载体和包被有TP抗原的固相载体的浓度可以例如为0.1mg/mL到2mg/mL。
在本发明实施方式中，包被有HIV-1/-2联合抗原的固相载体的浓度可以例如为0.1mg/mL到2mg/mL。
在示例性的实施方式中，包被有抗HBs抗原的抗体的固相载体(例如磁珠)和HIV p24抗体的固相载体的浓度为约0.2mg/mL，调节发光阈值为约50000(预设发光阈值)，则带有标记物的抗HBs抗原的抗体和带有标记物的抗HIV-1 p24抗体的浓度分别为约0.8μg/ml和约1.2μg/ml。
在示例性的实施方式中，带有标记物的抗人抗体的浓度为约1.0μg/mL，调节发光阈值为50000，则包被有HCV抗原的固相载体(例如磁珠)、包被有HIV-1/-2联合抗原的固相载体和包被有TP抗原的固相载体的浓度分别为约：0.2mg/mL、0.25mg/mL和0.2mg/mL。
在本发明中，带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体可以源于鼠、兔、山羊、绵羊、鸡，但本发明不限于此。
在本发明中，标记试剂中的抗体可以经由本领域常规技术(例如，化学键结合)的方法与标记物连接。
如图1所示，本发明实施方式提供了一种可选择性检测血液样本中不同传染性病原微生物的免疫分析仪。该免疫分析仪包括样本装置10、试剂装置20、反应装置30、光 测部件40和控制装置50。该免疫分析仪还可以包括显示部件(未示出)。
样本装置10用于承载待测试的样本，吸取样本后提供给反应装置30。样本装置10包括样本存储部件11和样本分注部件12。样本存储部件11用于存储待测样本。在一些实施方式中，样本存储部件11可以包括样本分配模块(SDM，Sample Delivery Module)及前端轨道。在另一些实施方式中，样本存储部件11也可以是样本盘，样本盘包括多个可以放置诸如样本管的样本位，样本盘通过转动其盘式结构，可以将样本调度到相应位置，例如供样本分注部件12吸取样本的位置。样本分注部件12用于吸取样本并排放到待加样的反应杯中。样本分注部件12例如可以包括样本针，样本针通过二维或三维的驱动机构来在空间上进行二维或三维的运动，从而样本针可以移动去吸取样本存储部件11所承载的样本，以及移动到待加样的反应杯，并向反应杯排放样本。
试剂装置20用于承载试剂，吸取试剂后提供给反应装置30。试剂装置20包括试剂存储部件13和试剂分注部件14。试剂存储部件13用于存储试剂盒。在一些实施方式中，试剂存储部件13可以为试剂盘，试剂盘呈圆盘状结构设置，具有多个用于承载试剂容器的位置，试剂存储部件13能够转动并带动其承载的试剂容器转动，用于将试剂容器转动到特定的位置，例如被试剂分注部件14吸取试剂的位置。试剂存储部件13的数量可以为一个或多个。试剂分注部件14用于吸取试剂盒中的试剂并排放到待加试剂的反应杯中。在一些实施方式中，试剂分注部件14可以包括试剂针，试剂针通过二维或三维的驱动机构来在空间上进行二维或三维的运动，从而试剂针可以移动去吸取试剂存储部件13所承载的试剂，以及移动到待加试剂的反应杯，并向反应杯排放试剂。
其中，试剂存储部件13用于存储试剂盒，该试剂盒包括固相试剂和标记试剂，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体的固相组分、包被有HCV抗原的固相组分、包被有抗HIV-1 p24抗体的固相组分和包被有HIV-1/-2联合抗原的固相组分，所述标记试剂包含标记抗HBs抗原的抗体的标记组分、标记抗人抗体的标记组分和标记抗HIV-1 p24抗体的标记组分。
反应装置30具有至少一个放置位，放置位用于放置反应杯并孵育反应杯中的反应液。例如，反应装置30可以为反应盘，其呈圆盘状结构设置，具有一个或多个用于放置反应杯的放置位，反应盘能够转动并带动其放置位中的反应杯转动，用于在反应盘内调度反应杯以及孵育反应杯中的反应液。
光测部件40用于对孵育完成的反应液进行光测定，得到样本的反应数据。例如光测部件40对待测的反应液的发光强度进行检测，通过定标曲线，计算样本中待测成分 的浓度等。在一些实施方式中，光测部件40分离设置于反应装置30的外面。
免疫分析仪还包括发光底物分注装置(未示出)。该发光底物分注装置与存储发光底物的容器连接并用于将发光底物注入到待加发光底物的反应杯中。
如图2，控制装置50至少包括：处理组件51、RAM 52、ROM 53、通信接口54、存储器56和I/O接口55，其中，处理组件51、RAM 52、ROM 53、通信接口54、存储器56和I/O接口55通过总线57进行通信。
处理组件可以为CPU、GPU或其它具有运算能力的芯片。
存储器56中装有操作系统和应用程序等供处理器组件51执行的各种计算机程序及执行该计算机程序所需的数据。另外，在样本检测过程中，如有需要本地存储的数据，均可以存储到存储器56中。
I/O接口55由比如USB、IEEE1394或RS-232C等串行接口、SCSI、IDE或IEEE1284等并行接口以及由D/A转换器和A/D转换器等组成的模拟信号接口构成。I/O接口55上连接有由键盘、鼠标、触摸屏或其它控制按钮构成的输入设备，用户可以用输入设备直接向控制装置50输入数据。另外，I/O接口55上还可以连接由具有显示功能的显示器，例如：液晶屏、触摸屏、LED显示屏等，控制装置50可以将处理的数据以图像显示数据输出到显示器上进行显示，例如：分析数据、仪器运行参数等。
通信接口54是可以是目前已知的任意通信协议的接口。通信接口54通过网络与外界进行通信。控制装置50可以通过通信接口54以一定的通信协议，与通过该网连接的任意装置之间传输数据。
其中，控制装置50配置用于接收测试指令，所述测试指令包括待测物质的类型，并且响应于所述测试指令执行下列步骤：
控制样本分注部件12将样本存储部件11中的待测样本加入到反应装置30上的反应杯中；
控制试剂分注部件14将试剂存储部件13中存储的试剂盒中与所述待测物质的类型所对应的固相组分加入到反应装置30上的反应杯中，以使所述待测样本与所加入的固相组分在所述反应杯中混合并孵育一段时间，使得加入的固相组分上包被的物质能与所述待测样本中的所述待测物质结合；
控制试剂分注部件14进一步将试剂盒中与所述待测物质的类型对应的标记组分加入所述反应杯，以使所加入的标记组分与所述反应杯中的混合物混合并孵育一段时间，使得所加入的标记组分能与所加入的固相组分上结合的所述待测物质结合；
控制发光底物分注装置将发光底物加入所述反应杯中；以及
根据光测部件40中所测得发光值与发光阈值的比值获得检测结果。
通过本发明实施方式提供的免疫分析仪，能够在一次检测中选择性地检测待测样本中的不同传染性病原微生物，提高检测灵活性，满足用户的不同场景需求。
在一些实施方式中，所述固相试剂中各个固相组分以单独分装的形式存在于所述试剂盒中。在该情况下，用户可以利用该免疫分析仪在一次检测中对多种传染性病原微生物进行初步筛查，也可以利用该免疫分析仪对单种传染性病原微生物进行筛查，只需要用户输入所需要检测的项目即可。该免疫分析仪可以根据用户的指令将与所需检测的项目对应的固相组分以及对应的标记组分加入到待测样本中。
在一些实施方式中，所述固相试剂中各个固相组分以预先混合的形式存在于所述试剂盒中。该试剂盒例如为上述根据本发明的试剂盒。在该情况下，由于固相试剂中的固相组分预先混合在一起，免疫分析仪根据用户的需求将混合的固相试剂和相应的标记组分加入待测样本，以实现不同的检测需求。
类似地，在一些实施方式中，所述标记试剂中的各个标记组分以单独分装的形式存在于所述试剂盒中，或者以预先混合的形式存在于所述试剂盒中。
实验材料：
抗HBs抗原抗体、HCV抗原、抗p24抗体、HIV-1/-2特异联合抗原和TP抗原来自于Meridian Life Science；
碱性磷酸酶来自于罗氏制药；
磁珠来自于Thermo Fisher；
与人抗体特异性结合的抗体来自于Jackson ImmunoResearch。
固相包被物的制备：
首先将抗原或抗体进行预处理，通过透析去除其缓冲基质中的保护组分。按照每毫克磁珠加入1-40μg的抗原/抗体的比例(优选5-30ug，更优选10-20ug)进行包被。在反应过程中磁珠表面的羧基在EDC/NHS催化下与抗原的氨基进行偶联。取20mg表面修饰有羧基的磁性微球，超声分散于10mM MES缓冲液中，加入80mg EDC和120mg NHS，超声混合均匀后，置于37℃摇床15min。之后处理后的磁珠中，按照比例加入抗原/抗体，混匀，并置于37℃摇床反应10-18h。清洗封闭后，制备得到包被有抗原或 抗体的磁性微球。
标记组分的制备：
将抗HBs抗原抗体、与人抗体特异性结合的抗体和抗p24抗体标记信号标记物。在本发明实施例中，信号标记物为碱性磷酸酶。选择50mM MES pH 6.0缓冲液分别将抗乙型肝炎表面抗原抗体信号标记物、与人抗体特异性结合抗体信号标记物、抗p24抗体信号标记物进行稀释，配制得标记组分。
检测步骤：
第一步，将样本和固相包被物添加到反应管中，在37℃孵育10分钟，使得固相包被物能与样本中对应的待测物质结合。在反应管内孵育完成后，结合在固相上的物质将置于一个磁场内被吸住，结合在磁珠固相上的物质被保留，而未结合的物质被冲洗除去。
第二步，将标记试剂加到反应管中孵育，进行混匀，在37℃孵育10分钟，与第一步形成的结合物结合复合物。在反应管内孵育完成后，该复合物被磁场吸住，而其他未结合的物质被清洗除去。
第三步，将AMPPD添加到反应管内，产生化学发光。再通过光电倍增管对反应所产生的光子数进行测量，以得到样本的化学发光信号值。
在本发明实施方式中，阴性符合率是指使用本发明实施方式的测试方法得到判断为阴性的样本个数占实际参与评估的阴性样本的比例，阳性符合率是指使用本发明实施方式的测试方法得到判断为阳性的样本个数占实际参与评估的阳性样本的比例；样本的真实阴阳性结果来自医院诊断结果。
实施例1确定血筛检测的发光阈值
选取具有明确临床诊断结果的样本，其中HBsAg检测样本1153例(阴性样本842例、阳性样本311例)、Anti-HCV检测样本1100例(阴性样本790例、阳性样本310例)、HIV-1 p24抗原检测样本1000例(阴性样本726例、阳性样本274例)、HIV-1/-2抗体检测样本1200例(阴性样本840例、阳性样本360例)、Anti-TP检测样本1100例(阴性样本761例、阳性样本339例)。
采用由“固相包被物的制备”所得到的包被有Anti-HBsAg的磁性微球作为固相试剂和由“标记组分的制备”所得到的带有碱性磷酸酶的Anti-HBsAg作为标记试剂，根据“检 测步骤”测试不同发光阈值下，HBsAg的阳性符合率和阴性符合率。采用同样的方式，分别测试了Anti-HCV、HIV-1 p24抗原和HIV-1/-2抗体的阳性符合率和阴性符合率，结果汇总于表1中。
表1.各病原微生物发光阈值的确定
Figure PCTCN2019130132-appb-000001
Figure PCTCN2019130132-appb-000002
由表1可知，当发光阈值选择为50000左右时，总体符合率最高，将试剂盒的发光阈值选择为50000。
实施例2各固相包被物浓度的确定以及各标记组分浓度的确定
对于用于检测HBsAg和HIV-1 p24抗原的相关组分来说，将包被有抗原的磁性微球浓度固定为约0.2mg/mL，调节其对应的标记组分，将发光阈值调配在50000，允许±5％偏差，结果如下表2所示。
表2
Figure PCTCN2019130132-appb-000003
由表2可知，HBsAg和HIV-1 p24抗原对应的标记组分的浓度分别为约0.8μg/ml和约1.2μg/mL。
对于用于检测Anti-HCV、HIV-1/-2抗体和Anti-TP的相关组分来说，其对应的标记组分均为带有碱性磷酸酶的与人抗体特异性结合的抗体(例如鼠抗体)，将标记组分浓度固定为约1μg/mL，对磁珠包被物的浓度进行调配，将发光阈值调配在50000，允许±5％偏差，结果如下表3所示。
表3
Figure PCTCN2019130132-appb-000004
如表3所示，Anti-HCV、HIV-1/-2抗体和Anti-TP对应的磁珠包被物的浓度分别为约0.2mg/mL、约0.25mg/mL和约0.2mg/mL。
实施例3配制用于检测不同传染性疾病的试剂组合
在本实施例中，配置了用于表4所示的实验的固相试剂和标记试剂。
表4
编号 检测项目
实验1 HBsAg+Anti-HCV+HIV-1 p24抗原+HIV-1/-2抗体
实验2 HBsAg+Anti-HCV+HIV-1 p24抗原+HIV-1/-2抗体+Anti-TP
对于固相试剂，按照“固相包被物的制备”分别将抗乙型肝炎表面抗原(HBsAg)抗体、HCV抗原、抗p24抗体、HIV-1/-2特异联合抗原和TP抗原包被于磁性微球。制备得包被抗HBsAg抗体的磁性微球、包被有HCV抗原的磁性微球、包被有抗p24抗体的磁性微球、包被有HIV-1/-2特异联合抗原的磁性微球和包被有TP抗原的磁性微球。选择50mM Tris pH 7.4缓冲液分别将各磁珠包被物进行稀释，得到0.2mg/mL的包被抗HBsAg抗体的磁性微球、0.2mg/mL的包被有HCV抗原的磁性微球、0.2mg/mL的包被 有抗p24抗体的磁性微球、0.25mg/mL的包被有HIV-1/-2特异联合抗原的磁性微球进行稀释和0.2mg/mL的包被有TP抗原的磁性微球。接下来，依照实验1～2所针对的项目对各磁珠包被物进行混合，分别制备得到实验1～2中的固相试剂。
对于标记试剂，按照“标记组分的制备”分别配制0.8μg/ml的带有碱性磷酸酶的抗HBsAg抗体、1μg/mL的带有碱性磷酸酶的与人抗体特异性结合的抗体和1.2μg/mL的带有碱性磷酸酶的抗HIV-1 p24抗体。接下来，依照实验1～2所针对的项目对各标记组分进行混合，分别制备得到实验1～2中的标记试剂。
实施例4使用实验1～2中的试剂组合进行测试
选取具有明确临床诊断结果的样本，共2763例。其中，所有检测项(HbsAg、Anti-HCV、HIV-1 p24抗原和HIV-1/-2抗体、anti-TP)阴性样本1000例，HbsAg阳性样本473例，anti-HCV阳性样本329例、HIV-1 p24抗原/HIV-1/-2抗体阳性样本529例，Anti-TP阳性样本432例。
使用实施例3所配制的实验1～2的试剂组合，按照上文“检测步骤”中的方法，利用本发明的免疫分析仪对本实施例中的样本进行测试。
在本发明实施例中，COI(Cutoff index)为测定样本的化学发光信号值(RLU)与阈值(Cutoff value)的比值，对定性检测方法而言，阈值(截断值)为判断测试结果为阳性或阴性的分界值。在本申请实施例中，样本中是否含有检测物，需要将样本的测试结果COI值与参考值(参考值为1.10)进行比较，如果大于或等于1.10，表示样本中检测物的一种或多种为阳性；如果小于0.90，表示样本中的检测物均为阴性。COI在0.90-1.10之间，结果为灰区(不确定)。
将测试的阳性和阴性结果与临床诊断结果进行比较，计算符合率，结果如下表5所示。
表5实验1～2测试的灵敏度和特异性
Figure PCTCN2019130132-appb-000005
如表5可知，实验1(即同时检测HbsAg、Anti-HCV、HIV-1 p24抗原和HIV-1/-2抗体)的阳性符合率为100％、阴性符合率为99.50％，总体符合率为99.79％。实验2(即同时检测HbsAg、Anti-HCV、HIV-1 p24抗原、HIV-1/-2抗体和Anti-TP)的阳性符合率为100％、阴性符合率为99.30％，总体符合率为99.75％。由此能够满足快速、有效地进行传染性疾病筛查的需求。

Claims (21)

  1. 一种试剂盒，包括：
    捕获混合试剂，其混合有包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体和包被在固相载体上的HIV-1/-2联合抗原；和
    标记试剂，其包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体。
  2. 根据权利要求1所述的试剂盒，其中，所述捕获混合试剂进一步混合有包被在固相载体上的TP抗原和包被在固相载体上的HBc抗原中的一种或更多种。
  3. 根据权利要求2所述的试剂盒，其中，所述捕获混合试剂混合有包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的TP抗原。
  4. 根据权利要求2所述的试剂盒，其中，所述捕获混合试剂混合有包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的HBc抗原。
  5. 根据权利要求1至4中任一项所述的试剂盒，其中，固相载体上所包被的各种抗体或抗原包被在同一固相载体上或分别包被在不同的固相载体上。
  6. 根据权利要求1至5中任一项所述的试剂盒，其中，所述捕获混合试剂和所述标记试剂中各组分的浓度被设计为使得，在同一个反应体系下采用所述捕获混合试剂中的每一种包被在固相载体上的组分和所述标记试剂中对应带标记物的组分单独对内部参考品进行检测时，所对应的发光阈值基本上相同。
  7. 根据权利要求1至6中任一项所述的试剂盒，其中，带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体以预先混合的标记试剂的形式存在于所述试剂盒中。
  8. 根据权利要求1至7中任一项所述的试剂盒，进一步包括：
    说明书，记载了当使用所述捕获混合试剂和所述标记试剂检测所对应的病原体时，将混合检测值与发光阈值的比大于等于1.1的情况确定为阳性结果。
  9. 根据权利要求8所述的试剂盒，其中，所述说明书进一步记载了当判断为阳性结果时，建议对包被固相载体上的各个组分所对应病原体的每一种单独作进一步检查。
  10. 一种免疫分析方法，包括以下步骤：
    将待测样本与固相试剂混合并孵育一段时间，使得所述固相试剂中包被在固相载体上的组分能与所述待测样本中对应的待测物质结合，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体和包被在固相载体上的HIV-1/-2联合抗原；
    对所述待测样本和所述固相试剂的混合物进行清洗，除去未结合的物质；
    在所述经清洗的混合物中加入标记试剂并孵育，使得所述标记试剂能与所述固相载体上结合的所述待测物质结合，形成复合物，所述标记试剂包含带有标记物的抗HBs抗原的抗体、带有标记物的抗人抗体和带有标记物的抗HIV-1 p24抗体；
    对所述复合物进行清洗，除去未结合的物质；
    在经清洗的复合物中加入发光底物，以检测所述待测样本中所述待测物质的混合检测值。
  11. 根据权利要求10所述的方法，其中，所述固相试剂进一步包含包被在固相载体上的TP抗原和包被在固相载体上的HBc抗原中的一种或更多种。
  12. 根据权利要求11所述的方法，其中，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的TP抗原；或者
    所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体、包被在固相载体上的HCV抗原、包被在固相载体上的抗HIV-1 p24抗体、包被在固相载体上的HIV-1/-2联合抗原和包被在固相载体上的HBc抗原。
  13. 根据权利要求10至12中任一项所述的方法，其中，所述固相试剂和所述标记试剂中各组分的浓度被设计为使得，在同一个反应体系下所述固相试剂中的每一种包被在固相载体上的组分和所述标记试剂中对应带标记物的组分单独对内部参考品进行检测时，所对应的发光阈值基本上相同。
  14. 根据权利要求10至13中任一项所述的方法，其中，所述固相试剂中包被在固相载体上的各个组分被分别加入到所述待测样本中进行混合并孵育，或者以预先混合的形式被加入到所述待测样本中进行混合并孵育。
  15. 根据权利要求10至14中任一项所述的方法，其中，所述标记试剂中带有标记 物的各个组分被分别加入到所述待测样本与所述固相试剂的混合物中进行混合并孵育，或者以预先混合的形式被加入到所述待测样本与所述固相试剂的混合物中进行混合并孵育。
  16. 根据权利要求10至15中任一项所述的方法，其中，当所述混合检测值与发光阈值的比大于等于1.1时确定为阳性结果。
  17. 根据权利要求16所述的方法，其中，当判断为阳性结果时，所述方法进一步包括对包被固相载体上的组分所对应病原体的每一种进行单独检测的步骤。
  18. 一种可选择性检测血液样本中不同传染性病原微生物的免疫分析仪，包括：
    样本装置，具有样本存储部件和样本分注部件，所述样本存储部件用于存储待测样本，所述样本分注部件用于吸取所述待测样本并排放到待加样的反应杯中；
    试剂装置，具有试剂存储部件和试剂分注部件，所述试剂存储部件用于存储试剂盒，所述试剂盒包括固相试剂和标记试剂，所述固相试剂包含包被在固相载体上的抗HBs抗原的抗体的固相组分、包被有HCV抗原的固相组分、包被有抗HIV-1 p24抗体的固相组分和包被有HIV-1/-2联合抗原的固相组分，所述标记试剂包含标记抗HBs抗原的抗体的标记组分、标记抗人抗体的标记组分和标记抗HIV-1 p24抗体的标记组分，所述试剂分注部件用于吸取所述试剂存储部件上存储的试剂盒中的固相试剂和标记试剂并排放到待加试剂的反应杯中；
    发光底物分注装置，与存储发光底物的容器连接并用于将发光底物注入到待加发光底物的反应杯中；
    反应装置，具有多个用于放置所述反应杯的放置位并用于孵育所述反应杯中的反应液；
    光测部件，用于对孵育完成的反应液进行光测定，以得到待测样本的检测结果；
    控制装置，与所述样本装置、所述试剂装置、所述发光底物分注装置和所述光测部件电连接，配置用于：
    接收测试指令，所述测试指令包括待测物质的类型；
    响应于所述测试指令：
    控制所述样本分注部件将所述样本存储部件中的待测样本加入到所述反应装置上的反应杯中；
    控制所述试剂分注部件将所述待测物质的类型所对应的固相组分加入到所述反应装置上的反应杯中，以使所述待测样本与所加入的固相组分在所述反应杯中 混合并孵育一段时间，使得加入的固相组分上包被的物质能与所述待测样本中的所述待测物质结合；
    控制所述试剂分注部件进一步将所述待测物质的类型所对应的标记组分加入所述反应杯，以使所加入的标记组分与所述反应杯中的混合物混合并孵育一段时间，使得所加入的标记组分能与所加入的固相组分上结合的所述待测物质结合；
    控制所述发光底物分注装置将发光底物加入所述反应杯中；以及
    根据所述光测部件中所测得发光值与发光阈值的比值获得检测结果。
  19. 根据权利要求18所述的免疫分析仪，其中，所述固相试剂中的各个固相组分以单独分装的形式存在于所述试剂盒中，或者以预先混合的形式存在于所述试剂盒中。
  20. 根据权利要求18或19所述的免疫分析仪，其中，所述标记试剂中的各个标记组分以单独分装的形式存在于所述试剂盒中，或者以预先混合的形式存在于所述试剂盒中。
  21. 根据权利要求18所述的免疫分析仪，其中，所述试剂盒为根据权利要求1～9中任一项所述的试剂盒。
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