WO2021134299A1 - 选择性检测不同待测物质的免疫分析仪、方法及试剂盒 - Google Patents

选择性检测不同待测物质的免疫分析仪、方法及试剂盒 Download PDF

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WO2021134299A1
WO2021134299A1 PCT/CN2019/130118 CN2019130118W WO2021134299A1 WO 2021134299 A1 WO2021134299 A1 WO 2021134299A1 CN 2019130118 W CN2019130118 W CN 2019130118W WO 2021134299 A1 WO2021134299 A1 WO 2021134299A1
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component
reagent
sample
antigen
labeling
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PCT/CN2019/130118
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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 CN201980101485.4A priority Critical patent/CN114599976A/zh
Priority to PCT/CN2019/130118 priority patent/WO2021134299A1/zh
Publication of WO2021134299A1 publication Critical patent/WO2021134299A1/zh

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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/02—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor using a plurality of sample containers moved by a conveyor system past one or more treatment or analysis stations

Definitions

  • the invention relates to the field of immunoassays, in particular to the selective detection of different types of test substances.
  • immunoassay methods for detecting test substances contained in samples have been developed.
  • the principle of such a method is generally: first use the capture material to bind the substance to be measured, and then use the labeling substance with the label to combine with the substance to be measured to form a complex of the capture substance-the substance to be measured-the label body, and then use optical
  • the detection device measures the intensity of the optical signal through the sensor to determine whether there is a substance to be tested.
  • immunoassay kits for realizing the above immunoassay methods have been developed.
  • test kit is implemented on an immunoassay analyzer.
  • the test of the sample to be tested by the immunoanalyzer is limited to the test items (that is, the specific test substance type) involved in the kit.
  • test items that is, the specific test substance type
  • the inventors have conducted research on the working mode of the immune analyzer and the matching kit to realize the present invention.
  • the present invention provides an immunoassay instrument that can selectively detect different test substances in a test sample, 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. At least one coating component, the labeling reagent includes a plurality of labeling components with a label, and the reagent dispensing part is used to suck the solid phase reagent and the labeling group in the kit stored on the reagent storage part Divide and discharge into the reaction cup where reagents are 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 including the type of the substance to be tested
  • the reagent dispensing component is controlled to further add at least one marking component corresponding to the type of the substance to be tested into the reaction cup, so that the added marking component is mixed with the mixture in the reaction cup and incubated For a period of time, the added labeling component can be combined with the test substance bound to at least one coating component coated on the solid carrier to form a coating component-test substance-labeling component Compound
  • the detection result is obtained according to the ratio of the luminescence value measured in the light measuring component to the luminescence threshold value.
  • the immune analyzer can respond to the instruction of the type of the substance to be tested on the basis of a kit, and select the reagent components corresponding to different types of the substance to be tested for detection.
  • the control reagent dispensing component adds the solid-phase reagent containing at least one coating component coated on the solid-phase carrier into the reaction cup, wherein at least one coating component can interact with the substance to be tested. Specific binding.
  • the immune analyzer further controls the reagent dispensing component to selectively add at least one labeled component corresponding to the type of the substance to be tested into the reaction cup.
  • the detection items can be selected according to user needs. Therefore, an immunoassay analyzer capable of implementing selective detection conveniently and flexibly is provided.
  • the solid-phase reagent contains at least one antigen coated on the solid-phase carrier; and the labeling reagent includes a labeling component containing a labeled anti-human IgG antibody and a labeled anti-human IgM antibody The marking components.
  • the immune analyzer of the first aspect of the present invention can selectively detect one or more of IgG and IgM anti-human antibodies of the pathogen corresponding to at least one antigen.
  • the human body mainly has 5 kinds of immunoglobulins: IgG, IgM, IgA, IgD and IgE, of which IgG is a major immunoglobulin in serum, and its content accounts for about 65 to 75% of immunoglobulins. It is a kind of anti-infection. Important substances. IgG antibody is produced late, maintained for a long time, disappeared slowly, and has a high concentration. It can be detected in the blood as an indicator of long-term infection. IgM is a pentamer, which is the largest molecule among immunoglobulins. IgM is the first to be produced and will be produced quickly once infected.
  • IgM maintains a short period of time and disappears quickly, and has an anti-infection effect at the initial stage of infection. Therefore, detection in the blood can be used as an indicator of recent infection.
  • the content of IgA, IgD and IgE in serum is relatively small.
  • Such as hepatitis B virus core antibody (Anti-HBc) and hepatitis A antibody (Anti-HAV) the main clinical detection of anti-human antibodies.
  • Anti-HBc hepatitis B virus core antibody
  • Anti-HAV hepatitis A antibody
  • its antibody subtypes will be distinguished, which is used to assess the patient's disease state and determine whether it is a past infection or an acute infection. In this case, it is necessary to selectively detect different subtypes of antibodies.
  • the corresponding solid-phase reagent when the selected antibody type to be detected is human IgG, the corresponding solid-phase reagent is a solid-phase carrier coated with at least one antigen, and the corresponding labeling component is an anti-human IgG antibody with a label.
  • the corresponding solid phase reagent when the selected antibody type to be detected is human IgM, the corresponding solid phase reagent is a solid phase carrier coated with at least one antigen, and the corresponding labeling component is a labeled anti-human IgM antibody.
  • the corresponding solid phase reagent is a solid phase carrier coated with at least one antigen
  • the corresponding labeling component is a labeled anti-human IgG antibody And labeled anti-human IgM antibody.
  • the solid-phase reagent contains at least one antigen coated on a solid-phase carrier, and the antigen is selected from the group consisting of HBV antigen (preferably HBc antigen), HAV antigen and HEV antigen; and the labeling reagent includes The labeled component of the labeled anti-human IgG antibody and the labeled component of the labeled anti-human IgM antibody.
  • HBV antigen preferably HBc antigen
  • HAV antigen HEV antigen
  • the labeling reagent includes The labeled component of the labeled anti-human IgG antibody and the labeled component of the labeled anti-human IgM antibody.
  • the solid-phase reagent contains at least one antigen coated on a solid-phase carrier, and the antigen is selected from the group consisting of Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus 1/ Type 2 antigen, parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen; and the labeling reagent includes a labeling component containing a labeled anti-human IgG antibody and a label containing a labeled anti-human IgM antibody Components.
  • the solid-phase reagent contains a plurality of capture antibodies coated on a solid-phase carrier, and the capture antibodies correspond to different biomarkers; and the labeling reagent includes a plurality of types corresponding to the different biomarkers. Label the antibody.
  • the immune analyzer of the first aspect of the present invention can selectively detect one or more of different biomarkers.
  • the solid-phase reagent includes a procalcitonin (PCT) capture antibody and a Presepsin capture antibody coated on a solid-phase carrier; and the labeling reagent includes a PCT-labeled antibody with a label. And Presepsin-labeled antibody with labeled substance.
  • PCT procalcitonin
  • the immune analyzer of the first aspect of the present invention can selectively detect PCT, Presepsin or PCT+Presepsin according to user requirements.
  • the solid-phase reagent includes multiple coating components coated on a solid-phase carrier.
  • the solid-phase carrier comprises antibodies against HBs antigen, HCV antigen, anti-HIV-1p24 antibody and HIV-1/-2 combined antigen coated on the solid-phase carrier; and
  • the labeling reagent includes a labeling component containing a labeled anti-HBs antigen antibody, a labeled component containing a labeled anti-human antibody, and a labeled component containing a labeled anti-HIV-1p24 antibody.
  • the immunoassay analyzer of the first aspect of the present invention can selectively screen the HBs antigen, anti-HCV antibody, HIV-1p24 antigen and HIV-1/-2 antibody in the sample to be tested (for example, blood sample)
  • the sample to be tested for example, blood sample
  • One or more of HBV, HCV and HIV infection in the individual from which the sample is derived can be determined.
  • the solid-phase carrier comprises an anti-HBs antigen antibody, HCV antigen, anti-HIV-1p24 antibody, HIV-1/-2 combined antigen, and TP antigen coated on the solid-phase carrier.
  • HBc antigen; and the labeling reagent includes a labeling component containing a labeled anti-HBs antigen antibody, a labeling component containing a labeled anti-human antibody, and a label containing a labeled anti-HIV-1p24 antibody Components.
  • the immune analyzer of the first aspect of the present invention can selectively screen the HBs antigen, anti-HCV antibody, HIV-1p24 antigen, and HIV-1/-2 antibody in the sample (for example, blood sample) to be tested. , One or more of anti-TP antibody and anti-HBc antibody, so as to determine whether the individual from which the sample is derived has one or more infections of HBV, HCV, HIV, and TP.
  • the solid-phase reagent comprises a plurality of coating components coated on a solid-phase carrier, and the plurality of coating components are present in the kit in the form of separate packaging.
  • the solid-phase reagent includes a plurality of coating components coated on a solid-phase carrier, and the plurality of coating components are present in the kit in a pre-mixed form.
  • the present invention provides an immunoassay instrument that can selectively detect different test substances in a test sample, 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 plurality of types coated on a solid-phase carrier.
  • the labeling reagent contains at least one labeling component with a label, and the reagent dispensing component is used to suck the coating component and the label in the kit stored on the reagent storage component The reagents are discharged into the reaction cup where the reagents are 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 including the type of the substance to be tested
  • Control the reagent dispensing component to add at least one coating component corresponding to the type of the substance to be tested into the reaction cup on the reaction device, so that the sample to be tested and the added coating
  • the coatings in the components are mixed in the reaction cup and incubated for a period of time, so that the added coating components can be combined with the test substance in the test sample;
  • the reagent dispensing component is controlled to further add the 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 at least one of the labeling reagents is labeled
  • the component can be combined with the test substance bound to the added coating component to form a complex of the coating component-the test substance-the labeling component;
  • the detection result is obtained according to the ratio of the luminescence value measured in the light measuring component to the luminescence threshold value.
  • the immune analyzer can respond to the instruction of the type of the substance to be tested according to a kit, and select reagent components corresponding to different types of the substance to be tested for detection.
  • the immune analyzer responds to the command to control the reagent dispensing component to selectively add at least one coating component corresponding to the type of the substance to be tested into the reaction cup.
  • the control reagent dispensing component adds a labeling reagent containing at least one labeling component with a label to the reaction cup, wherein the at least one labeling component can specifically bind to the substance to be tested.
  • the solid-phase reagent contains an anti-human IgG antibody coated on a solid-phase carrier and a solid-phase reagent containing an anti-human IgM antibody coated on a solid-phase carrier; and the labeling reagent contains a band At least one antigen of the marker.
  • the immune analyzer of the second aspect of the present invention can selectively detect one or more of human IgG and human IgM of the pathogen corresponding to at least one antigen.
  • the corresponding solid phase reagent when the selected antibody type to be detected is human IgG, the corresponding solid phase reagent is a solid phase carrier coated with an anti-human IgG antibody, and the corresponding labeling component is an antigen with a label.
  • the corresponding solid-phase reagent when the selected antibody type to be detected is human IgM, the corresponding solid-phase reagent is a solid-phase carrier coated with anti-human IgM antibodies, and the corresponding labeling component is an antigen with a label.
  • the selected antibody types to be detected are human IgG and human IgM
  • the corresponding solid-phase reagents are solid-phase carriers coated with anti-human IgG antibodies and solid-phase carriers coated with anti-human IgM antibodies, and the corresponding label group Divided into labeled antigens.
  • the solid-phase reagent comprises a plurality of coating components coated on a solid-phase carrier, and the plurality of coating components are present in the kit in the form of separate packaging.
  • the solid-phase reagent includes a plurality of coating components coated on a solid-phase carrier, and the plurality of coating components are present in the kit in a pre-mixed form.
  • the present invention provides an immunoassay method for selectively detecting a substance to be tested in a sample, including the following steps:
  • the sample to be tested is mixed with a solid phase reagent containing at least one coating component coated on a solid carrier and incubated for a period of time, so that the at least one coating component can interact with the sample to be tested in the sample to be tested.
  • At least one corresponding labeling component is selected from the labeling reagent, and the at least one labeling component is added to the washed mixture and incubated, so that the added labeling component can be combined with the package.
  • the test substance bound to at least one coating component on the solid phase carrier binds to form a complex, wherein the labeling reagent includes a plurality of labeling components with a label;
  • a luminescent substrate is added to the washed complex to detect the detection value of the test substance in the test sample.
  • the solid phase reagent is mixed and incubated with the sample to be tested, wherein the solid phase reagent contains at least one coating component coated on the solid phase carrier, and the solid phase At least one coating component in the reagent can specifically bind to the substance to be tested.
  • a labeling component is selected from the labeling reagent, and the labeling component is mixed with the mixture of the sample and the solid phase reagent and incubated to selectively detect one or more labels
  • the type of substance to be tested corresponding to the component. That is to say, by using a single solid phase reagent and one or more labeling components in the selective labeling reagent, the present invention realizes a convenient and low-cost selective immunoassay.
  • the solid-phase reagent includes multiple coating components coated on a solid-phase carrier, and the multiple coating components are added to the sample to be tested separately or pre-mixed The method is added to the sample to be tested.
  • the present invention provides an immunoassay method for selectively detecting the substance to be tested in a sample, including the following steps:
  • At least one coating component corresponding to the solid phase reagent is selected, and the sample to be tested is mixed with the at least one coating component and incubated for a period of time, so that the coating component
  • the component can be combined with the test substance in the test sample, wherein the solid-phase reagent includes multiple coating components on a solid-phase carrier;
  • a luminescent substrate is added to the washed complex to detect the detection value of the test substance in the test sample.
  • one or more coating components are selected from the solid-phase reagent and the sample and the sample to be tested are mixed and incubated according to the requirements of the type of the substance to be tested. .
  • the labeling reagent is mixed and incubated with the mixture of the sample and the coating component, wherein the labeling reagent contains at least one labeling component with a label, and the at least one labeling component can specifically bind to the substance to be tested ,
  • the present invention realizes a convenient and low-cost selective immunoassay.
  • the labeling reagent comprises a plurality of labeling components with a label, and the plurality of labeling components are added to the washed mixture separately or in a pre-mixed manner. The cleaned mixture.
  • the present invention provides a kit including:
  • the capture mixed reagent includes at least one coating component, especially multiple coating components, coated on a solid carrier;
  • the labeling reagent contains multiple labeling components with labels present in the kit in the form of separate aliquots.
  • the kit of the present invention can be used in conjunction with the immune analyzer of the first aspect of the present invention and/or the method of the third aspect of the present invention.
  • the capture mixture reagent contains at least one antigen coated on a solid-phase carrier, and the antigen is selected from the group consisting of HBV antigen (preferably HBc antigen), HAV antigen and HEV antigen;
  • the labeling reagent includes a labeling component containing a labeled anti-human IgG antibody and a labeling component containing a labeled anti-human IgM antibody.
  • the capture mixture reagent contains at least one antigen coated on a solid carrier, and the antigen is 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 labeling reagent includes a labeling component containing a labeled anti-human IgG antibody and a labeled anti The labeling component of human IgM antibody.
  • the present invention provides a kit including:
  • Capture reagents including multiple coating components coated on a solid carrier in the kit in the form of separate aliquots;
  • Labeling mixed reagent at least one labeling component with a label, especially multiple labeling components.
  • kit of the present invention can be used in conjunction with the immune analyzer of the second aspect of the present invention and/or the method of the fourth aspect of the present invention.
  • 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 term "coating component” refers to the substance coated on the solid-phase carrier, which can bind to the substance to be tested in the sample to be tested; in the case that the coating component is an antibody, It can be called a capture antibody.
  • labeling component refers to a substance labeled with a label, which can bind to the substance to be tested in the sample to be tested; when the labeling component is an antibody, it can be called a label Antibody.
  • 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 coating components coated on a solid phase carrier, and the at least two coating components coated on the solid phase carrier are The mixed form is present in the kit; in the case where the solid phase reagent contains at least two coating components in mixed form and coated on the solid phase carrier, such solid phase reagent can be called “capture mixed Reagents”.
  • the term “labeling mixed reagent” means that it contains at least two labeling components with a label, and the at least two labeling components with a label are present in the kit in a mixed form; the labeling reagent contains at least two labeling components. In the case where there are at least two labeling components with a label, such labeling reagent can be called a labeling mixed reagent.
  • the solid-phase carrier can be coated to produce the capture mixed reagent in the following manner:
  • each substance to be coated can be separately coated on a different solid-phase carrier, and then The coated solid-phase carriers are mixed.
  • the anti-HBs antigen antibody, HCV antigen, anti-HIV-1p24 antibody and HIV-1/-2 combined antigen can be separately coated on the 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 is mixed.
  • antibodies to HBs antigen, HCV antigen, anti-HIV-1p24 antibody and HIV-1/-2 combined antigen can be formed into a group, and this group can be coated with the same solid phase carrier .
  • 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, and down-conversion luminescence.
  • 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.
  • the antigens of the embodiments of the present invention may exist in the form of multimers, recombinant antigens, natural antigens, antigen fragments, or antigen peptides, for example.
  • 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.
  • the antibodies may be derived from mice, rabbits, goats, sheep, and chickens, but the present invention is not limited thereto.
  • anti-human immunoglobulin antibody can be used interchangeably, meaning that they can be combined with human IgG, or/and human IgM, Or/and antibodies that specifically bind to other human antibody subtypes (such as IgA, IgE, etc.).
  • the labeling component in the labeling reagent can be connected to the label via a conventional technique in the art (for example, chemical bonding).
  • the ratio of the 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 each component in the solid phase reagent and the labeling reagent is designed such that each of the solid phase reagents is used to coat the solid phase in the same reaction system.
  • the coating component on the carrier and the corresponding labeled component in the labeling reagent separately detect the internal reference product, the corresponding luminescence threshold is optimal, preferably substantially the same.
  • the coating component and labeling component corresponding to a certain substance to be tested are used to separately detect the internal reference defined in ISO 18113-1:2009, and the concentration of the labeling component is maintained as A constant value is used to adjust the concentration of the coating component to adjust the luminescence threshold to a preset value; or, to maintain the concentration of the coating component at a constant value, to adjust the concentration of the labeling component to adjust the luminescence threshold to the preset value.
  • 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 non-existence of the measurement.
  • substantially the same means that the relative deviation is within ⁇ 10%, such as within ⁇ 5%, ⁇ 2%, or ⁇ 1%.
  • the embodiment of the present invention provides an immunoassay analyzer that can selectively detect different test substances in a test 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 component 13 is used to store reagent kits.
  • the kit includes a solid-phase reagent and a labeling reagent, the solid-phase reagent includes at least one coating component coated on a solid-phase carrier, and the labeling reagent includes a plurality of labeled reagents. Mark the components.
  • the kit includes a solid-phase reagent and a labeling reagent. The solid-phase reagent includes a plurality of coating components coated on a solid-phase carrier, and the labeling reagent includes at least one label-bearing reagent. Mark the components.
  • 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 optical 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 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 reagent in the reagent kit stored in the reagent storage component 13 to the reaction cup on the reaction device 30, so that the sample to be tested and the added solid phase reagent are in the reaction cup. Mixing and incubating for a period of time, so that at least one coating component on the solid-phase carrier 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 coating component in the added solid phase reagent;
  • 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.
  • 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 at least one coating component corresponding to the type of the substance to be tested in the reagent kit stored in the storage component 13 into the reaction cup, so that the sample to be tested and the at least one coating component A coating component is mixed in the reaction cup and incubated for a period of time, so that the coating component on the solid-phase carrier can be combined with the test substance 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 added labeling reagent is mixed with the mixture in the reaction cup and incubated for a period of time, so that the labeling group in the added labeling reagent
  • the component can be combined with the test substance bound to the coating component in the added solid phase reagent;
  • 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 embodiments of the present invention can selectively detect different types of substances to be tested based on a kit based on user requirements, improve detection flexibility, and meet user requirements in different scenarios.
  • HAV antigen from Meridian Life Science
  • Anti-human antibodies such as anti-human IgM antibodies and anti-human IgG antibodies are from Jackson ImmunoReasearch;
  • Alkaline phosphatase comes from Roche Pharmaceuticals
  • the magnetic beads come from Thermo Fisher.
  • 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 0.5-40 ⁇ g of antigen added per mg of magnetic beads (preferably 1-30 ⁇ g).
  • 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.
  • EDC/NHS electrospray mediated saline
  • the antigen added to the processed magnetic beads according to the proportion, mix them, and place them on a shaker at 37°C for 10-18 hours.
  • magnetic microspheres coated with antigen are prepared. Choose a 50mM Tris pH 7.4 buffer to dilute the antigen-coated magnetic microspheres to prepare a solid phase reagent. Among them, the concentration of the magnetic microspheres coated with the antigen is 0.7 mg/mL.
  • the anti-human IgM antibody and anti-human antibody are labeled with signal markers.
  • the signal marker is alkaline phosphatase. Choose 50mM MES pH 6.0 buffer to dilute the anti-human IgM antibody signal marker and anti-human antibody signal marker respectively to prepare labeled components.
  • the sample and solid phase reagent are added to the reaction tube, and incubated at 37°C for 10 minutes, so that the antigen on the solid phase surface 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 second step select the required labeled components and add them to the reaction tube to incubate, mix, and incubate 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.
  • the luminescence signal value is divided by the threshold value to obtain the COI value. Compare the COI value of the test result of the sample with the reference value (reference value 1.10). If it is greater than or equal to 1.1, it means that one or more of the test substances in the sample is positive; if it is less than 0.90, it means that the test substances in the sample are all Is negative. The COI is between 0.90 and 1.10, and the result is a gray zone (indeterminate).
  • 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.
  • Example 2 Based on a kit, selective detection of IgM antibodies and IgG antibodies against HBc
  • Preparation of solid-phase coating use the magnetic microspheres coated with HBc antigen obtained from "Preparation of solid-phase coating" as the solid-phase component;
  • Preparation of labeling component 1 According to "Preparation of labeling component", use the prepared anti-human IgM antibody with alkaline phosphatase, and match it with the solid phase component to determine the internal reference. Select the dilution corresponding to the luminescence threshold of 50000 (allowing ⁇ 10% deviation) as the dilution of labeled component 1;
  • Preparation of labeling component 2 According to "Preparation of labeling component", use the prepared anti-human IgG antibody with alkaline phosphatase, and match it with the solid phase component to determine the internal reference. The dilution corresponding to the luminescence threshold of 70,000 (allowing ⁇ 10% deviation) is selected as the dilution of labeled component 2.
  • Example 4 Based on a kit, selective detection of IgM antibodies and IgG antibodies against HAV
  • Preparation of solid-phase coating use the magnetic microspheres coated with HAV antigen obtained from "Preparation of solid-phase coating" as the solid-phase component;
  • Preparation of labeling component 1 According to "Preparation of labeling component", use the prepared anti-human IgM antibody with alkaline phosphatase, and match it with the solid phase component to determine the internal reference. Select the dilution corresponding to the luminescence threshold of 40,000 (allowing ⁇ 10% deviation) as the dilution of labeled component 1;
  • Preparation of labeling component 2 According to "Preparation of labeling component", use the prepared anti-human IgG antibody with alkaline phosphatase, and match it with the solid phase component to determine the internal reference. The dilution corresponding to the luminescence threshold of 70,000 (allowing ⁇ 10% deviation) is selected as the dilution of labeled component 2.
  • the luminescence signal value is divided by the threshold value to obtain the COI value. Compare the COI value of the test result of the sample with the reference value (reference value 1.10). If it is greater than or equal to 1.1, it means that one or more of the test substances in the sample is positive; if it is less than 0.90, it means that the test substances in the sample are all Is negative. The COI is between 0.90 and 1.10, and the result is a gray zone (indeterminate).

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Abstract

可选择性检测待测样本中的不同待测物质的免疫分析仪，该免疫分析仪配套一个试剂盒可根据用户需求，实现不同待测物质的类型的选择性检测。还涉及免疫分析方法及试剂盒。

Description

选择性检测不同待测物质的免疫分析仪、方法及试剂盒 技术领域
本发明涉及免疫分析领域，具体涉及不同待测物质的类型的选择性检测。
背景技术
近年来，已开发了用于检测包含在样本中的待测物质的免疫分析方法。这样的方法的原理一般为：先利用捕获物与待测物质结合，再用带有标记物的标记体与待测物质结合，形成捕获物-待测物质-标记体的复合物，之后利用光学检测装置，经由感测器测得光学信号的强度，即可判断是否存在待测物质。同时，已开发了用于实现上述免疫分析方法的免疫检测试剂盒。
通常，试剂盒对待测物质检测在免疫分析仪上实施。在这一过程中，免疫分析仪对待测样本的检测仅局限于该试剂盒所涉及的检测项目(即特定的检测物质类型)中。这样一来，用户无法根据自己的检测需求来选择合适的检测项目，从而在临床应用方面带来了诸多限制。
据此，在使用免疫分析仪进行免疫分析时，存在着按照用户需求，灵活地选择检测项目的强烈需求。
发明内容
为了解决当前免疫分析仪无法基于同一试剂盒实现选择性分析不同项目的问题，本发明人对免疫分析仪的工作模式及配套的试剂盒进行了研究，从而实现本发明。
在第一方面，本发明提供了一种可选择性检测待测样本中的不同待测物质的免疫分析仪，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至少一种包被组分，所述标记试剂包含多种带标记物的标记组分，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组分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固相试剂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述固相试剂在所述反应杯中混合并孵育一段时间，使得包被在固相载体上的至少一种包被组分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至少一种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包被在固相载体上的至少一种包被组分上结合的所述待测物质结合，形成包被组分-待测物质-标记组分的复合物；
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。
需要说明的是，根据本发明第一方面的免疫分析仪在一种试剂盒的基础上就能够响应待测物质的类型的指令，选择不同的待测物质类型对应的试剂组分进行检测。在检测过程中，控制试剂分注部件将含包被在固相载体上的至少一种包被组分的固相试剂加入到反应杯中，其中至少一种包被组分能与待测物质特异性结合。接下来，免疫分析仪响应指令进一步控制试剂分注部件以选择性地将与待测物质的类型所对应的至少一种标记组分加入到该反应杯中。通过形成的包被组分-待测物质-标记组分的复合物，实现按用户需求选择检测项目。由此提供了一种能够便捷、灵活地实现选择性检测的免疫分析仪。
在一些实施方式中，固相试剂含包被在固相载体上的至少一种抗原；而标记试剂包括含带标记物的抗人IgG抗体的标记组分和含带标记物的抗人IgM抗体的标记组分。
通过采用这样的方式，本发明第一方面的免疫分析仪能够对至少一种抗原所对应的病原体的IgG和IgM抗人抗体中的一种或更多种进行选择性检测。
这种选择性是十分有必要的。人体主要有IgG、IgM、IgA、IgD和IgE 5种免疫球蛋白，其中IgG是血清中一种主要的免疫球蛋白，含量占免疫球蛋白的65～75％左右，是机体抗感染的一种重要物质。IgG抗体产生晚、维持时间长、消失慢、浓度高，在血液中检测到可作为远期感染指标。IgM为五聚体，是免疫球蛋白中分子最大者。IgM产生最早，一经感染会快速产生。但IgM维持时间短、消失快，在感染初期抗感染起作用，因此在血液中检测到可作为近期感染指标。IgA、IgD和IgE在血清中的含量相对较少。在临床应用中，经常需要对抗体的不同亚型进行检测，并根据检测结果进行临床判断。如乙肝病毒核心抗体(Anti-HBc)和甲肝抗体(Anti-HAV)，在临床上主要检测抗人抗体。但在某些应用场景会对其抗体亚型进行区分，用于评估患者发病状态，确定其为既往感染或是急性感染。在这种情况下，就需要选择性检测抗体的不同亚型。
例如，当选择的待检测的抗体类型为人IgG时，对应的固相试剂为包被有至少一种抗原的固相载体，对应的标记组分为带标记物的抗人IgG抗体。又例如，当选择的待检测的抗体类型为人IgM时，对应的固相试剂为包被有至少一种抗原的固相载体，对应的标记组分为带标记物的抗人IgM抗体。再例如，当选择的待检测的抗体类型为人IgG和人IgM时，对应的固相试剂为包被有至少一种抗原的固相载体，对应的标记组分为带标记物的抗人IgG抗体和带标记物的抗人IgM抗体。
在示例性的实施方式中，固相试剂含包被在固相载体上的至少一种抗原，所述抗原选自HBV抗原(优选为HBc抗原)、HAV抗原和HEV抗原；而标记试剂包括含带标记物的抗人IgG抗体的标记组分和含带标记物的抗人IgM抗体的标记组分。
在另一示例性的实施方式中，固相试剂含包被在固相载体上的至少一种抗原，所述抗原选自弓形虫抗原、风疹病毒抗原、巨细胞病毒抗原、单纯疱疹病毒1/2型抗原、细小病毒B19抗原、柯萨奇病毒抗原和带状疱疹病毒抗原；而标记试剂包括含带标记物的抗人IgG抗体的标记组分和含带标记物的抗人IgM抗体的标记组分。
在另一些实施方式中，固相试剂含包被在固相载体上的多种捕获抗体，所述捕获抗体与不同生物标志物对应；而标记试剂包括与所述不同生物标志物对应的多种标记抗体。
通过采用这样的方式，本发明第一方面的免疫分析仪能够对不同生物标志物中的一种或更多种进行选择性检测。
在示例性的实施方式中，固相试剂含包被在固相载体上的降钙素原(procalcitonin,PCT)捕获抗体和Presepsin捕获抗体；而所述标记试剂包括含带标记物的PCT标记抗体和含带标记物的Presepsin标记抗体。
通过采用这样的方式，本发明第一方面的免疫分析仪能够根据用户需求对PCT、Presepsin或者PCT+Presepsin进行选择性检测。
在又一些实施方式中，所述固相试剂包含包被在固相载体上的多种包被组分。
在一个示例性的实施方式中，所述固相载体包含包被在固相载体上的抗HBs抗原的抗体、HCV抗原、抗HIV-1p24抗体和HIV-1/-2联合抗原；而所述标记试剂包括含带标记物的抗HBs抗原的抗体的标记组分、含带标记物的抗人抗体的标记组分和含带标记物的抗HIV-1p24抗体的标记组分。
通过采用这样的方式，本发明第一方面的免疫分析仪能够选择性地筛查待测样本(例如，血液样本)中HBs抗原、抗HCV抗体、HIV-1p24抗原和HIV-1/-2抗体的一种或更多种，从而判断样本所来源于的个体是否存在HBV、HCV和HIV中的一种或多种感染。
在另一个示例性的实施方式中，所述固相载体包含包被在固相载体上的抗HBs抗原的抗体、HCV抗原、抗HIV-1p24抗体、HIV-1/-2联合抗原、TP抗原和HBc抗原；而所述标记试剂包括含带标记物的抗HBs抗原的抗体的标记组分、含带标记物的抗人抗体的标记组分和含带标记物的抗HIV-1p24抗体的标记组分。
通过采用这样的方式，本发明第一方面的免疫分析仪能够选择性地筛查待测样本(例如，血液样本)中HBs抗原、抗HCV抗体、HIV-1p24抗原、HIV-1/-2抗体、抗TP抗体和抗HBc抗体的一种或更多种，从而判断样本所来源于的个体是否存在HBV、HCV、HIV、TP中的一种或多种感染。
在一些实施方式中，所述固相试剂包含包被在固相载体上的多种包被组分，所述多种包被组分以单独分装的形式存在于所述试剂盒中。
在一些实施方式中，所述固相试剂包含包被在固相载体上的多种包被组分，所述多种包被组分以预先混合的形式存在于所述试剂盒中。
在第二方面，本发明提供了一种可选择性检测待测样本中的不同待测物质的免疫分析仪，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，所述固相试剂包含多种包被在固相载体上的包被组分，所述标记试剂包含带标记物的至少一种标记组分，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包被组分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至少一种包被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包被组分中的包被物在所述反应杯中混合并孵育一段时间，使得所加入的包被组分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标记试剂加入所述反应杯，以使所述标记试剂与所述反应杯中的混合物混合并孵育一段时间，使得所述标记试剂中的至少一种标记组分能与所加入的包被组分上结合的所述待测物质结合，形成包被组分-待测物质-标记组分的复合物；
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。
需要说明的是，根据本发明第二方面的免疫分析仪根据一种试剂盒就能够响应待测物质的类型的指令，选择与不同的待测物质类型对应的试剂组分进行检测。在检测过程 中，免疫分析仪响应指令控制试剂分注部件选择性地将与待测物质的类型所对应的至少一种包被组分加入到反应杯中。接下来，控制试剂分注部件将含带有标记物的至少一种标记组分的标记试剂加入到该反应杯中，其中至少一种标记组分能与待测物质特异性结合。通过形成的包被组分-待测物质-标记组分的复合物，实现按用户需求检测。由此提供了一种能够便捷、灵活地实现选择性检测的免疫分析仪。
在一些实施方式中，固相试剂含包被在固相载体上的抗人IgG抗体的固相试剂和含包被在固相载体上的抗人IgM抗体的固相试剂；而标记试剂含带标记物的至少一种抗原。
通过采用这样的方式，本发明第二方面的免疫分析仪能够对至少一种抗原所对应的病原体的人IgG和人IgM中的一种或更多种选择性检测。
例如，当选择的待检测的抗体类型为人IgG时，对应的固相试剂为包被抗人IgG抗体的固相载体，对应的标记组分为带标记物的抗原。又例如，当选择的待检测的抗体类型为人IgM时，对应的固相试剂为包被抗人IgM抗体的固相载体，对应的标记组分为带标记物的抗原。再例如，当选择的待检测的抗体类型为人IgG和人IgM时，对应的固相试剂为包被抗人IgG抗体的固相载体和包被抗人IgM抗体的固相载体，对应的标记组分为带标记物的抗原。
在一些实施方式中，所述固相试剂包含包被在固相载体上的多种包被组分，所述多种包被组分以单独分装的形式存在于所述试剂盒中。
在一些实施方式中，所述固相试剂包含包被在固相载体上的多种包被组分，所述多种包被组分以预先混合的形式存在于所述试剂盒中。
在第三方面，本发明提供了一种选择性检测样本中的待测物质的免疫分析方法，包括以下步骤：
设定待测物质的类型；
将待测样本与包含包被在固相载体上的至少一种包被组分的固相试剂混合并孵育一段时间，使得至少一种包被组分能与所述待测样本中的待测物质结合；
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；
根据待测物质的类型，从标记试剂中选择所对应的至少一种标记组分，向经清洗的混合物中加入所述至少一种标记组分并孵育，使得所加入的标记组分能与包被在固相载体上的至少一种包被组分上结合的所述待测物质结合形成复合物，其中，所述标记试剂包含多种带标记物的标记组分；
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检测值。
需要说明的是，在本发明第三方面的方法中，固相试剂与待测样本进行混合并孵育，其中固相试剂含有包被在固相载体上的至少一种包被组分，固相试剂中至少一种包被组分能与待测物质特异性结合。接下来，根据待测物质的类型的需求，从标记试剂中选择标记组分，将标记组分与样本和固相试剂的混合物进行混合并孵育，以选择性地检测一种或更多种标记组分所对应的待测物质的类型。也就是说，通过使用单一的固相试剂及选择标记试剂中的一种或更多种标记组分，本发明实现了便捷、成本较低的选择性免疫分析。
在一些实施方式中，所述固相试剂包含包被在固相载体上的多种包被组分，所述多种包被组分被分别加入到所述待测样本中或以预先混合的方式被加入到所述待测样本中。
本发明第一方面中对于“固相试剂”、“标记试剂”、“标记组分”、“包被组分”的说明和限定同样可以用于本发明的第三方面中。
在第四方面，本发明提供了一种选择性检测样本中的待测物质的免疫分析方法，包括以下步骤：
设定待测物质的类型；
根据待测物质的类型，从固相试剂中选择所对应的至少一种包被组分，将待测样本与所述至少一种包被组分混合并孵育一段时间，使得所述包被组分能与所述待测样本中的所述待测物质结合，其中，所述固相试剂包含被在固相载体上的多种包被组分；
对所述待测样本和所述至少一种包被组分的混合物进行清洗，除去未结合的物质；
向经清洗的混合物中加入包含带有标记物的至少一种标记组分的标记试剂并孵育，使得所加入的标记试剂中的标记组分与所述包被组分上结合的所述待测物质结合，形成复合物；
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检测值。
需要说明的是，在本发明第四方面的方法中，根据待测物质的类型的需求，从固相试剂中选择一种或更多种包被组分与样本与待测样本进行混合并孵育。接下来，将标记 试剂与样本和包被组分的混合物进行混合并孵育，其中标记试剂含有带标记物的至少一种标记组分，该至少一种标记组分能与待测物质特异性结合，以选择性地检测一种或更多种包被组分所对应的待测物质的类型。也就是说，通过选择固相试剂中的一种或多种包被组分及使用单一的标记试剂中，本发明实现了便捷、成本较低的选择性免疫分析。
在一些实施方式中，所述标记试剂包含带标记物的多种标记组分，所述多种标记组分被分别加入到所述经清洗的混合物中或以预先混合的方式被加入到所述经清洗的混合物中。
本发明第二方面中对于“固相试剂”、“标记试剂”、“固相组分”、“包被组分”的说明和限定同样可以用于本发明的第四方面中。
在第五方面，本发明提供一种试剂盒，包括：
捕获混合试剂，含包被在固相载体上的至少一种包被组分、尤其是多种包被组分；
标记试剂，含以单独分装的形式存在于试剂盒中的带标记物的多种标记组分。
本发明的试剂盒可与本发明第一方面的免疫分析仪和/或本发明第三方面的方法配合使用。
在示例性的实施方式中，捕获混合试剂含包被在固相载体上的至少一种抗原，所述抗原选自所述抗原选自HBV抗原(优选为HBc抗原)、HAV抗原和HEV抗原；而标记试剂包括含带标记物的抗人IgG抗体的标记组分和含带标记物的抗人IgM抗体的标记组分。
在另一示例性的实施方式中，捕获混合试剂含包被在固相载体上的至少一种抗原，所述抗原选自所述抗原选自弓形虫抗原、风疹病毒抗原、巨细胞病毒抗原、单纯疱疹病毒1/2型抗原、细小病毒B19抗原、柯萨奇病毒抗原和带状疱疹病毒抗原；而标记试剂包括含带标记物的抗人IgG抗体的标记组分和含带标记物的抗人IgM抗体的标记组分。
同样，本发明第一方面中对于“固相试剂”、“标记试剂”、“标记组分”、“包被组分”的说明和限定同样可以用于本发明的第五方面中。
在第六方面，本发明提供一种试剂盒，包括：
捕获试剂，含以单独分装的形式存在于试剂盒中的包被在固相载体上的多种包被组分；
标记混合试剂，带标记物的至少一种标记组分、尤其是多种标记组分。
本发明的试剂盒可与本发明第二方面的免疫分析仪和/或本发明第四方面的方法配合使用。
同样，本发明第二方面中对于“固相试剂”、“标记试剂”、“固相组分”、“包被组分”的说明和限定同样可以用于本发明的第六方面中。
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。
在本发明实施方式中，术语“包被组分”是指固相载体上所包被的物质，其能与待测样本中的待测物质结合；在包被组分为抗体的情况下，其可称为捕获抗体。
在本发明实施方式中，术语“标记组分”是指标记有标记物的物质，其能与待测样本中的待测物质结合；在标记组分为抗体的情况下，其可称为标记抗体。
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包被组分，并且包被在固相载体上的至少两种包被组分以混合的形式存在于试剂盒中；在固相试剂包含以混合形式存在的、包被在固相载体上的至少两种包被组分的情况下，这样的固相试剂可称为“捕获混合试剂”。术语“标记混合试剂”表示，其包含带有标记物的至少两种标记组分，并且带有标记物的至少两种标记组分以混合的形式存在于试剂盒中；在标记试剂包含以混合形式存在的、带有标记物的至少两种标记组分的情况下，这样的标记试剂可称为标记混合试剂。
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以产生捕获混合试剂：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混合。例如，可以分别将抗HBs抗原的抗体、HCV抗原、抗HIV-1p24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-1p24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。
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。
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原例如可以多聚体、重组抗原、天然抗原、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，抗体可以源于鼠、兔、山羊、绵羊、鸡，但本发明不限于此。
在本发明实施方式中，“抗人免疫球蛋白抗体”、“抗人抗体”和“与人抗体特异性结合的抗体”可以互换使用，是指可以与人IgG，或/和人IgM，或/和人其它抗体亚型(如IgA、IgE等)特异性结合的抗体。
在本发明实施方式中，标记试剂中的标记组分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。
在本发明实施方式中，通过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采用所述固相试剂中的每一种包被在固相载体上的包被组分和所述标记试剂中相应带标记物的标记组分单独对内部参考品进行检测时，所对应的发光阈值最优，优选基本上相同。例如，在确定的反应体系下，采用某一种待测物质对应的包被组分和标记组分单独对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，调节包被组分的浓度，调配发光阈值至预设值；或者，保持包被组分的浓度为一恒定值，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，“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。
如图1所示，本发明实施方式提供了可选择性检测待测样本中的不同待测物质的免 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。在一个实施方式中，试剂盒包括固相试剂和标记试剂，所述固相试剂含包被在固相载体上的至少一种包被组分，而所述标记试剂包括多种带标记物的标记组分。在另一个实施方式中，试剂盒包括固相试剂和标记试剂，所述固相试剂包括多种包被在固相载体上的包被组分，而标记试剂含带有标记物的至少一种标记组分。
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。
在一种情况下，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中存储的试剂盒中固相试剂加入到反应装置30上的反应杯中，以使所述待测样本与所加入的固相试剂在所述反应杯中混合并孵育一段时间，使得固相载体上的至少一种包被组分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试剂中的包被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。
在另一种情况下，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将存储部件13中存储的试剂盒中与所述待测物质的类型对应的至少一种包被组分加入所述反应杯，以使所述待测样本与所述至少一种包被组分在所述反应杯中混合并孵育一段时间，使得固相载体上的包被组分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进一步将试剂盒中标记试剂加入所述反应杯，以使所加入的标记试剂与所述反应杯中的混合物混合并孵育一段时间，使得所加入的标记试剂中的标记组分能与所加入的固相试剂中的包被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，能够在基于一种试剂盒，根据用户需求选择性地检测不同类型的待测物质，提高检测灵活性，满足用户的不同场景需求。
实验材料：
乙肝病毒核心抗原、HAV抗原、自于Meridian Life Science；
抗人抗体，例如抗人IgM抗体、抗人IgG抗体来自于Jackson ImmunoReasearch；
碱性磷酸酶来自于罗氏制药；
磁珠来自于Thermo Fisher。
固相包被物的制备：
首先将抗原进行前处理，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0.5-40μg的抗原的比例(优选1-30μg)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。之后在处理后的磁珠中，按照比例加入抗原，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的磁性微球。选择50mM Tris pH 7.4缓冲液将包被抗原的磁性微球进行稀释，配制得固相试剂。其中，包被抗原的磁性微球浓度为0.7mg/mL。
标记组分的制备：
将抗人IgM抗体、抗人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缓冲液分别将抗人IgM抗体信号标记物、抗人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，将样本和固相试剂添加到反应管中，在37℃孵育10分钟，使得固相表面的抗原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，选择所需的标记组分加到反应管中孵育，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。
将发光信号值除以阈值，得到COI值。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1.1，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至1.10之间，结果为灰区(不确定)。
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 HBV选择性检测的发光阈值确定
选取具有明确临床诊断结果的样本，其中Anti-HBc IgM检测样本350例(阴性样本200例、阳性样本150例)、Anti-HBc IgG抗体检测样本600例(阴性样本370例、阳性样本230例)。
采用由“固相包被物的制备”所得到的包被有HBc抗原的磁性微球作为固相组分和由“标记组分的制备”所得到的带有碱性磷酸酶的抗人IgM抗体、带有碱性磷酸酶的抗人IgG抗体作分别为标记组分，根据“检测步骤”测试不同发光阈值下，HBc抗原的阳性符合率和阴性符合率。结果汇总于表1中。
表1 HBV选择性检测的发光阈值的确定
Figure PCTCN2019130118-appb-000001
由表1可知，对于Anti-HBc IgM检测，发光值在50000左右时，阳性符合率为100％、阴性符合率为99.5％，其总体符合率最高(99.71％)。对于Anti-HBc IgG检测，发光值在70000左右时，阳性符合率为100％、阴性符合率为99.73％，其总符合率最高(99.83％)。
实施例2基于一个试剂盒，选择性检测针对HBc的IgM抗体以及IgG抗体
固相包被物的制备：采用由“固相包被物的制备”所得到的包被有HBc抗原的磁性微球作为固相组分；
标记组分1的制备：按照“标记组分的制备”，使用制备得到带有碱性磷酸酶的抗人IgM抗体，将其与固相组分搭配测定内部参考品。选择发光阈值在50000(允许±10％偏 差)对应的稀释度为标记组分1的稀释度；
标记组分2的制备：按照“标记组分的制备”，使用制备得到带有碱性磷酸酶的抗人IgG抗体，将其与固相组分搭配测定内部参考品。选择发光阈值在70000(允许±10％偏差)对应的稀释度为标记组分2的稀释度。
当需要检测HBc的IgM抗体时，选择固相组分搭配标记组分1按照“检测步骤”测得发光信号值。
当需要检测HBc的IgG抗体时，选择固相组分搭配标记组分2按照“检测步骤”测得发光信号值。
实施例3 HAV选择性检测的发光阈值确定
选取具有明确临床诊断结果的样本，其中Anti-HAV IgM检测样本400例(阴性样本250例、阳性样本150例)、Anti-HAV IgG抗体检测样本500例(阴性样本330例、阳性样本170例)。
采用由“固相包被物的制备”所得到的包被有HAV抗原的磁性微球作为固相组分和由“标记组分的制备”所得到的带有碱性磷酸酶的抗人IgM抗体、带有碱性磷酸酶的抗人IgG抗体作为标记试剂，根据“检测步骤”测试不同发光阈值下，HAV抗原的阳性符合率和阴性符合率。结果汇总于表2中。
表2 HAV选择性检测的发光阈值的确定
Figure PCTCN2019130118-appb-000002
由表2可知，对于Anti-HAV IgM检测，发光值在40000左右时，阳性符合率为100％、阴性符合率为99.60％，其总体符合率最高(99.75％)。对于Anti-HAV IgG检测，发光值在70000左右时，阳性符合率为100％、阴性符合率为99.70％，其总符合率最高(99.80％)。
实施例4基于一个试剂盒，选择性检测针对HAV的IgM抗体以及IgG抗体
固相包被物的制备：采用由“固相包被物的制备”所得到的包被有HAV抗原的磁性微球作为固相组分；
标记组分1的制备：按照“标记组分的制备”，使用制备得到带有碱性磷酸酶的抗人IgM抗体，将其与固相组分搭配测定内部参考品。选择发光阈值在40000(允许±10％偏差)对应的稀释度为标记组分1的稀释度；
标记组分2的制备：按照“标记组分的制备”，使用制备得到带有碱性磷酸酶的抗人IgG抗体，将其与固相组分搭配测定内部参考品。选择发光阈值在70000(允许±10％偏差)对应的稀释度为标记组分2的稀释度。
当需要检测HAV的IgM抗体时，选择固相组分和标记组分1搭配按照“检测步骤”测得发光信号值。
当需要检测HAV的IgG抗体时，选择固相组分和标记组分2搭配按照“检测步骤”测得发光信号值。
将发光信号值除以阈值，得到COI值。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1.1，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至1.10之间，结果为灰区(不确定)。

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  1. 一种可选择性检测待测样本中的不同待测物质的免疫分析仪，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至少一种包被组分，所述标记试剂包含多种带标记物的标记组分，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组分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固相试剂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述固相试剂在所述反应杯中混合并孵育一段时间，使得包被在固相载体上的至少一种包被组分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至少一种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包被在固相载体上的至少一种包被组分上结合的所述待测物质结合，形成包被组分-待测物质-标记组分的复合物；
    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。
  2. 权利要求1所述的免疫分析仪，其中，所述至少一种包被组分为至少一种抗原，所述多种标记试剂包括带标记物的抗人IgG抗体和带标记物的抗人IgM抗体，从而能够检测所述待测样本中是否存在与所述抗原对应的人IgG和人IgM中的一种或更多种。
  3. 权利要求2所述的免疫分析仪，其中，所述抗原选自HBV抗原、优选为HBc抗原、HAV抗原、HEV抗原、弓形虫抗原、风疹病毒抗原、巨细胞病毒抗原、单纯疱疹病毒1/2型抗原、细小病毒B19抗原、柯萨奇病毒抗原和带状疱疹病毒抗原。
  4. 权利要求1所述的免疫分析仪，其中，所述至少一种包被组分包括不同生物标志物对应的多种捕获抗体，所述标记试剂包括与所述不同生物标志物对应的多种标记抗体，从而能够检测所述待测样本中是否存在所述不同生物标志物中的一种或更多种。
  5. 权利要求4所述的免疫分析仪，其中，所述至少一种包被组分包括PCT捕获抗体和Presepsin捕获抗体，所述标记试剂包括PCT标记抗体和Presepsin标记抗体，从而能够检测所述待测样本中是否存在生物标志物PCT和Presepsin中的一种或更多种。
  6. 权利要求1至5中任一项所述的免疫分析仪，其中，所述固相试剂包含包被在固相载体上的多种包被组分。
  7. 权利要求6所述的免疫分析仪，其中，所述多种包被组分包括抗HBs抗原的抗体、HCV抗原、抗HIV-1 p24抗体和HIV-1/-2联合抗原，所述标记试剂包括带标记物的抗HBs抗原的抗体、带标记物的抗人抗体和带标记物的抗HIV-1 p24抗体，从而能够检测所述待测样本中是否存在HBs抗原、抗HCV抗体、HIV-1 p24抗原和HIV-1/-2抗体中的一种或更多种。
  8. 权利要求6所述的免疫分析仪，其中，所述多种包被组分包括抗HBs抗原的抗体、HCV抗原、抗HIV-1 p24抗体、HIV-1/-2联合抗原、TP抗原和HBc抗原，所述标记试剂包括带标记物的抗HBs抗原的抗体、带标记物的抗人抗体和带标记物的抗HIV-1p24抗体，从而能够检测所述待测样本中是否存在HBs抗原、抗HCV抗体、HIV-1 p24抗原、HIV-1/-2抗体、抗TP抗体和抗HBc抗体中的一种或更多种。
  9. 权利要求1至8中任一项所述的免疫分析仪，其中，所述固相试剂包含包被在固相载体上的多种包被组分，所述多种包被组分以单独分装的形式或者以预先混合的形式存在于所述试剂盒中。
  10. 一种可选择性检测待测样本中的不同待测物质的免疫分析仪，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，所述固相试剂包含多种包被在固相载体上的包被组分，所述标记试剂包含带标记物的至少一种标记组分，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包被组分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至少一种包被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包被组分在所述反应杯中混合并孵育一段时间，使得所加入包被组分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标记试剂加入所述反应杯，以使所述标记试剂与所述反应杯中的混合物混合并孵育一段时间，使得所述标记试剂中的至少一种标记组分能与所加入的包被组分上结合的所述待测物质结合，形成包被组分-待测物质-标记组分的复合物；
    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。
  11. 权利要求10所述的免疫分析仪，其中，所述包被组分包括抗人IgG抗体和抗人IgM抗体，所述至少一种标记组分为抗原，从而能够检测所述待测样本中是否存在与所述抗原对应的人IgG和人IgM中的一种或更多种。
  12. 权利要求10或11所述的免疫分析仪，其中，所述标记试剂包含带标记物的多 种标记组分，所述多种标记组分以单独分装的形式或者以预先混合的形式存在于所述试剂盒中。
  13. 一种选择性检测样本中的待测物质的免疫分析方法，包括以下步骤：
    设定待测物质的类型；
    将待测样本与包含包被在固相载体上的至少一种包被组分的固相试剂混合并孵育一段时间，使得至少一种包被组分能与所述待测样本中的待测物质结合；
    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；
    根据待测物质的类型，从标记试剂中选择所对应的至少一种标记组分，向经清洗的混合物中加入所述至少一种标记组分并孵育，使得所加入的标记组分能与包被在固相载体上的至少一种包被组分上结合的所述待测物质结合形成复合物，其中，所述标记试剂包含多种带标记物的标记组分；
    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检测值。
  14. 权利要求13所述的方法，其中，所述至少一种包被组分为抗原，所述标记组分包括抗人IgG抗体和抗人IgM抗体，从而检测所述待测物质中是否存在与所述抗原对应的人IgG和人IgM中的一种或更多种。
  15. 权利要求13所述的方法，其中，所述至少一种包被组分包括不同生物标志物对应的抗体，所述标记组分包括与所述不同生物标志物对应的抗体，从而能够检测所述待测样本中是否存在所述不同生物标志物中的一种或更多种。
  16. 权利要求13至15中任一项所述的方法，其中，所述固相试剂包含包被在固相载体上的多种包被组分，所述多种包被组分被分别加入到所述待测样本中或以预先混合的方式被加入到所述待测样本中。
  17. 一种选择性检测样本中的待测物质的免疫分析方法，包括以下步骤：
    设定待测物质的类型；
    根据待测物质的类型，从固相试剂中选择所对应的至少一种包被组分，将待测样本与所述至少一种包被组分混合并孵育一段时间，使得所述包被组分能与所述待测样本中的所述待测物质结合，其中，所述固相试剂包含包被在固相载体上的多种包被组分；
    对所述待测样本和所述至少一种包被组分的混合物进行清洗，除去未结合的物质；
    向经清洗的混合物中加入包含带有标记物的至少一种标记组分的标记试剂并孵育， 使得所加入的标记试剂中的标记组分能与所述包被组分上结合的所述待测物质结合，形成复合物；
    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检测值。
  18. 权利要求17所述的方法，其中，所述包被组分包括抗人IgG抗体和抗人IgM抗体，所述至少一种标记组分为抗原，从而能够检测所述待测样本中是否存在与所述抗原对应的人IgG和IgM中的一种或更多种。
  19. 权利要求17或18所述的方法，其中，所述标记试剂包含带标记物的多种标记组分，所述多种标记组分被分别加入到所述经清洗的混合物中或以预先混合的方式被加入到所述经清洗的混合物中。
  20. 一种试剂盒，包括：
    捕获混合试剂，含包被在固相载体上的多种包被组分；
    标记试剂，含以单独分装的形式存在于试剂盒中的带标记物的多种标记组分。
  21. 权利要求20所述的试剂盒，其中，所述多种包被组分选自HBV抗原、HAV抗原、HEV抗原、弓形虫抗原、风疹病毒抗原、巨细胞病毒抗原、单纯疱疹病毒1/2型抗原、细小病毒B19抗原、柯萨奇病毒抗原和带状疱疹病毒抗原，所述标记组分包括抗人IgG抗体和抗人IgM抗体。
  22. 权利要求20所述的试剂盒，其中，所述多种包被组分包括不同的HBV抗原片段，所述标记组分包括抗人IgG抗体和抗人IgM抗体。
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