CN114599976A

CN114599976A - Immunoanalyzer, method and kit for selective detection of different substances to be tested

Info

Publication number: CN114599976A
Application number: CN201980101485.4A
Authority: CN
Inventors: 于丽娜; 李可; 何建文
Original assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Current assignee: Shenzhen Mindray Bio Medical Electronics Co Ltd
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2022-06-07
Also published as: WO2021134299A1

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。An immunoanalyzer that can selectively detect different substances to be tested in a sample to be tested. The immunoanalyzer is equipped with a kit to achieve selective detection of different types of substances to be tested according to user needs. It also relates to immunoassay methods and kits.

Description

Immunoassay instrument, method and kit for selectively detecting different substances to be detected

Technical Field

The invention relates to the field of immunoassay, in particular to selective detection of the types of different substances to be detected.

Background

In recent years, an immunoassay method for detecting a substance to be measured contained in a sample has been developed. The principle of such a method is generally: the capture object is firstly combined with the substance to be detected, then the marker with the marker is combined with the substance to be detected to form a capture object-substance to be detected-marker compound, and then the optical detection device is used to measure the intensity of an optical signal through the sensor, so that whether the substance to be detected exists can be judged. Meanwhile, an immunoassay kit for implementing the above immunoassay method has been developed.

Typically, the kit will perform the detection of the substance to be detected on an immunoassay analyzer. In this process, the detection of the sample to be detected by the immunoassay analyzer is limited to the detection items (i.e., specific detection substance types) involved in the kit. Thus, the user cannot select the appropriate test items according to the test requirement, which brings many limitations in clinical application.

Accordingly, there is a strong demand for flexibly selecting a test item according to a user's demand when an immunoassay analyzer is used for immunoassay.

Disclosure of Invention

In order to solve the problem that the current immunoassay analyzer cannot selectively analyze different projects based on the same kit, the inventor researches the working mode of the immunoassay analyzer and a matched kit, thereby realizing the invention.

In a first aspect, the present invention provides an immunoassay analyzer for selectively detecting different test substances in a test sample, comprising:

the sample device is provided with a sample storage component and a sample separate injection component, the sample storage component is used for storing a sample to be tested, and the sample separate injection component is used for sucking the sample to be tested and discharging the sample to be tested into a reaction cup to be added with sample;

a reagent apparatus having a reagent storage part for storing a reagent cartridge including a solid phase reagent containing at least one coating component coated on a solid phase carrier and a labeled reagent containing a plurality of labeled components, and a reagent dispensing part for aspirating the solid phase reagent and the labeled component in the reagent cartridge stored on the reagent storage part and discharging them into a reaction cup to which the reagent is to be added;

a luminescent substrate dispensing device which is connected with a container for storing luminescent substrates and is used for injecting the luminescent substrates into a reaction cup to which the luminescent substrates are added;

a reaction device having a plurality of placement positions for placing the reaction cups and for incubating the reaction solution in the reaction cups;

the light measurement component is used for performing light measurement on the incubated reaction liquid to obtain a detection result of the sample to be detected;

a control device electrically connected to the sample device, the reagent device, the luminescent substrate dispensing device, and the photometric element, and configured to:

receiving a test instruction, wherein the test instruction comprises the type of a substance to be tested;

in response to the test instruction:

controlling the sample dispensing component to add the sample to be detected in the sample storage component into a reaction cup on the reaction device;

controlling the reagent dispensing component to add the solid-phase reagent into a reaction cup on the reaction device, so that the sample to be tested and the solid-phase reagent are mixed in the reaction cup and incubated for a period of time, and at least one coating component coated on a solid-phase carrier can be combined with the substance to be tested in the sample to be tested;

controlling the reagent dispensing component to further add at least one labeling component corresponding to the type of the substance to be detected into the reaction cup, so that the added labeling component is mixed with the mixture in the reaction cup and incubated for a period of time, and the added labeling component can be combined with the substance to be detected combined on at least one coating component coated on a solid phase carrier to form a coating component-substance to be detected-labeling component compound;

controlling the light-emitting substrate dispensing device to add a light-emitting substrate into the reaction cup; and

and obtaining a detection result according to the ratio of the luminous value measured in the optical measurement component to the luminous threshold value.

It should be noted that the immunoassay analyzer according to the first aspect of the present invention can select reagent components corresponding to different types of substances to be detected for detection in response to an instruction of the types of the substances to be detected on the basis of one kit. During the detection process, the reagent dispensing component is controlled to add a solid phase reagent containing at least one coating component coated on a solid phase carrier into the reaction cup, wherein at least one coating component can be specifically combined with the substance to be detected. Next, the immunoassay analyzer further controls a reagent dispensing member in response to the instruction to selectively add at least one labeled component corresponding to the type of the substance to be measured into the reaction cuvette. Through the formed complex of the coating component, the substance to be detected and the labeling component, the detection items can be selected according to the requirements of users. Therefore, the immunoassay analyzer capable of conveniently and flexibly realizing selective detection is provided.

In some embodiments, the solid phase reagent comprises at least one antigen coated on a solid support; and the labeling reagent comprises a labeling component containing a labeled anti-human IgG antibody and a labeling component containing a labeled anti-human IgM antibody.

In this manner, the immunoassay device of the first aspect of the present invention is capable of selectively detecting one or more of IgG and IgM anti-human antibodies of a pathogen to which at least one antigen corresponds.

Such selectivity is highly desirable. The human body mainly comprises 5 immune globulin of IgG, IgM, IgA, IgD and IgE, wherein the IgG is the main immune globulin in serum, the content of the IgG accounts for about 65-75% of the immune globulin, and the IgG is an important substance for resisting infection of the body. The IgG antibody is produced late, maintained for a long time, disappears slowly, has high concentration, and can be detected in blood as a long-term infection index. IgM is a pentamer, the largest molecule in immunoglobulins. IgM production is the earliest and occurs rapidly upon infection. However, IgM has a short duration and a rapid disappearance, and acts as an anti-infective agent at the initial stage of infection, and thus is detected in blood as an indicator of recent infection. IgA, IgD and IgE are present in relatively small amounts in the serum. In clinical application, different subtypes of antibodies are often required to be detected, and clinical judgment is performed according to the detection result. Such as hepatitis B virus core antibody (Anti-HBc) and hepatitis A antibody (Anti-HAV), Anti-human antibodies are mainly detected clinically. However, in some application scenarios, the antibody subtypes can be distinguished, and the antibody subtypes can be used for evaluating the morbidity state of a patient and determining the patient to be infected before or acutely. In this case, it is desirable to selectively detect different subtypes of antibodies.

For example, when the type of antibody to be detected is selected to be human IgG, 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 IgG antibody. For another example, when the type of antibody to be detected is selected to be 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. For another example, when the selected antibody types to be detected are human IgG and human IgM, the corresponding solid-phase reagent is a solid-phase carrier coated with at least one antigen, and the corresponding labeling components are a labeled anti-human IgG antibody and a labeled anti-human IgM antibody.

In an exemplary embodiment, the solid phase reagent comprises at least one antigen selected from the group consisting of an HBV antigen (preferably HBc antigen), an HAV antigen and an HEV antigen; and the labeling reagent comprises a labeling component containing labeled anti-human IgG antibody and a labeling component containing labeled anti-human IgM antibody.

In another exemplary embodiment, the solid phase reagent comprises at least one antigen selected from the group consisting of a toxoplasma antigen, a rubella virus antigen, a cytomegalovirus antigen, a herpes simplex virus 1/2 type antigen, a parvovirus B19 antigen, a coxsackie virus antigen, and a herpes zoster virus antigen coated on a solid support; and the labeling reagent comprises a labeling component containing a labeled anti-human IgG antibody and a labeling component containing a labeled anti-human IgM antibody.

In other embodiments, the solid phase reagent comprises a plurality of capture antibodies corresponding to different biomarkers coated on a solid phase support; and the labeling reagent comprises a plurality of labeled antibodies corresponding to the different biomarkers.

In this way, the immunoassay of the first aspect of the invention is capable of selectively detecting one or more of the different biomarkers.

In an exemplary embodiment, the solid phase reagent comprises a Procalcitonin (PCT) capture antibody and a Presepsin capture antibody coated on a solid phase support; and the labeling reagent comprises a PCT labeled antibody containing a label and a Presepsin labeled antibody containing a label.

In this way, the immunoassay analyzer of the first aspect of the present invention can selectively detect PCT, Presepsin or PCT + Presepsin according to the needs of the user.

In still other embodiments, the solid phase reagent comprises a plurality of coating components coated on a solid support.

In an exemplary embodiment, the solid support comprises an anti-HBs antigen antibody, an HCV antigen, an anti-HIV-1 p24 antibody, and an HIV-1/-2 combination antigen coated on the solid support; and the labeling reagent comprises a labeling component containing a labeled anti-HBs antigen antibody, a labeling component containing a labeled anti-human antibody and a labeling component containing a labeled anti-HIV-1 p24 antibody.

In this manner, the immunoassay of the first aspect of the present invention can selectively screen a sample (e.g., a blood sample) for one or more of HBs antigen, anti-HCV antibody, HIV-1p24 antigen, and HIV-1/-2 antibody, thereby determining whether an individual from which the sample is derived has an infection with one or more of HBV, HCV, and HIV.

In another exemplary embodiment, the solid support comprises an anti-HBs antigen antibody, an HCV antigen, an anti-HIV-1 p24 antibody, an HIV-1/-2 combination antigen, a TP antigen, and an HBc antigen coated on the solid support; and the labeling reagent comprises a labeling component containing a labeled anti-HBs antigen antibody, a labeling component containing a labeled anti-human antibody and a labeling component containing a labeled anti-HIV-1 p24 antibody.

In this manner, the immunoassay of the first aspect of the present invention can selectively screen a test sample (e.g., a blood sample) for one or more of HBs antigen, anti-HCV antibody, HIV-1p24 antigen, HIV-1/-2 antibody, anti TP antibody, and anti-HBc antibody, thereby determining whether the individual from which the sample is derived has one or more of HBV, HCV, HIV, and TP infections.

In some embodiments, the solid phase reagent comprises a plurality of coating components coated on a solid support, the plurality of coating components being present in the kit in separate aliquots.

In some embodiments, the solid phase reagent comprises a plurality of coating components coated on a solid support, the plurality of coating components being present in the kit in a pre-mixed form.

In a second aspect, the present invention provides an immunoassay instrument for selectively detecting different test substances in a test sample, comprising:

a reagent apparatus having a reagent storage part for storing a reagent cartridge including a solid phase reagent containing a plurality of coating components coated on a solid phase carrier and a labeled reagent containing at least one labeled component with a label, and a reagent dispensing part for aspirating the coating components and the labeled reagent in the reagent cartridge stored on the reagent storage part and discharging into a reaction cup to which the reagent is to be added;

a reaction device having a plurality of placement positions for placing the reaction cuvette and for incubating a reaction solution in the reaction cuvette;

in response to the test instruction:

controlling the reagent dispensing component to add at least one coating component corresponding to the type of the substance to be detected into a reaction cup on the reaction device, so that the sample to be detected and the coating in the added coating component are mixed in the reaction cup and incubated for a period of time, and the added coating component can be combined with the substance to be detected in the sample to be detected;

controlling the reagent dispensing component to further add the labeled reagent into the reaction cup, so that the labeled reagent is mixed with the mixture in the reaction cup and incubated for a period of time, so that at least one labeled component in the labeled reagent can be combined with the substance to be detected combined with the added coating component to form a coating component-substance to be detected-labeled component complex;

controlling the luminescent substrate dispensing device to add a luminescent substrate into the reaction cup; and

It should be noted that the immunoassay analyzer according to the second aspect of the present invention selects reagent components corresponding to different types of substances to be tested for detection in accordance with instructions from one kit in response to the types of substances to be tested. In the detection process, the immunoassay analyzer responds to the instruction to control the reagent dispensing component to selectively add at least one coating component corresponding to the type of the substance to be detected into the reaction cup. Next, the reagent dispensing control unit adds a labeled reagent containing at least one labeled component with a label to the reaction cuvette, wherein the at least one labeled component is capable of specifically binding to the substance to be measured. The formed complex of the coating component, the substance to be detected and the labeling component realizes the detection according to the requirements of users. Therefore, the immunoassay analyzer capable of conveniently and flexibly realizing selective detection is provided.

In some embodiments, the solid phase reagent comprises a solid phase reagent comprising anti-human IgG antibodies coated on a solid phase support and a solid phase reagent comprising anti-human IgM antibodies coated on a solid phase support; and the labeling reagent contains at least one antigen with a label.

In this manner, the immunoassay of the second aspect of the present invention can selectively detect one or more of human IgG and human IgM of a pathogen to which at least one antigen corresponds.

For example, when the antibody type to be detected is selected to be 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 a labeled antigen. For another example, when the type of antibody to be detected is selected to be human IgM, the corresponding solid-phase reagent is a solid-phase carrier coated with anti-human IgM antibody, and the corresponding labeling component is a labeled antigen. For another example, when the selected antibody types to be detected are human IgG and human IgM, the corresponding solid-phase reagents are a solid-phase carrier coated with anti-human IgG antibody and a solid-phase carrier coated with anti-human IgM antibody, and the corresponding labeling component is a labeled antigen.

In a third aspect, the present invention provides an immunoassay method for selectively detecting a substance to be detected in a sample, comprising the steps of:

setting the type of a substance to be detected;

mixing a sample to be tested with a solid phase reagent comprising at least one coating component coated on a solid phase carrier and incubating for a period of time such that the at least one coating component is capable of binding to a substance to be tested in the sample to be tested;

washing the mixture of the sample to be tested and the solid phase reagent to remove unbound substances;

selecting at least one corresponding labeling component from a labeling reagent according to the type of a substance to be detected, adding the at least one labeling component to the washed mixture, and incubating so that the added labeling component can be combined with the substance to be detected combined on at least one coating component coated on a solid phase carrier to form a complex, wherein the labeling reagent comprises a plurality of labeling components with labels;

washing the complex to remove unbound material;

and adding a luminescent substrate into the washed complex to detect the detection value of the substance to be detected in the sample to be detected.

It should be noted that, in the method of the third aspect of the present invention, a solid phase reagent is mixed with a sample to be tested and incubated, wherein the solid phase reagent contains at least one coating component coated on a solid phase carrier, and at least one coating component in the solid phase reagent is capable of specifically binding to a substance to be tested. Next, according to the requirement of the type of the substance to be detected, a labeled component is selected from the labeled reagent, and the labeled component is mixed with the mixture of the sample and the solid phase reagent and incubated to selectively detect the type of the substance to be detected corresponding to one or more labeled components. That is, the present invention enables convenient, low cost selective immunoassays by using a single solid phase reagent and one or more marker components of a selective labeling reagent.

In some embodiments, the solid phase reagent comprises a plurality of coating components coated on a solid support, the plurality of coating components being added to the sample to be tested separately or in a pre-mixed manner.

The descriptions and definitions of "solid phase reagent", "labeling component", "coating component" in the first aspect of the present invention can be applied to the third aspect of the present invention as well.

In a fourth aspect, the present invention provides an immunoassay method for selectively detecting a substance to be detected in a sample, comprising the steps of:

setting the type of a substance to be detected;

selecting at least one corresponding coating component from a solid phase reagent according to the type of a substance to be detected, mixing a sample to be detected with the at least one coating component, and incubating for a period of time so that the coating component can be combined with the substance to be detected in the sample to be detected, wherein the solid phase reagent comprises a plurality of coating components coated on a solid phase carrier;

washing the mixture of the sample to be tested and the at least one coating component to remove unbound material;

adding a labeling reagent containing at least one labeling component with a label to the washed mixture and incubating, so that the labeling component in the added labeling reagent is combined with the substance to be tested combined on the coating component to form a complex;

washing the complex to remove unbound material;

It should be noted that, in the method of the fourth aspect of the present invention, one or more coating components selected from the solid phase reagent are mixed with the sample to be tested and incubated according to the requirement of the type of the substance to be tested. Next, a labeling reagent is mixed with the mixture of the sample and the coating components and incubated, wherein the labeling reagent contains at least one labeled component with a label, and the at least one labeled component can specifically bind to the substance to be detected, so as to selectively detect the type of the substance to be detected corresponding to the one or more coating components. That is, by selecting one or more coating components in the solid phase reagent and using a single labeled reagent, the present invention enables convenient, low cost selective immunoassay.

In some embodiments, the labeling reagent comprises a plurality of labeled components that are added separately to the washed mixture or added to the washed mixture in a pre-mixed manner.

The descriptions and definitions of "solid-phase reagent", "labeling reagent", "solid-phase component", "coating component" in the second aspect of the present invention can be applied to the fourth aspect of the present invention as well.

In a fifth aspect, the present invention provides a kit comprising:

a capture cocktail reagent comprising at least one coating component, in particular a plurality of coating components, coated on a solid support;

a labeling reagent comprising a plurality of labeled components in the kit in separate aliquots.

The kit of the invention may be used in conjunction with the immunoassay analyzer of the first aspect of the invention and/or the method of the third aspect of the invention.

In an exemplary embodiment, the capture cocktail of reagents comprises at least one antigen selected from the group consisting of an HBV antigen (preferably HBc antigen), an HAV antigen, and an HEV antigen coated on a solid support; and the labeling reagent comprises a labeling component containing a labeled anti-human IgG antibody and a labeling component containing a labeled anti-human IgM antibody.

In another exemplary embodiment, the capture cocktail of reagents comprises at least one antigen selected from the group consisting of a toxoplasma antigen, a rubella virus antigen, a cytomegalovirus antigen, a herpes simplex virus 1/2 type antigen, a parvovirus B19 antigen, a coxsackie virus antigen, and a herpes zoster virus antigen coated on a solid support; and the labeling reagent comprises a labeling component containing a labeled anti-human IgG antibody and a labeling component containing a labeled anti-human IgM antibody.

Also, the descriptions and definitions of "solid phase reagent", "labeling component", "coating component" in the first aspect of the present invention can be applied to the fifth aspect of the present invention as well.

In a sixth aspect, the present invention provides a kit comprising:

a capture reagent comprising a plurality of coating components coated on a solid support in a kit in a separate package;

the labeling cocktail reagent, at least one labeling component, especially a plurality of labeling components, with a label.

The kit of the invention may be used in conjunction with the immunoassay analyzer of the second aspect of the invention and/or the method of the fourth aspect of the invention.

Also, the descriptions and definitions of "solid-phase reagent", "labeling reagent", "solid-phase component", "coating component" in the second aspect of the present invention can be applied to the sixth aspect of the present invention as well.

Drawings

FIG. 1 shows a schematic diagram of an immunoassay system according to an embodiment of the present invention;

fig. 2 shows a schematic configuration diagram of a control device according to an embodiment of the present invention.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. If there is a conflict, the present specification will control.

In the present embodiment, the term "coating component" refers to a substance coated on a solid support, which is capable of binding to a substance to be tested in a sample to be tested; where the coating component is an antibody, it may be referred to as a capture antibody.

In the present embodiment, the term "labeling component" refers to a substance labeled with a label, which is capable of binding to a substance to be measured in a sample to be measured; in the case where the labeling component is an antibody, it may be referred to as a labeled antibody.

In embodiments of the invention, the terms "solid phase support", "solid support" and "solid support" are used interchangeably and refer to a solid surface to which an antigen or antibody can be attached. The solid phase carrier to be used in the present invention is not particularly limited, and commercially available solid phase carriers and any solid phase carrier usable for immunoassay can be used in the present invention. Exemplary solid supports may be magnetic beads (e.g., carboxyl magnetic beads), microplate, plastic plate, plastic tube, latex beads, agarose beads, glass, nitrocellulose membrane, nylon membrane, silica plate, or microchip, but the present invention is not limited thereto.

In the present embodiment, the term "capture mixing reagent" means that it comprises at least two coating components coated on a solid support, and the at least two coating components coated on the solid support are present in the kit in a mixed form; where the solid phase reagent comprises at least two coating components coated on a solid support in a mixed form, such a solid phase reagent may be referred to as a "capture cocktail reagent". The term "label mixing reagent" means that it comprises at least two label components carrying a label, and that the at least two label components carrying the label are present in the kit in a mixed form; in the case where the labeling reagent contains at least two labeling components carrying a label in a mixed form, such labeling reagent may be referred to as a labeling mixed reagent.

In embodiments of the invention, the coating on the solid support to produce the capture cocktail can be performed by: in one aspect, each substance to be coated can be coated separately on a different solid support, and the coated solid supports can then be mixed. For example, an antibody against HBs antigen, HCV antigen, anti HIV-1p24 antibody, and HIV-1/-2 combined antigen can be coated on solid carriers, respectively, and then mixed together. On the other hand, it is also possible to divide the substances to be coated into one or more groups each containing one or more substances to be coated, coat each group with a different solid carrier, and then mix the coats, for example, an antibody to HBs antigen, HCV antigen, anti-HIV-1 p24 antibody, and HIV-1/-2 combined antigen may be made into one group, and the group may be coated with the same solid carrier.

Labels useful in embodiments of the invention are well known to those skilled in the art, and include, for example, enzymes such as alkaline phosphatase (ALP), oxidase, microperoxidase, horseradish peroxidase, beta-galactosidase, glucose oxidase, and glucose 6-phosphate dehydrogenase; fluorescent substances such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, fluorescein, rhodamine, europium and green fluorescent protein; chemiluminescent substances such as luminol, isoluminol, phenanthridinium, and acridinium esters; coenzyme such as NAD; biotin;³⁵S、 ¹⁴C、 ³²P、 ¹³¹i and¹²⁵i, etc., but the present invention is not limited thereto.

One skilled in the art will be able to select an appropriate luminescent substrate, depending on the type of label used, to generate a detectable signal. For example, when alkaline phosphatase is used as the label, 3- (2-spiroadamantane) -4-methoxy-4- (3-phosphoryloxy) -phenyl-1, 2-dioxetane is used as a luminescent substrate, which is decomposed by alkaline phosphatase to remove a phosphate group, thereby producing an unstable intermediate product, which produces a methyl-metaborate anion through intramolecular electron transfer, and produces chemiluminescence when the methyl-metaborate anion in an excited state returns from the excited state to the ground state. And measuring the number of photons generated in the reaction by a photomultiplier, wherein the amount of the generated photons is in direct proportion to the content of the detection object in the sample.

The embodiment of the invention is suitable for ELISA, chemiluminescence, electrochemiluminescence, POCT, immunochromatography, up-conversion luminescence, down-conversion luminescence and other methods.

In the scope of the present invention, the term roc (receiver operating characteristic) curve refers to a curve obtained by dividing the diagnostic test result into several critical points, plotting the sensitivity corresponding to each critical point as ordinate and the specificity as abscissa. The ROC curve is an effective tool for comprehensively and accurately evaluating a diagnostic test. Another function of the ROC curve is to determine the optimal threshold for detection. The ROC curve method determines that the critical point is the best by selecting a point on the curve as close to the upper left as possible under most conditions of the critical point. In application, according to the ROC curve, the sensitivity and specificity result of each tangent point are combined, and the tangent point which is as close to the upper left Youden index (Youden index) on the curve as possible is selected as the optimal critical point, so that the sensitivity and specificity of the test are high, and the misdiagnosis rate and the missed diagnosis rate are low.

When used to prepare solid phase reagents and/or labeled reagents, the antigens of embodiments of the invention may be present, for example, in the form of multimers, recombinant antigens, natural antigens, antigen fragments, or antigenic peptides.

When used to prepare solid phase reagents and/or labelled reagents. The antibody according to the embodiment of the present invention may be present in the form of, for example, a monoclonal antibody, a polyclonal antibody, a recombinant antibody, a chimeric antibody, a humanized antibody, or an antigen-binding fragment of an antibody.

In the present embodiment, the antibody may be derived from mouse, rabbit, goat, sheep, chicken, but the present invention is not limited thereto.

In the present embodiments, "anti-human immunoglobulin antibody", "anti-human antibody" and "antibody that specifically binds to a human antibody" are used interchangeably and refer to an antibody that specifically binds to human IgG, or/and human IgM, or/and other antibody subtypes of human (e.g., IgA, IgE, etc.).

In embodiments of the invention, the labeling component of the labeling reagent may be attached to the label by methods that are conventional in the art (e.g., chemical bonding).

In the embodiment of the present invention, the result is determined to be positive or negative by the ratio of the detection value to the luminescence threshold value (COI value). For example, when the ratio is 1.1 or more, the result is judged to be positive, indicating that the detection result of at least one biomarker is positive. When the ratio is between 0.9 and 1.1, judging that the result is a gray area and determining that the gray area is not positive or negative; and when the ratio is less than 0.9, judging that the result is negative, and indicating that the detection results of the specific biomarkers are negative.

In an embodiment of the present invention, the concentrations of the components in the solid phase reagent and the labeled reagent are designed such that the corresponding luminescence thresholds are optimal, preferably substantially the same, when the internal reference is detected separately in the same reaction system using the coating component coated on the solid phase carrier in each of the solid phase reagents and the corresponding labeled component with the label in the labeled reagent. For example, under a determined reaction system, an internal reference product defined in ISO 18113-1:2009 is separately detected by adopting a coating component and a marking component corresponding to a certain substance to be detected, the concentration of the marking component is kept at a constant value, the concentration of the coating component is adjusted, and a light-emitting threshold value is adjusted to a preset value; or, keeping the concentration of the coating component at a constant value, adjusting the concentration of the marking component, and adjusting the luminescence threshold value to a preset value.

In an embodiment of the invention, an "internal reference" refers to the standard and basis for the determination of the composition of the system, which has the definition as in international standard ISO 18113-1:2009 and is available according to this standard. The internal reference is a sample used for verifying the product performance by a medical instrument producer, and is the most important determination standard and basis for selecting, preparing, identifying and determining raw material quality standards, determining a product production process, and reacting system compositions, reaction conditions and the like. For qualitative items, an identified sample of an internal business reference is a sample of a numerical or quantitative value that determines the presence or absence of a particular disease, condition, or boundary between measured presence and absence.

In embodiments of the invention, "substantially the same" means that the relative deviation is within ± 10%, such as within ± 5%, within ± 2%, or within ± 1%.

As shown in fig. 1, the embodiment of the present invention provides an immunoassay analyzer capable of selectively detecting different substances to be detected in a sample to be detected. The immunoassay analyzer comprises a sample device 10, a reagent device 20, a reaction device 30, a photometric component 40 and a control device 50. The immunoassay analyzer may further include a display unit (not shown).

The sample device 10 is used for carrying a sample to be tested, and the sample is provided to the reaction device 30 after being sucked. The sample apparatus 10 includes a sample storage unit 11 and a sample dispensing unit 12. The sample storage unit 11 is used for storing a sample to be measured. In some embodiments, the Sample storage unit 11 may include a Sample Delivery Module (SDM) and a front track. In other embodiments, the sample storage unit 11 may also be a sample tray, the sample tray includes a plurality of sample sites for placing samples such as sample tubes, and the sample tray can dispatch the samples to corresponding positions by rotating the tray structure, for example, the position for the sample dispensing unit 12 to suck the samples. The sample dispensing member 12 is used to suck a sample and discharge the sample into a reaction cup to be loaded. The sample dispensing member 12 may include, for example, a sample needle that performs a two-dimensional or three-dimensional motion in space by a two-dimensional or three-dimensional driving mechanism, so that the sample needle can be moved to aspirate a sample carried by the sample storage member 11 and to a cuvette to be loaded and discharge the sample to the cuvette.

The reagent device 20 is used for carrying a reagent, and sucking the reagent and supplying the reagent to the reaction device 30. The reagent apparatus 20 includes a reagent storage unit 13 and a reagent dispensing unit 14. The reagent storage part 13 is used for storing the reagent cartridge. In some embodiments, the reagent storage component 13 may be a reagent disk, which is configured in a disk-shaped structure and has a plurality of positions for holding reagent containers, and the reagent storage component 13 can rotate and drive the reagent containers held by the reagent storage component to rotate to a specific position, for example, a position for sucking reagent by the reagent dispensing component 14. The number of the reagent storage part 13 may be one or more. The reagent dispensing unit 14 is used for sucking up a reagent in a reagent cartridge and discharging the reagent into a reaction cup to which the reagent is to be added. In some embodiments, the reagent dispensing unit 14 may include a reagent needle that performs a two-dimensional or three-dimensional motion in space by a two-dimensional or three-dimensional driving mechanism, so that the reagent needle may move to aspirate a reagent carried by the reagent storage unit 13 and to a cuvette to which the reagent is to be added and discharge the reagent to the cuvette.

Wherein the reagent storage part 13 is used for storing the reagent cartridge. In one embodiment, a kit comprises a solid phase reagent comprising at least one coating component coated on a solid support and a labeling reagent comprising a plurality of labeled labeling components. In another embodiment, a kit comprises a solid phase reagent comprising a plurality of coating components coated on a solid support and a labeling reagent comprising at least one labeling component bearing a label.

The reaction device 30 has at least one placing position for placing the reaction cup and incubating the reaction solution in the reaction cup. For example, the reaction device 30 may be a reaction tray, which is configured in a disc-shaped structure and has one or more placing positions for placing reaction cups, and the reaction tray can rotate and drive the reaction cups in the placing positions to rotate, so as to schedule the reaction cups in the reaction tray and incubate the reaction solution in the reaction cups.

The photometric device 40 is used to perform photometric measurement on the incubated reaction solution to obtain reaction data of the sample. For example, the light measuring unit 40 detects the light emission intensity of the reaction solution to be measured, and calculates the concentration of the component to be measured in the sample from the calibration curve. In some embodiments, the photometric component 40 is separately disposed outside of the reaction device 30.

The immunoassay analyzer also includes a luminescent substrate dispensing device (not shown). The luminescent substrate dispensing apparatus is connected to a container storing a luminescent substrate and is used for dispensing the luminescent substrate into a reaction cuvette to which the luminescent substrate is to be added.

As shown in fig. 2, 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, wherein the processing component 51, the RAM 52, the ROM 53, the communication interface 54, the memory 56, and the I/O interface 55 communicate over a bus 57.

The processing component may be a CPU, GPU or other chip with computing capabilities.

The memory 56 stores therein various computer programs such as an operating system and an application program to be executed by the processor unit 51, and data necessary for executing the computer programs. In addition, data stored locally during the sample testing process, if desired, may be stored in the memory 56.

The I/O interface 55 is constituted by 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, an a/D converter, and the like. The I/O interface 55 is connected to an input device including a keyboard, a mouse, a touch panel, or other control buttons, and a user can directly input data to the control apparatus 50 using the input device. In addition, a display having a display function, such as: liquid crystal screen, touch screen, LED display screen, etc., the control device 50 may output the processed data as image display data to a display for displaying, for example: analytical data, instrument operating parameters, etc.

The communication interface 54 is an interface that may be any communication protocol known today. The communication interface 54 communicates with the outside through a network. Control device 50 may communicate data with any device connected through the network via communication interface 54 using a communications protocol.

In one case, the control device 50 is configured to receive test instructions, the test instructions including the type of substance to be tested, and in response to the test instructions to perform the steps of:

controlling the sample dispensing component 12 to add the sample to be tested in the sample storage component 11 into a reaction cup on the reaction device 30;

controlling the reagent dispensing component 14 to add the solid phase reagent in the kit stored in the reagent storage component 13 into a reaction cup on the reaction device 30, so that the sample to be tested and the added solid phase reagent are mixed in the reaction cup and incubated for a period of time, so that at least one coating component on the solid phase carrier can be combined with the substance to be tested in the sample to be tested;

the reagent dispensing control part 14 further adds a labeling component corresponding to the type of the substance to be detected in the reagent kit to the reaction cup, so that the added labeling component is mixed with the mixture in the reaction cup and incubated for a period of time, so that the added labeling component can be combined with the substance to be detected combined on the coating component in the added solid-phase reagent;

controlling a luminescent substrate dispensing device to add a luminescent substrate into the reaction cup; and

the detection result is obtained from the ratio of the light emission value measured in the light measuring section 40 to the light emission threshold value.

In another case, the control device 50 is configured to receive test instructions, the test instructions including the type of substance to be tested, and in response to the test instructions to perform the steps of:

controlling a reagent dispensing component 14 to add at least one coating component corresponding to the type of the substance to be detected in the kit stored in the storage component 13 into the reaction cup, so that the sample to be detected and the at least one coating component are 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 substance to be detected in the sample to be detected;

the reagent dispensing control part 14 further adds a labeled reagent in the reagent kit to the reaction cup, so that the added labeled reagent is mixed with the mixture in the reaction cup and is incubated for a period of time, so that the labeled component in the added labeled reagent can be combined with the substance to be detected combined with the coating component in the added solid phase reagent;

The immunoassay analyzer provided by the embodiment of the invention can selectively detect different types of substances to be detected according to the requirements of users based on one kit, thereby improving the detection flexibility and meeting the requirements of the users in different scenes.

Experimental materials:

hepatitis B virus core antigen, HAV antigen, from Meridian Life Science;

anti-human antibodies, such as anti-human IgM antibodies, anti-human IgG antibodies are from Jackson immunorereasearch;

alkaline phosphatase is derived from Roche pharmaceutical;

magnetic beads were from Thermo Fisher.

Preparation of solid-phase coating:

the antigen is first pre-treated and its protective components in the buffer matrix are removed by dialysis. The coating is carried out in a proportion of 0.5-40. mu.g of antigen per mg of magnetic beads (preferably 1-30. mu.g). And in the reaction process, carboxyl on the surface of the magnetic bead is coupled with amino of the antigen under the catalysis of EDC/NHS. Taking 20mg of magnetic microspheres modified with carboxyl on the surface, ultrasonically dispersing the magnetic microspheres in 10mM MES buffer solution, adding 80mg of EDC and 120mg of NHS, ultrasonically mixing the mixture uniformly, and placing the mixture in a shaking table at 37 ℃ for 15 min. Then adding the antigen into the treated magnetic beads according to the proportion, mixing uniformly, and placing the mixture in a shaking table at 37 ℃ for reaction for 10-18 h. And after cleaning and sealing, preparing the magnetic microspheres coated with the antigen. The antigen-coated magnetic microspheres were diluted with 50mM Tris pH 7.4 buffer to prepare a solid phase reagent. Wherein, the concentration of the magnetic microspheres coated with the antigen is 0.7 mg/mL.

Preparation of a marker component:

labeling the anti-human IgM antibody and the anti-human antibody with a signal marker. In embodiments of the invention, the signal marker is alkaline phosphatase. And respectively diluting the anti-human IgM antibody signal marker and the anti-human antibody signal marker by selecting 50mM MES pH 6.0 buffer solution to prepare a marker component.

A detection step:

in the first step, the sample and the solid phase reagent are added into the reaction tube and incubated at 37 ℃ for 10 minutes, so that the antigen on the solid phase surface can be combined with the corresponding substance to be detected in the sample. After the incubation in the reaction tube is completed, the substance bound to the solid phase is attracted by placing it in a magnetic field, the substance bound to the solid phase of the magnetic beads is retained, and the unbound substance is washed away.

In the second step, the desired labeling components are selected and added to the reaction tube for incubation, mixed and incubated at 37 ℃ for 10 minutes to bind to the conjugate formed in the first step. After incubation in the reaction tube is complete, the complex is attracted by the magnetic field and other unbound material is washed away.

Third, AMPPD is added to the reaction tube to produce chemiluminescence. And measuring the number of photons generated by the reaction through a photomultiplier to obtain a chemiluminescence signal value of the sample.

And dividing the luminous signal value by a threshold value to obtain a COI value. Comparing the COI value of the test result of the sample with a reference value (the reference value is 1.10), and if the COI value is greater than or equal to 1.1, indicating that one or more of the detection objects in the sample are positive; if less than 0.90, it indicates that all the test substances in the sample are negative. COI is between 0.90 and 1.10, the result is a gray zone (indeterminate).

In the embodiment of the present invention, the negative coincidence rate refers to the proportion of the number of samples determined to be negative obtained by using the test method of the embodiment of the present invention to the negative samples actually participating in the evaluation, and the positive coincidence rate refers to the proportion of the number of samples determined to be positive obtained by using the test method of the embodiment of the present invention to the positive samples actually participating in the evaluation; the true negative and positive results of the sample are from hospital diagnostic results.

Example 1 luminescence threshold determination for selective detection of HBV

Samples with definite clinical diagnosis results are selected, wherein 350 Anti-HBc IgM detection samples (200 negative samples and 150 positive samples) and 600 Anti-HBc IgG antibody detection samples (370 negative samples and 230 positive samples).

The magnetic microsphere coated with the HBc antigen obtained by the preparation of the solid phase coating is used as a solid phase component, and the anti-human IgM antibody with alkaline phosphatase and the anti-human IgG antibody with alkaline phosphatase obtained by the preparation of the marking component are used as marking components respectively, and the positive coincidence rate and the negative coincidence rate of the HBc antigen are tested under different luminescence thresholds according to the detection step. The results are summarized in Table 1.

TABLE 1 determination of luminescence threshold for selective detection of HBV

As can be seen from Table 1, when the Anti-HBc IgM detection value is around 50000, the positive coincidence rate is 100%, the negative coincidence rate is 99.5%, and the total coincidence rate is the highest (99.71%). For the Anti-HBc IgG detection, when the luminescence value is about 70000, the positive coincidence rate is 100%, the negative coincidence rate is 99.73%, and the total coincidence rate is the highest (99.83%).

Example 2 Selective detection of IgM and IgG antibodies against HBc based on a kit

Preparation of solid-phase coating: the magnetic microsphere coated with HBc antigen obtained by the preparation of the solid phase coating is used as a solid phase component;

preparation of marker fraction 1: according to the preparation of the labeled component, the anti-human IgM antibody with alkaline phosphatase is prepared and used, and the anti-human IgM antibody is matched with the solid-phase component to determine an internal reference substance. Selecting the dilution with the luminescence threshold value of 50000 (allowing deviation of +/-10%) as the dilution of the marker component 1;

preparation of marker component 2: the anti-human IgG antibody with alkaline phosphatase prepared according to the preparation of the labeled component is used and matched with the solid phase component to determine the internal reference substance. The dilution corresponding to an emission threshold at 70000 (allowing a ± 10% deviation) was selected as the dilution of marker component 2.

When the IgM antibody of HBc needs to be detected, the solid phase component is selected to match with the marker component 1, and the luminescent signal value is measured according to the detection step.

When the IgG antibody of HBc needs to be detected, the solid phase component is selected to match with the labeled component 2, and the luminescent signal value is measured according to the detection step.

EXAMPLE 3 luminescence threshold determination for HAV Selective detection

Samples with definite clinical diagnosis results were selected, wherein 400 Anti-HAV IgM detection samples (250 negative samples and 150 positive samples) and 500 Anti-HAV IgG antibody detection samples (330 negative samples and 170 positive samples).

The HAV antigen-coated magnetic microspheres obtained by the preparation of the solid phase coating are used as solid phase components, and the anti-human IgM antibody with alkaline phosphatase and the anti-human IgG antibody with alkaline phosphatase obtained by the preparation of the marking components are used as marking reagents, and the positive coincidence rate and the negative coincidence rate of the HAV antigen are tested under different luminescence thresholds according to the detection step. The results are summarized in Table 2.

TABLE 2 determination of luminescence threshold for HAV Selective detection

As shown in Table 2, the Anti-HAV IgM detection showed that the luminescence value was around 40000, the positive coincidence rate was 100%, the negative coincidence rate was 99.60%, and the total coincidence rate was the highest (99.75%). For Anti-HAV IgG detection, when the luminescence value is around 70000, the positive coincidence rate is 100%, the negative coincidence rate is 99.70%, and the total coincidence rate is the highest (99.80%).

Example 4 Selective detection of IgM antibodies and IgG antibodies against HAV based on a kit

Preparation of solid-phase coating: adopting HAV antigen-coated magnetic microspheres obtained by 'preparation of solid-phase coating' as solid-phase components;

preparation of marker fraction 1: according to the preparation of the labeled component, the anti-human IgM antibody with alkaline phosphatase is prepared and used, and the anti-human IgM antibody is matched with the solid-phase component to determine an internal reference substance. Selecting the dilution corresponding to the light-emitting threshold value of 40000 (tolerance of +/-10% deviation) as the dilution of the marker component 1;

When the IgM antibodies of HAV are to be detected, the solid phase component and the labeled component 1 are selected in combination to measure the luminescent signal value according to the "detection step".

When IgG antibody of HAV is to be detected, the solid phase component and the labeled component 2 are selected in combination to measure the luminescent signal value according to the "detection procedure".

And dividing the luminous signal value by a threshold value to obtain a COI value. Comparing the COI value of the test result of the sample with a reference value (the reference value is 1.10), and if the COI value is more than or equal to 1.1, indicating that one or more of the detection objects in the sample are positive; if less than 0.90, it indicates that all the test substances in the sample are negative. COI is between 0.90 and 1.10, the result is a gray zone (indeterminate).

Claims

PCT domestic application, the claims have been published.