CN114641689A

CN114641689A - Immunoassay instrument, method and kit for detecting HCV

Info

Publication number: CN114641689A
Application number: CN201980102015.XA
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: 2022-06-17
Also published as: WO2021134302A1

Abstract

The immunoassay instrument can selectively detect the HCV antigen and/or the anti-HCV antigen antibody in a sample to be detected, and the immunoassay instrument is matched with a kit to realize the selective detection of different HCV projects according to the requirements of users. Also relates to an immunoassay method and a kit.

Description

Immunoassay instrument, method and kit for detecting HCV

Technical Field

The present invention relates to the field of immunoassays, and in particular to the detection of HCV.

Background

The Hepatitis C Virus (HCV) was isolated and named in 1989 by Michael Houghto, chronic infection of HCV may cause chronic inflammatory necrosis of liver and liver fibrosis, and some patients may develop liver cirrhosis and even hepatocellular carcinoma, which is a serious social and public health problem. According to the world health organization, the global infection rate of HCV is about 3%, and about 1.7 million people are estimated to be infected with HCV.

In clinical applications, for HCV infection, detection of antibodies and/or antigens thereof may be involved. In which the detection of anti-HCV antibodies often indicates the presence of a past history of infection in a patient, but does not identify whether the patient is an infected patient or a patient who has recovered from the past infection. Detection of HCV antigens can indicate the infection status of a patient and can monitor the course of treatment for the patient, and is a diagnostic and useful complementary tool. In addition, the detection of HCV antigen can effectively shorten the window period and avoid early infection of diseases.

Currently, detection kits for HCV have been developed for specific test items. However, when a sample to be tested is tested using such a kit on an immunoassay analyzer, only a specific item to which the kit is directed can be used. Since the use frequency of anti-HCV antibodies, HCV antigens, and the mixed detection of antibodies and antigens is not the same, hospitals have to prepare a large number of different kinds of kits to cope with different needs, and an increase in medical costs is inevitable.

Accordingly, there is a strong demand for flexibly and conveniently selecting a test item according to a user's demand when detecting HCV using an immunoassay analyzer.

Disclosure of Invention

The problem that when the current immunoassay analyzer detects HCV based on the same kit, an appropriate detection item (i.e., only HCV antigen, only anti-HCV antigen antibody, and a mixture of HCV antigen and anti-HCV antigen antibody) cannot be selected as required is solved. 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 instrument for selectively detecting HCV antigens and/or antibodies against HCV antigens 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 device having a reagent storage means for storing a reagent kit comprising a solid phase reagent and a labeled reagent, the solid phase reagent comprising a first solid phase component comprising an anti-HCV antigen antibody coated on a solid phase carrier and a second solid phase component comprising an HCV antigen coated on a solid phase carrier, the labeled reagent comprising a first labeled component comprising a labeled anti-HCV antigen antibody and a second labeled component comprising a labeled anti-human antibody, the reagent storage means being for aspirating the solid phase component and the labeled component in the reagent kit stored on the reagent storage means 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 dispensing the luminescent substrates into a reaction cup to be added with the luminescent substrates;

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

the light measurement component is used for performing light measurement on the incubated reaction solution 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 at least one solid-phase 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 at least one solid-phase component are mixed in the reaction cup and incubated for a period of time, and the coating coated on the solid-phase carrier can be combined with the substance to be detected in the sample to be detected;

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 label in the added labeling component can be combined with the substance to be detected combined on the coating body coated on the solid phase carrier to form a coating body-substance to be detected-label body compound;

controlling the luminescent substrate dispensing device to add a luminescent 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 is to be noted that the immunoassay analyzer according to the first aspect of the present invention selects different substances to be tested based on a kit capable of responding to an instruction containing the type of the substances to be tested. For example, when the detection of HCV antigen (i.e., the substance to be detected is HCV antigen) is selected, the reagent dispensing component first adds only the first solid-phase component or at least the first solid-phase component into the reaction cup, and the antibody against HCV antigen coated on the solid-phase carrier in the solid-phase reagent can specifically bind to HCV antigen in the sample to be detected; next, the immunoassay analyzer further controls the reagent dispensing part in response to the instruction to select at least the first marker component or only the first marker component to be added to the reaction cup, and to realize detection of the HCV antigen by forming a complex of an antibody against the HCV antigen-an antibody against the HCV antigen. For another example, when an antibody against HCV antigen (i.e., an antibody against HCV antigen to the substance to be detected) is selected for detection, the reagent dispensing unit first adds only the second solid-phase component or at least the second solid-phase component to the reaction cuvette, and the HCV antigen coated on the solid-phase carrier in the solid-phase reagent can specifically bind to the antibody against HCV antigen in the sample to be detected; next, the immunoassay analyzer further controls the reagent dispensing part in response to the instruction to select at least the second marker component or only the second marker component to be added to the reaction cup, and detection of the antibody against the HCV antigen is achieved by forming an HCV antigen-anti-HCV antigen antibody-anti-human antibody complex. For another example, when the mixed detection of the HCV antigen and the anti-HCV antigen (i.e., the substance to be detected is the HCV antigen and the antibody against the HCV antigen) is selected, the reagent dispensing component first adds the first solid-phase component and the second solid-phase component into the cuvette, the antibody against the HCV antigen coated on the solid-phase carrier in the solid-phase reagent can specifically bind to the HCV antigen in the sample to be detected, and the HCV antigen coated on the solid-phase carrier can specifically bind to the antibody against the HCV antigen in the sample to be detected; next, the immunoassay analyzer further controls the reagent dispensing part in response to the instruction to select the first marker component and the second marker component to be added to the cuvette, and to realize mixed detection of the HCV antigen and the antibody against the HCV antigen by forming a complex of antibody-HCV antigen-antibody against the HCV antigen, HCV antigen-antibody against the HCV antigen-anti-human antibody.

With the immunoassay analyzer according to the first aspect of the present invention, whether an individual needs to detect HCV antigens, detect antibodies against HCV antigens, mix and detect HCV antigens and antibodies against HCV antigens, or a combination of the above-mentioned detection items, it is possible to flexibly and conveniently achieve the detection by controlling the reagent dispensing part to add corresponding reagent components according to instructions. Meanwhile, extra reagent boxes do not need to be stored in the reagent storage part of the immunoassay analyzer, so that testers do not need to be equipped with various reagent boxes, and the waste of the reagent boxes caused by different use frequencies is avoided, so that the detection cost is reduced.

In some embodiments, the anti-HCV antigen antibody and HCV antigen coated on a solid support in a solid phase reagent are present in separate aliquots; in the labeled reagent, the anti-HCV antigen antibody and the anti-human antibody with the label are present in a separate package or a premixed form.

In some embodiments, the anti-HCV antigen antibody coated on the solid support in the solid-phase reagent and the HCV antigen are present in a pre-mixed form; in the labeled reagent, the labeled anti-HCV antigen antibody and the anti-human antibody are present in a separately dispensed form. By using a single solid phase reagent and one or more marker components of a selectable marker reagent, a convenient, low cost selective immunoassay is achieved.

In a second aspect, the present invention provides an immunoassay method for detecting HCV, comprising the steps of:

mixing a sample to be tested with anti-HCV antigen antibodies and HCV antigens coated on a solid phase carrier and incubating for a period of time, so that the anti-HCV antigen antibodies and HCV antigens coated on the solid phase carrier can be combined with 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;

adding a labeled anti-HCV antigen antibody and a labeled anti-human antibody to the washed mixture and incubating so that the labeled anti-HCV antigen antibody and the labeled anti-human antibody can bind to the test substance bound to the anti-HCV antigen antibody and the HCV antigen coated on the solid phase carrier to form a complex;

washing the complex to remove unbound material;

and adding a luminescent substrate into the washed complex to detect the mixed detection value of the anti-HCV antigen antibody and the HCV antigen in the sample to be detected.

In the method of the second aspect of the present invention, a solid phase reagent comprising an anti-HCV antigen antibody and an HCV antigen coated on a solid phase carrier, which are capable of specifically binding to an alternative analyte, i.e., an HCV antigen and an anti-HCV antigen antibody, respectively, is mixed with a sample to be tested and incubated. Next, the labeled component is mixed with the mixture of the specimen and the solid-phase reagent and incubated to mix the detection HCV antigen and the antibody against the HCV antigen. In other words, the method of the invention can finally obtain a mixed detection result, and can carry out mixed detection on the HCV antigen and the anti-HCV antigen antibody in the sample, thereby improving the detection efficiency.

In some embodiments, the anti-HCV antigen antibody and HCV antigen coated on the solid support are added to the test sample separately, mixed and incubated.

In some embodiments, the anti-HCV antigen antibody and HCV antigen coated on the solid support are added to the test sample in a pre-mixed form for mixing and incubation.

In some embodiments, labeled anti-HCV antigen and anti-human antibodies are added separately to the washed mixture for mixing and incubation.

In some embodiments, the labeled anti-HCV antigen antibody and anti-human antibody are added to the washed mixture in a pre-mixed form for mixing and incubation.

In a third aspect, the present invention provides an immunoassay method for selectively detecting HCV, comprising the steps of:

selecting at least one of a first solid phase component and a second solid phase component from a solid phase reagent according to the type of a substance to be tested, mixing a sample to be tested with the selected solid phase component and incubating for a period of time so that at least one coating coated on a solid phase carrier can bind to the substance to be tested in the sample to be tested;

washing the mixture of the sample to be tested and the selected solid phase component to remove unbound material;

selecting at least one of a first labeling component and a second labeling component from a labeling reagent according to the type of a substance to be detected, adding the selected labeling component to the washed mixture, and incubating so that a label in the added labeling component can bind to the substance to be detected bound to at least one coating body coated on the solid phase carrier to form a complex;

washing the complex to remove unbound material;

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

wherein the first solid phase component is an anti-HCV antigen antibody coated on a solid phase carrier, the second solid phase component is an HCV antigen coated on a solid phase carrier, the first labeling component is a labeled anti-HCV antigen antibody, and the second labeling component is a labeled anti-human antibody.

It should be noted that, in the method of the third aspect of the present invention, the first solid phase component and/or the second solid phase component is selected from the solid phase reagent according to the requirement of the type of the substance to be measured, and the selected solid phase component is mixed with the sample to be measured and incubated, so that at least one coating coated on the solid phase carrier can bind to the substance to be measured. Next, a first labeled component and/or a second labeled component is selected from the labeled reagent according to the requirement of the type of the substance to be detected, and the selected labeled component is mixed and incubated with the mixture of the specimen and the solid phase reagent to selectively detect HCV antigen, detect an antibody against HCV antigen, and mix-detect HCV antigen and an antibody against HCV antigen.

In a fourth aspect, the present invention provides a kit for detecting HCV, comprising:

a solid phase reagent comprising an anti-HCV antigen antibody and an HCV antigen coated on a solid phase carrier;

a labeling agent comprising a labeled anti-HCV antigen antibody and an anti-human antibody.

In some embodiments, the anti-HCV antigen antibody and HCV antigen coated on a solid support in a solid phase reagent are present in separate aliquots.

In some embodiments, the anti-HCV antigen antibody and HCV antigen coated on a solid support in the solid phase reagent are present in a pre-mixed form.

In some embodiments, the anti-HCV antigen antibody and the anti-human antibody with a label in the labeling reagent are present in separate dispensed forms.

In some embodiments, the anti-HCV antigen antibody with a label and the anti-human antibody in the labeling reagent are present in a pre-mixed form.

The kit provided in the fourth aspect of the present invention is applicable to the immunoassay analyzer provided in the first aspect of the present invention and the immunoassay methods provided in the second and third aspects of the present invention.

In the first to fourth aspects of the present invention, specifically, the anti-human antibody is selected from at least one of an anti-human IgG antibody and an anti-human IgM antibody.

In the first to fourth aspects of the present invention, specifically, the concentrations of the solid-phase reagent and the labeling reagent are designed such that, when an internal reference (for example, an internal reference defined according to ISO18113-1:2009 standard) is detected separately using an coating body coated on a solid-phase carrier with each of the solid-phase reagents and a labeling body with a corresponding label in the labeling reagent under the same reaction system, the corresponding luminescence thresholds are substantially the same.

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.

As described above, in the detection of HCV, antibodies, antigens, and a mixture of antibodies and antigens each have a range of applications, some detection items are used for screening, and some tests are used only in a specific scenario. Meanwhile, the items are used with different frequencies in clinic.

The above-mentioned current situation brings great inconvenience to the testing party, especially some hospitals with few number of patients. Some items with smaller test volumes still have a large number of tests unused at the end of the effective life of the kit, resulting in increased medical costs. On the other hand, the testing party needs to purchase a plurality of kits and their associated calibrators and quality control products at the same time, which is very inconvenient.

In contrast, the invention designs a method for selectively detecting antigen and/or antibody, namely, based on the same kit, antigen detection, antibody detection or mixed detection of antigen and antibody can be selectively realized. The user can select one or more items to detect according to own requirements.

In one embodiment of the present invention, the term "coating" 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.

In the present embodiment, the term "label" refers to a substance labeled with a label, which is capable of binding to a test substance in a test sample.

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 an embodiment of the present invention, antibodies against HCV antigens and HCV antigens coated on a solid support can be produced by: in one aspect, antibodies to HCV antigens and HCV antigens can be coated separately on separate solid supports, and the coated solid supports can then be mixed. Alternatively, antibodies to HCV antigens can be mixed with HCV antigens prior to coating with the solid support.

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 can be used as a light-emitting substrate, which is decomposed by alkaline phosphatase to remove a phosphate group, thereby producing an unstable intermediate product, which generates a methyl-metaborate anion through intramolecular electron transfer, and which generates chemiluminescence when the methyl-metaborate anion in an excited state returns from the excited state to a 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 characteristics) curve refers to a curve obtained by dividing the diagnostic test result into several critical points, and plotting the sensitivity corresponding to each critical point as the ordinate and the specificity as the 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 a solid phase reagent, the antigens of embodiments of the invention can 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 embodiment, "anti-human antibody" refers to an antibody that can specifically bind to a human antibody.

In the embodiment of the present invention, the label in the labeling reagent may be linked to the label by a method that is conventional in the art (e.g., chemical bond binding).

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 result of detection of at least one test substance 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, the judgment result is negative, which indicates that the detection results of all the substances to be detected 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 internal reference substance is detected by using each component coated on the solid phase carrier in the solid phase reagent and the corresponding labeled component in the labeled reagent separately in the same reaction system, and the corresponding luminescence thresholds are substantially the same. For example, in a certain reaction system, a solid-phase component and a marker component corresponding to a certain substance to be detected are adopted to independently detect an internal reference substance defined in ISO18113-1:2009, the concentration of the marker component is kept at a constant value, the concentration of the solid-phase component is adjusted, and a luminescence threshold value is adjusted to a preset value; or, keeping the concentration of the solid phase component at a constant value, adjusting the concentration of the labeling 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 ISO18113-1:2009 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 ± 3%, within ± 2%, or within ± 1%.

As shown in fig. 1, the present invention provides an immunoassay instrument that can selectively detect HCV antigens and/or antibodies against HCV antigens in a test sample. 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 also include a display component (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 unit 12 is used for sucking up a sample and discharging 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, the kit comprises a solid phase reagent and a labeling reagent. The solid phase reagent comprises a first solid phase component comprising an anti-HCV antigen antibody coated on a solid phase carrier and a second solid phase component comprising an HCV antigen coated on a solid phase carrier. The labeling reagent includes a first labeling component including a labeled anti-HCV antigen antibody and a second labeling component including a labeled anti-human antibody.

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 photometric device 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 and an a/D converter. 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.

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 at least one solid-phase component corresponding to the type of the substance to be detected in the kit into a reaction cup on the reaction device 30, so that the sample to be detected and the added at least one solid-phase component are mixed in the reaction cup and incubated for a period of time, and the coating on the solid-phase carrier can be combined with the substance to be detected in the sample to be detected;

the reagent dispensing control component 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 label in the added labeling component can be combined with the substance to be detected combined on the coating body 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.

The immunoassay analyzer provided by the embodiment of the invention can selectively detect different 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:

antibodies against HCV antigen were from Meridian Life Science;

the anti-human antibody is from Jackson ImmunoResearch;

HCV antigen is from Meridian Life Science;

alkaline phosphatase is derived from Roche pharmaceutical;

magnetic beads were from Thermo Fisher.

Preparation of solid-phase coating:

the antigen/antibody is first pre-treated and its protective components in the buffer matrix are removed by dialysis. The coating is carried out in a ratio of 1 to 40. mu.g of antigen/antibody per mg of magnetic beads, preferably 5 to 30. mu.g. And coupling carboxyl on the surface of the magnetic bead with amino of the antigen/antibody under the catalysis of EDC/NHS in the reaction process. 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/antibody 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/antibody. The antigen/antibody coated magnetic microspheres were diluted to 0.3mg/mL using 50mM Tris pH 7.4 buffer.

Preparation of the marker component:

the anti-human antibody and the anti-HCV antigen antibody are labeled with a signal label. In embodiments of the invention, the signal marker is alkaline phosphatase. The anti-human antibody signal marker and the anti-HCV antigen antibody signal marker were diluted with 50mM MES pH 6.0 buffer, respectively, to prepare a labeled fraction.

A detection step:

in the first step, the sample and the solid phase reagent are added to the reaction tube and incubated at 37 ℃ for 10 minutes so that the antigen/antibody on the solid phase surface can bind to 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, while 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 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).

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

Example 1 luminescence threshold determination for HCV detection

Samples with definite clinical diagnosis results were selected, including HCV antigen detection samples 563 (317 negative samples and 246 positive samples), and Anti-HCV antibody detection samples 600 (320 negative samples and 280 positive samples).

The HCV antigen-coated magnetic microspheres obtained by the preparation of the solid phase coating are used as solid phase components, the alkaline phosphatase-containing anti-human antibodies obtained by the preparation of the labeled components are used as labeled components, and the positive coincidence rate and the negative coincidence rate of the anti-HCV antigen antibodies are detected under different luminescence thresholds according to the detection step. Similarly, the magnetic microspheres coated with the anti-HCV antigen obtained from "preparation of solid-phase coating" were used as solid-phase components, and the anti-HCV antigen antibody with alkaline phosphatase obtained from "preparation of labeled component" was used as labeled component, and the positive and negative coincidence rates of the HCV antigen were determined according to the "detection procedure" at different luminescence thresholds. The results are summarized in Table 1.

TABLE 1

As is clear from table 1, when the emission threshold was selected to be around 70000, the total coincidence rate was the highest, and the emission threshold of the reagent kit was selected to be 70000.

EXAMPLE 2 diagnostic efficacy Studies of HCV Selective detection

Preparation of solid-phase reagents: the magnetic microspheres coated with HCV antigen and the magnetic microspheres coated with anti-HCV antigen antibody obtained by the preparation of the solid phase coating are used as solid phase reagents;

preparation of marker fraction 1: according to the preparation of the labeled component, the prepared anti-human antibody with alkaline phosphatase is used and matched with the corresponding solid phase component to determine the internal reference substance. Selecting the dilution corresponding to the luminescence threshold at 70000 (allowing + -3% deviation) as the dilution of the marker component 1;

preparation of marker component 2: according to the "preparation of labeled fraction", an antibody against HCV antigen with alkaline phosphatase is prepared and used, and the antibody is used in combination with a corresponding solid-phase fraction to determine an internal reference. The dilution corresponding to an emission threshold at 70000 (allowing a ± 3% deviation) was selected as the dilution of marker component 2.

Samples with definite clinical diagnosis results were selected for 1000 cases in total. Among them, 670 HCV antibody and antigen negative samples, and 330 HCV antibody or antigen positive samples (among them, 318 HCV antibody positive samples, and 211 HCV antigen positive samples).

When the antibody against the HCV antigen needs to be detected, the solid-phase reagent is selected to match with the labeled component 1, and the luminescence signal value is measured according to the detection step.

When the HCV antigen needs to be detected, the solid-phase reagent is selected to match with the labeled component 2, and the luminescence signal value is measured according to the detection step.

When the HCV antigen-antibody and the HCV antigen need to be detected in a mixed manner, the solid-phase reagent is selected to match with the labeled components 1 and 2, and the luminous signal value is measured according to the detection step.

The results are shown in table 2 below.

TABLE 2

As can be seen from table 2, when the anti-CV antibody was detected alone, the positive coincidence rate was 100%, the negative coincidence rate was 99.06%, and the overall coincidence rate was 99.70%; when HCV antigen is detected independently, the positive coincidence rate is 100%, the negative coincidence rate is 99.53%, and the total coincidence rate is 99.90%; the HCV antigen and the antibody are detected in a mixed mode, the positive coincidence rate is 100%, the negative coincidence rate is 98.48%, and the total coincidence rate is 99.95%. The embodiment of the invention can realize the selective detection of the HCV antigen and/or the anti-HCV antigen in the sample to be detected.

Claims

An immunoassay instrument for selectively detecting an HCV antigen and/or an antibody against the HCV antigen in a sample to be tested, 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 device having a reagent storage means for storing a reagent kit comprising a solid phase reagent and a labeled reagent, the solid phase reagent comprising a first solid phase component comprising an anti-HCV antigen antibody coated on a solid phase carrier and a second solid phase component comprising an HCV antigen coated on a solid phase carrier, the labeled reagent comprising a first labeled component comprising a labeled anti-HCV antigen antibody and a second labeled component comprising a labeled anti-human antibody, the reagent storage means being for aspirating the solid phase component and the labeled component in the reagent kit stored on the reagent storage means 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 at least one solid-phase 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 at least one solid-phase component are mixed in the reaction cup and incubated for a period of time, and the coating coated on the solid-phase carrier can be combined with the substance to be detected in the sample to be detected;

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 label in the added labeling component can be combined with the substance to be detected combined on the coating body coated on the solid phase carrier to form a coating body-substance to be detected-label body compound;

controlling the luminescent substrate dispensing device to add a luminescent 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.
The immunoassay analyzer of claim 1, wherein in the solid-phase reagent, the anti-HCV antigen antibody and HCV antigen coated on the solid-phase carrier are present in a separately dispensed form;

in the labeled reagent, the anti-HCV antigen antibody and the anti-human antibody with the label are present in a separate package or a premixed form.
The immunoassay analyzer of claim 1, wherein in the solid-phase reagent, the anti-HCV antigen antibody and HCV antigen coated on the solid-phase carrier are present in a pre-mixed form;

in the labeled reagent, the labeled anti-HCV antigen antibody and the anti-human antibody are present in a separately dispensed form.
The immunoassay analyzer of any one of claims 1 to 3, wherein the concentrations of the respective substances in the solid-phase reagent and the labeled reagent are designed so that the luminescence thresholds of the respective internal reference substances are substantially the same when the internal reference substances are detected separately using the coating substance coated on the solid-phase carrier of each of the solid-phase reagents and the labeled substance corresponding to the labeled substance in the labeled reagent in the same reaction system.
The immunoassay analyzer of any one of claims 1 to 4, wherein the anti-human antibody is selected from at least one of an anti-human IgG antibody and an anti-human IgM antibody.
An immunoassay method for detecting HCV comprising the steps of:

mixing a sample to be tested with anti-HCV antigen antibodies and HCV antigens coated on a solid phase carrier and incubating for a period of time, so that the anti-HCV antigen antibodies and HCV antigens coated on the solid phase carrier can be combined with 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;

adding a labeled anti-HCV antigen antibody and a labeled anti-human antibody to the washed mixture and incubating so that the labeled anti-HCV antigen antibody and the labeled anti-human antibody are bound to the test substance bound to the anti-HCV antigen antibody and the HCV antigen, respectively, coated on the solid carrier, to form a complex;

washing the complex to remove unbound material;

and adding a luminescent substrate into the washed complex to detect the mixed detection value of the anti-HCV antigen and the HCV antigen in the sample to be detected.
The method of claim 6, wherein the anti-HCV antigen antibody and HCV antigen coated on the solid carrier are separately added to the test sample to be mixed and incubated, or are added to the test sample in a pre-mixed form to be mixed and incubated.
The method of claim 6 or 7, wherein an anti-HCV antigen antibody and an anti-human antibody with a label are added to the washed mixture separately for mixing and incubation, or are added to the washed mixture in a pre-mixed form for mixing and incubation.
The method according to any one of claims 6 to 8, wherein the concentrations of the solid-phase reagent and the labeled reagent are designed so that the internal reference substances are detected with each of the solid-phase reagent-coated solid-phase carrier-coated coating bodies and the labeled reagent-coated labeling bodies separately in the same reaction system, and the luminescence thresholds are substantially the same.
A kit for detecting HCV, comprising:

a solid phase reagent comprising an anti-HCV antigen antibody and an HCV antigen coated on a solid phase carrier;

a labeling agent comprising a labeled anti-HCV antigen antibody and an anti-human antibody.
The kit of claim 10, wherein said anti-human antibody is selected from at least one of an anti-human IgG antibody and an anti-human IgM antibody.
The kit according to claim 10 or 11, wherein the concentrations of the solid-phase reagent and the labeled reagent are set so that the internal reference substances are detected with each of the solid-phase reagent-coated solid-phase carrier and the labeled reagent-coated labeled antibody alone in the same reaction system at substantially the same luminescence threshold.
The kit according to any one of claims 10 to 12, wherein, in the solid-phase reagent, the anti-HCV antigen antibody and HCV antigen coated on a solid-phase carrier are present in a separate package or a premixed form.
The kit according to any one of claims 10 to 13, wherein the labeled anti-HCV antigen antibody and the anti-human antibody are present in the labeled reagent in a separate package or a premixed form.