WO2021134304A1 - Kit, method and immunoassay analyzer for screening torch infection - Google Patents

Abstract

A kit for screening TORCH infection, comprising: a capture mixture in which at least two antigens coated on a solid-phase carrier and selected from the following antigens are mixed: a toxoplasma gondii antigen, a rubella virus antigen, a cytomegalovirus antigen, a herpes simplex virus type 1/2 antigen, a parvovirus B19 antigen, a coxsackievirus antigen, and a herpes zoster virus antigen; and an anti-human IgM antibody with a marker. The kit can test IgM antibodies caused by a variety of different pathogenic microorganisms in a sample to be tested, and assesses a risk according to a mixed test result. In addition, the present invention further relates to an immunoassay analysis method and immunoassay analyzer for screening TORCH infection.

Description

Kit, method and immune analyzer for screening TORCH infection Technical field

The present invention relates to the field of immunoassays, in particular to the analysis method of Torch screening.

Background technique

TORCH refers to the abbreviation of a group of pathogenic microorganisms in English. T stands for Toxoplasma gondii or Toxoplasma gondii; O stands for other pathogenic microorganisms, such as herpes zoster virus, parvovirus B19, Coxsackie virus, etc.; R stands for rubella virus; C stands for cytomegalovirus; and H stands for herpes simplex virus Ⅰ Type and Type II. This group of pathogens can be transmitted to the fetus through the placenta, causing perinatal infections, and there is a risk of miscarriage, stillbirth, premature delivery, congenital malformations, and intellectual disability. Therefore, antibody testing for TORCH infection has gradually become a routine pregnancy test project.

In the TORCH test, a negative IgG antibody of this group of pathogens indicates that the subject has not been infected or infected but has not produced antibodies; a negative IgM antibody of this group of pathogens indicates that the subject has no active infection, but does not exclude potential infections . If the IgG antibody of a certain pathogen is positive, it means that the subject has been infected by this pathogen in the past or has been vaccinated; if the IgM antibody of a certain pathogen is positive, it indicates that the subject has an active infection of this virus in the near future. Acute infection, higher risk.

At present, due to the many inspection items of TORCH, the cost and time-consuming of comprehensive inspection are relatively high, which brings difficulties to the popularization of TORCH screening.

Accordingly, in the field of pregnancy screening, there is a strong demand for low-cost and high-efficiency detection of TORCH infection.

Summary of the invention

In order to improve the current high cost and time-consuming problem of TORCH inspection. Based on the detection characteristics of TORCH infection, after research, the present invention proposes a new mixed detection mode.

In the first aspect, the present invention provides a kit including:

A capture mixture, which comprises at least two antigens selected from the group consisting of Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen; and

Labeled anti-human IgM antibody.

It should be noted that the kit of the present invention coats at least two antigens required for Torch detection on a solid-phase carrier, and also includes a labeled anti-human IgM antibody, so that it can simultaneously detect the antigens in Torch detection. Multiple IgM for mixed detection. In other words, using the kit of the present invention can finally obtain a mixed detection result, which can simultaneously evaluate the infection risk of multiple pathogenic microorganisms in TORCH, thereby improving the efficiency of screening. That is to say, when the subject is tested using the kit provided in the first aspect of the present invention, a mixed test result is obtained. If the mixed test result is negative, it means that the subject has no activity of at least two pathogenic microorganisms. Sexual infection; if the mixed test result is positive, it indicates that the subject has an active infection of at least one pathogenic microorganism in the near future, and it is recommended to further screen for the infection of each pathogenic microorganism separately. This can greatly improve the efficiency of preliminary screening.

In some embodiments, the at least two antigens are coated on the same solid phase carrier.

In other embodiments, the at least two antigens are respectively coated on different solid carriers.

In a specific embodiment, the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed so that, in the same reaction system, each coating is used in the solid phase. When the antigen on the carrier and the anti-human IgM antibody single internal reference (for example, the internal reference defined in the ISO 1183-1:2004 standard) are tested, the corresponding luminescence threshold is basically the same.

In an exemplary embodiment, the luminescence threshold is determined by an ROC curve.

In a specific embodiment, the capture mixture comprises Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus type 1/2 antigen coated on a solid phase carrier.

In a specific embodiment, the capture mixture comprises Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, and parvovirus B19 antigen coated on a solid phase carrier.

In a specific embodiment, the capture mixture comprises parvovirus B19 antigen, Coxsackie virus antigen, and herpes zoster virus antigen coated on a solid phase carrier.

In a specific embodiment, the capture mixture comprises Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, and Coxsackie virus antigen coated on a solid phase carrier. And herpes zoster virus antigen.

The kit of the present invention may further include instructions, which record that when the corresponding pathogen is detected using at least two antigens on the capture mixture and the labeled anti-human IgM antibody, the combined detection value and luminescence The case where the ratio of the threshold value is greater than or equal to 1.1 is determined as a positive result.

In a specific embodiment, the instructions further state that when a positive result is determined, it is recommended that the pathogen corresponding to each of the at least two antigens be further examined.

In the second aspect, the present invention provides an immunoassay method, including the following steps:

The sample to be tested is coated with at least two of the toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen, and herpes zoster virus antigen. Mixing and incubating a solid-phase carrier of three antigens for a period of time, so that the at least two antigens coated on the solid-phase carrier can bind to the IgM antibodies corresponding to the at least two antigens in the sample to be tested;

Washing the mixture of the sample to be tested and the solid phase carrier to remove unbound substances;

Add the labeled anti-human IgM antibody to the washed mixture and incubate, so that the labeled anti-human IgM antibody can bind to the at least two antigens on the solid phase carrier The corresponding IgM antibody binds to form a complex;

Cleaning the complex to remove unbound substances;

A luminescent substrate is added to the washed complex to detect the mixed detection value of IgM antibodies corresponding to the at least two antigens in the sample to be tested.

In some embodiments, the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed so that, in the same reaction system, each coating on the solid-phase carrier is used. When the above antigen and the anti-human IgM antibody are tested on the internal reference product alone, the corresponding luminescence threshold is basically the same.

In some embodiments, the at least two antigens coated on the solid-phase carrier are added to the sample to be tested, mixed and incubated, or added to the sample to be tested in a pre-mixed form Mix and incubate.

In a specific embodiment, the at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus type 1/2 antigen.

In a specific embodiment, the at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, and parvovirus B19 antigen.

In a specific embodiment, the at least two antigens are parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen.

In a specific embodiment, the at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen and herpes zoster virus antigen .

In a specific embodiment, when it is judged as a positive result, the method further includes the step of individually detecting the pathogen corresponding to each of the at least two antigens.

In the third aspect, the present invention provides a sample analyzer that can selectively detect different types of IgM antibodies in blood samples, including:

The sample device has a sample storage component and a sample dispensing component, the sample storage component is used to store the sample to be tested, and the sample dispensing component is used to suck the sample to be tested and discharge it into the reaction cup to be added;

The reagent device has a reagent storage component and a reagent dispensing component. The reagent storage component is used to store a reagent kit. The reagent kit includes a solid-phase reagent and a labeling reagent. The solid-phase reagent includes a coating with Toxoplasma gondii antigen and rubella. A solid phase component of at least two antigens among viral antigens, cytomegalovirus antigens, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen and herpes zoster virus antigen, and the labeling reagent comprises A labeled anti-human IgM antibody, the reagent dispensing part is used for sucking the solid phase components and labeling reagents in the kit stored on the reagent storage part and discharging them into the reaction cup where the reagent is to be added;

The luminescent substrate dispensing device is connected to the container storing the luminescent substrate and is used to inject the luminescent substrate into the reaction cup where the luminescent substrate is to be added;

The reaction device has a plurality of placement positions for placing the reaction cup and is used for incubating the reaction solution in the reaction cup;

The optical measurement component is used to perform optical measurement on the reaction solution after the incubation to obtain the detection result of the sample to be tested;

The control device is electrically connected to the sample device, the reagent device, the luminescent substrate dispensing device and the light measuring component, and is configured to:

Receiving a test instruction, where the test instruction includes the type of IgM antibody to be tested;

In response to the test instruction:

Controlling the sample dispensing component to add the sample to be tested in the sample storage component to the reaction cup on the reaction device;

Control the reagent dispensing component to add the solid phase component corresponding to the type of IgM antibody to be tested into the reaction cup on the reaction device, so that the sample to be tested and the solid phase component are in the Mixing in the reaction cup and incubating for a period of time, so that the antigen coated on the solid phase component can bind to the test IgM antibody in the test sample;

The reagent dispensing component is controlled to further add a labeling reagent to the reaction cup, so that the labeling reagent is mixed with the mixture in the reaction cup and incubated for a period of time, so that the labeling reagent can be combined with the solid phase component Binding to the IgM antibody to be tested;

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

The detection result is obtained according to the ratio of the luminescence value and the luminescence threshold value measured in the light measuring component.

In some embodiments, the at least two antigens coated on the solid phase component are present in the kit in the form of separate aliquots, or in a pre-mixed form in the kit.

In some embodiments, the kit is the kit provided in the first aspect of the present invention.

In the fourth aspect, the use of the solid phase reagent and the labeling reagent of the present invention in preparing a kit for Torch detection is provided.

Using the above scheme to implement mixed detection of multiple IgM in the sample to be tested, the present invention realizes the evaluation of multiple infection risks based on the obtained one detection result, thereby greatly improving the efficiency of Torch detection and evaluation, shortening the average detection time, and thereby reducing The cost of testing is conducive to the promotion of Torch screening. On the other hand, the embodiments of the present invention can selectively screen the items involved in the Torch detection as required, which further improves the scope and flexibility of the detection.

Description of the drawings

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

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

Detailed ways

Hereinafter, the present invention will be described in detail with reference to specific implementations and examples, and the advantages and various effects of the present invention will be presented more clearly. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present invention, but not to limit the present invention.

Throughout the specification, unless otherwise specified, the terms used herein should be understood as the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present invention belongs. If there is a conflict, this manual takes precedence.

As mentioned above, in view of the current Torch detection items and high cost, the present invention provides a multiple IgM antibody evaluation method based on capture mixture, which can realize multiple infection risks based on only one mixed detection result. The evaluation has improved the efficiency of screening. For example, when the result of the method of the present invention is negative, it indicates that all the pathogen IgM items of the mixed test are negative and the risk is low; when the result is positive, it indicates that at least one of the pathogen IgM items of the mixed test is positive, and it is recommended to proceed further an examination. Accordingly, the screening efficiency of Torch detection is improved and the detection time is shortened, thereby solving the problems of high cost of Torch detection and limited promotion.

In the embodiment of the present invention, the terms "solid support", "solid support", "solid support" and "solid support" can be used interchangeably, which refers to a solid surface to which antigens or antibodies can be attached. There is no particular limitation on the solid-phase carrier used in the present invention, and commercial solid-phase carriers and any solid-phase carrier that can be used in immunoassays can be used in the present invention. Exemplary solid phase carriers can be magnetic beads (such as carboxyl magnetic beads), enzyme-labeled plates, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates, or micro Chip, but the present invention is not limited to this.

In the embodiment of the present invention, the term "capture mixture" means that it contains at least two antigens coated on a solid-phase carrier, and the at least two antigens coated on the solid-phase carrier are present in the mixed form. In the kit.

In the embodiment of the present invention, at least two antigens can be coated on a solid-phase carrier in the following manner: on the one hand, each antigen can be separately coated on a different solid-phase carrier, and then each antigen coated The solid phase carrier is mixed. For example, the parvovirus B19 antigen, the Coxsackie virus antigen, and the herpes zoster virus antigen can be separately coated on a solid phase carrier, and then mixed together. On the other hand, at least two antigens can also be divided into one or more groups, each group contains one or more antigens, each group of antigens are respectively coated with a different solid phase carrier, and then the coating It is mixed with a solid-phase carrier with antigen, for example, herpes simplex virus type 1/2 antigen is usually coated on the same solid-phase carrier during the coating process.

The markers that can be used in the embodiments of the present invention are well known to those skilled in the art, such as alkaline phosphatase (ALP), peroxidase, microperoxidase, horseradish peroxidase, β-half Enzymes such as lactosidase, glucose oxidase and glucose 6-phosphate dehydrogenase; fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, fluorescein, rhodamine, europium and green fluorescent protein, etc. Fluorescent substances; chemiluminescent substances such as luminol, isoluminol, phenanthridinium and acridinium esters; coenzymes such as NAD; biotin; radioactive substances such ^{as 35} S, ¹⁴ C, ³² P, ¹³¹ I and ^{125 I} , But the present invention is not limited to this.

Those skilled in the art can select a suitable luminescent substrate according to the type of label used to generate a detectable signal. For example, when alkaline phosphatase is used as a label, 3-(2-helicaladamantane)-4-methoxy-4-(3-phosphooxy)-phenyl-1,2-dioxide can be used. Cycloethane is used as a luminescent substrate. The substrate will be decomposed by alkaline phosphatase to remove a phosphate group and generate an unstable intermediate product. The intermediate product generates methyl meta-oxybenzoate anion through intramolecular electron transfer. When the methyl isobenzoate anion in the excited state returns to the ground state from the excited state, chemiluminescence is generated. The number of photons generated in the reaction is measured by a photomultiplier tube, and the amount of photons generated is proportional to the content of the detected substance in the sample.

The embodiments of the present invention are applicable to various methods such as ELISA, chemiluminescence, electrochemiluminescence, POCT, immunochromatography, up-conversion luminescence, down-conversion luminescence, etc. The antigen used is a recombinant antigen or a natural antigen.

In the scope of the present invention, the term ROC (receiver operating characteristic) curve refers to the curve obtained by dividing the diagnostic test result into several critical points, with the sensitivity corresponding to each critical point as the ordinate and the specificity as the abscissa. . ROC curve is an effective tool for comprehensive and accurate evaluation of diagnostic tests. Another function of the ROC curve is to determine the optimal threshold for detection. ROC curve method to determine the critical point In most cases, select the point on the curve as close to the upper left as possible to determine the critical point as the best. In application, according to the ROC curve, combined with the sensitivity and specificity results of each tangent point, select the tangent point on the curve that is as close as possible to the maximum Youden index on the upper left side as the best critical point, so as to make the test sensitivity and specificity The degree of diagnosis is relatively high, and the rate of misdiagnosis and missed diagnosis is relatively small.

Adopting the detection method of the embodiment of the present invention allows a personalized Torch screening scheme to be formulated for the individual to be tested, thereby providing a more flexible screening method. For example, when the individual to be tested needs to be screened routinely, a solid phase carrier coated with Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus type 1/2 antigen can be used; For a comprehensive test, you can use solids coated with Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, Coxsackie virus antigen, and herpes zoster virus antigen. Phase carrier; For example, when the individual to be tested has previously received one or more of the Torch tests, a solid carrier coated with the antigens corresponding to the remaining items can be used, which can flexibly provide the test subject Personalized item selection, which broadens the scope of application during testing.

When used to prepare a solid phase reagent, the antigen of the embodiment of the present invention may exist in the form of, for example, a multimer, a recombinant antigen, an antigen fragment, or an antigen peptide.

When used to prepare solid phase reagents and/or labeling reagents. The antibodies of the embodiments of the present invention may exist in the form of monoclonal antibodies, polyclonal antibodies, recombinant antibodies, chimeric antibodies, humanized antibodies, and antigen-binding fragments of antibodies, for example.

In the embodiment of the present invention, "mixed detection value", "mixed detection result" and "one detection result" can be used interchangeably, and refer to the detection result obtained by using the kit, method and system of the present invention. For example, when alkaline phosphatase is used as a marker, the detection result is a luminescence value.

In the embodiment of the present invention, the ratio of the mixed detection value to the luminescence threshold (COI value) is used to determine whether the result is positive or negative. For example, when the ratio is greater than or equal to 1.1, the judgment result is positive, indicating that the IgM antibody test result corresponding to at least one pathogen is positive. When the ratio is between 0.9 and 1.1, the judgment result is a gray area, which is neither positive or negative; when the ratio is less than 0.9, the judgment result is negative, indicating that the IgM antibodies for the item are all negative.

In the embodiment of the present invention, the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed so that each coating on the solid-phase carrier is used in the same reaction system. When the antigen on the carrier and the anti-human IgM antibody individually detect the internal reference, the corresponding luminescence threshold is basically the same.

In the embodiment of the present invention, "internal reference" refers to the standard and basis for determining the composition of the reaction system, which has the definition as defined in the international standard ISO 18113-1:2009 and can be obtained according to the standard. Internal reference materials are samples used by medical device manufacturers to verify product performance. They are the most important criteria and basis for product selection, preparation, identification, and determination of raw material quality standards, product production process determination, reaction system composition, reaction conditions, etc. . For qualitative items, the identification sample of the internal reference product of the enterprise is a sample that judges the value or quantity of a specific disease, state, or the boundary between the existence and nonexistence of the measurement.

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

The concentration of the labeled anti-human IgM antibody is not particularly limited in the present invention, and an exemplary concentration may be about 0.5 μg/mL to about 5 μg/mL.

In the embodiment of the present invention, the solid phase carrier coated with Toxoplasma gondii antigen, the solid phase carrier coated with rubella virus antigen, the solid phase carrier coated with cytomegalovirus antigen, and the herpes simplex virus 1/ The concentration of the solid phase carrier of the type 2 antigen, the solid phase carrier coated with the parvovirus B19 antigen, and the solid phase carrier coated with the Coxsackie virus antigen may be, for example, 0.1 to 0.3 mg/mL.

In the embodiment of the present invention, the concentration of the solid phase carrier coated with the herpes zoster virus antigen may be, for example, 0.1 to 0.4 mg/mL.

In an exemplary embodiment, the concentration of the labeled anti-human IgM antibody is about 1.0 μg/mL, and the luminescence threshold is adjusted to 30,000, then the solid phase carrier (such as magnetic microspheres) coated with the Toxoplasma antigen, Solid-phase carrier coated with rubella virus antigen, solid-phase carrier coated with cytomegalovirus antigen, solid-phase carrier coated with herpes simplex virus type 1/2 antigen, solid-phase carrier coated with parvovirus B19 antigen The concentration of the solid-phase carrier coated with the herpes zoster virus antigen and the solid-phase carrier coated with the Coxsackie virus antigen are respectively: 0.15mg/mL, 0.15mg/mL, 0.15mg/mL, 0.12 mg/mL, 0.2mg/mL, 0.15mg/mL, 0.12mg/mL.

In the present invention, the labeled anti-human IgM antibody can be derived from mice, rabbits, goats, sheep, and chickens, but the present invention is not limited thereto.

As shown in FIG. 1, the embodiment of the present invention provides an immunoassay instrument that can selectively detect the type of IgM antibody in a blood sample. The immunoassay analyzer includes a sample device 10, a reagent device 20, a reaction device 30, a light measuring component 40, and a control device 50. The immune analyzer may also include a display part (not shown).

The sample device 10 is used to carry the sample to be tested, aspirate the sample and provide it to the reaction device 30. The sample device 10 includes a sample storage part 11 and a sample dispensing part 12. The sample storage component 11 is used to store samples to be tested. In some embodiments, the sample storage component 11 may include a sample distribution module (SDM, Sample Delivery Module) and a front-end track. In other embodiments, the sample storage component 11 may also be a sample tray. The sample tray includes a plurality of sample positions such as sample tubes. By rotating the tray structure of the sample tray, the sample can be scheduled to the corresponding position, for example, for The position where the sample dispensing part 12 sucks the sample. The sample dispensing component 12 is used to aspirate the sample and discharge it into the reaction cup to be added. The sample dispensing component 12 may include, for example, a sample needle. The sample needle performs a two-dimensional or three-dimensional movement in space through a two-dimensional or three-dimensional drive mechanism, so that the sample needle can move to aspirate the sample carried by the sample storage component 11, and Move to the reaction cup to be added, and discharge the sample into the reaction cup.

The reagent device 20 is used to carry reagents, and the reagents are sucked and supplied to the reaction device 30. The reagent device 20 includes a reagent storage part 13 and a reagent dispensing part 14. The reagent storage part 13 is used to store a reagent cartridge. In some embodiments, the reagent storage component 13 may be a reagent tray. The reagent tray is arranged in a disc-shaped structure and has multiple positions for carrying reagent containers. The reagent storage component 13 can rotate and drive the reagent container it carries to rotate. It is used to rotate the reagent container to a specific position, for example, a position where the reagent is sucked by the reagent dispensing part 14. The number of reagent storage parts 13 may be one or more. The reagent dispensing part 14 is used for sucking the reagents in the reagent box and discharging them into the reaction cup to be added with the reagents. In some embodiments, the reagent dispensing part 14 may include a reagent needle, and the reagent needle can move in a two-dimensional or three-dimensional space through a two-dimensional or three-dimensional drive mechanism, so that the reagent needle can move to absorb the reagent storage part 13 The loaded reagent, and move to the reaction cup where the reagent is to be added, and discharge the reagent into the reaction cup.

Wherein, the reagent storage part 13 is used to store a reagent kit, which includes a solid-phase reagent and a labeling reagent, and the solid-phase reagent includes the coating with Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, and herpes simplex virus 1/ A solid phase component of at least two antigens of type 2 antigen, parvovirus B19 antigen, Coxsackie virus antigen, and herpes zoster virus antigen, and the labeling reagent includes an anti-human IgM antibody with a label.

The reaction device 30 has at least one placement position for placing the reaction cup and incubating the reaction solution in the reaction cup. For example, the reaction device 30 may be a reaction disc, which is arranged in a disc-shaped structure and has one or more placement positions for placing reaction cups. The reaction disc can rotate and drive the reaction cup in its placement position to rotate for The reaction cup is arranged in the reaction tray and the reaction solution in the incubation reaction cup is incubated.

The optical measurement component 40 is used to perform optical measurement on the reaction solution after the incubation to obtain reaction data of the sample. For example, the light measuring component 40 detects the luminous intensity of the reaction solution to be measured, and calculates the concentration of the component to be measured in the sample through a calibration curve. In some embodiments, the optical sensing component 40 is separately arranged outside the reaction device 30.

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

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. Among them, the processing component 51, RAM 52, ROM 53, communication interface 54, memory 56 and the I/O interface 55 communicate through the bus 57.

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

The memory 56 is loaded with various computer programs such as an operating system and application programs for the processor component 51 to execute, and data required to execute the computer programs. In addition, during the sample detection process, any data that needs to be stored locally can be stored in the memory 56.

The I/O interface 55 is composed of a serial interface such as USB, IEEE1394 or RS-232C, a parallel interface such as SCSI, IDE or IEEE1284, and an analog signal interface composed of a D/A converter and an A/D converter. An input device composed of a keyboard, a mouse, a touch screen or other control buttons is connected to the I/O interface 55, and the user can use the input device to directly input data to the control device 50. In addition, the I/O interface 55 can also be connected to a display with display function, such as: LCD screen, touch screen, LED display screen, etc., and the control device 50 can output the processed data as image display data to the display for display, for example : Analyze data, instrument operating parameters, etc.

The communication interface 54 is an interface that can be any currently known communication protocol. The communication interface 54 communicates with the outside world through the network. The control device 50 can transmit data to any device connected through the network through the communication interface 54 in a certain communication protocol.

Wherein, the control device 50 is configured to receive a test instruction, the test instruction includes the type of the IgM antibody to be tested, and the following steps are executed in response to the test instruction:

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

The control reagent dispensing component 14 adds the solid phase component corresponding to the type of IgM antibody to be tested from the reagent kit stored in the reagent storage component 13 into the reaction cup on the reaction device 30, so that the to-be-tested The sample and the solid phase component are mixed in the reaction cup and incubated for a period of time, so that the antigen coated on the solid phase component can bind to the test IgM antibody in the test sample;

The control reagent dispensing component 14 further adds the labeled reagent in the kit to the reaction cup, so that the labeled reagent is mixed with the mixture in the reaction cup and incubated for a period of time, so that the labeled reagent can be combined with the solid Binding of the IgM antibody to be tested bound on the phase component;

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

The detection result is obtained according to the ratio of the luminescence value measured in the light measuring component 40 to the luminescence threshold value.

The immune analyzer provided by the embodiment of the present invention can selectively detect any one or more of the Torch detection items in one detection, which improves the flexibility of Torch detection and meets the needs of users in different scenarios.

In some embodiments, in the solid phase reagent, at least two antigens coated on the solid phase component are present in the kit in the form of separate aliquots. For example, the solid phase reagent includes a solid phase component coated with a Toxoplasma antigen and a solid phase component coated with a rubella virus antigen, which are separated from each other. In this case, the user can use the immune analyzer to perform preliminary screening of multiple items in the Torch test item in one test, or use the immune analyzer to screen each item of the Torch test item one by one. The user needs to input the items that need to be detected. The immunoassay analyzer can add the solid phase components corresponding to the items to be tested and the labeling reagents to the sample to be tested according to the user's instructions.

When the solid-phase reagent contains at least two antigens coated on the solid-phase component separately, the control device 50 is configured to control the reagent dispensing part 14 to separate different solid-phase components in the solid-phase reagent. Add to the reaction cup.

In some embodiments, in the solid phase reagent, at least two antigens coated on the solid phase component are present in the kit in a pre-mixed form. The kit is, for example, the above-mentioned kit according to the present invention.

Experimental Materials:

Toxoplasma anti-antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1 antigen, and herpes simplex virus type 2 antigen are from Meridian Life Science;

Alkaline phosphatase comes from Roche Pharmaceuticals;

The magnetic beads come from Thermo Fisher;

Human IgM antibodies specifically bind antibodies from Jackson ImmunoResearch.

Preparation of solid phase coating:

First, the antigen is pre-treated, and the protective components in the buffer matrix are removed by dialysis. The coating is carried out at a ratio of 0.5-40ug (preferably 1-30ug, more preferably 10-20ug) of antigen added per mg of magnetic beads. During the reaction, the carboxyl groups on the surface of the magnetic beads are coupled with the amino groups of the antigen under the catalysis of EDC/NHS. In the embodiment of the present invention, 20 mg of magnetic microspheres modified with carboxyl groups on the surface were taken, dispersed in 10 mM MES buffer by ultrasonic, 80 mg EDC and 120 mg NHS were added, and after ultrasonic mixing, they were placed on a shaker at 37°C for 15 minutes. Then add the antigen to the processed magnetic beads according to the proportion, mix them, and place them on a shaker at 37°C for 10-18 hours. After washing and sealing, magnetic microspheres coated with antigen are prepared.

Preparation of labeling reagent :

The mouse, rabbit, goat, sheep, chicken and other antibodies that specifically bind to human IgM antibodies are labeled with signal markers. In the embodiment of the present invention, the signal marker is alkaline phosphatase. The alkaline phosphatase was diluted with 50mM MES (2-morpholineethanesulfonic acid) pH=6.0 buffer. The concentration of the signal marker after dilution is in the range of about 0.5 μg/mL to about 5 μg/mL. In the embodiment of the present invention, the concentration of the signal marker after dilution is about 1 μg/mL, and the labeling reagent is prepared.

Detection steps:

In the first step, the sample and solid-phase coating are added to the reaction tube, and incubated at 37°C for 10 minutes, so that the solid-phase coating can bind to the corresponding IgM antibody in the sample. After the incubation in the reaction tube is completed, the substance bound to the solid phase will be placed in a magnetic field and be attracted, the substance bound to the solid phase of the magnetic beads is retained, and the unbound substance is washed and removed.

In the second step, add the labeling reagent to the reaction tube, mix it, and incubate at 37°C for 10 minutes to combine with the conjugate formed in the first step to form a complex. After the incubation in the reaction tube is completed, the complex is attracted by the magnetic field, and other unbound substances are washed and removed.

The third step is to add AMPPD to the reaction tube to generate chemiluminescence. The number of photons produced by the reaction is measured by a photomultiplier tube to obtain the chemiluminescence signal value of the sample.

In the embodiment of the present invention, the negative coincidence rate refers to the ratio of the number of samples judged to be negative using the test method of the embodiment of the present invention to the negative samples actually participating in the evaluation, and the positive coincidence rate refers to the test using the embodiment of the present invention The method obtains the proportion of the number of positive samples judged to be positive to the positive samples actually participating in the evaluation; the true negative and positive results of the samples come from the diagnosis results of the hospital.

Embodiment 1 Determining the detection threshold

Select samples with clear clinical diagnosis results, including 1153 samples for toxoplasma IgM antibody testing (852 negative samples, 301 positive samples), 1123 samples for rubella virus IgM antibody testing (843 negative samples, 280 positive samples), 1422 cases of cytomegalovirus IgM antibody test samples (1031 cases of negative samples, 391 cases of positive samples), 1152 cases of IgM antibody test samples of herpes simplex virus type 1/2 (842 cases of negative samples, 310 cases of positive samples), parvovirus B19IgM antibody 1217 cases of test samples (861 cases of negative samples, 356 cases of positive samples), 1,050 cases of IgM antibody test samples of herpes zoster virus (801 cases of negative samples, 249 cases of positive samples), 1,050 cases of IgM antibody test samples of Coxsackie virus (negative samples) 798 samples and 252 positive samples).

The above-mentioned "solid phase coating preparation" method was used to prepare magnetic microspheres coated with Toxoplasma antigen, magnetic microspheres coated with rubella virus antigen, magnetic microspheres coated with cytomegalovirus antigen, and magnetic microspheres coated with cytomegalovirus antigen. Herpes simplex virus type 1/2 antigen (HSV-1 and HSV-2 mixed at 1:1) magnetic microspheres, magnetic microspheres coated with parvovirus B19 antigen, magnetic microspheres coated with herpes zoster virus antigen Balls, magnetic microspheres coated with Coxsackie virus antigens, and labeling reagents prepared by the above-mentioned "preparation of labeling reagents" method, and testing the positive and negative coincidence rates of each pathogen sample at different luminescence thresholds according to the "detection steps" Compliance rate, the results are shown in Table 1 below.

Table 1. Determination of the threshold of each pathogen

It can be seen from Table 1 that when the luminescence threshold is selected to be about 30,000, the overall coincidence rate is the highest, and the luminescence threshold of the kit is selected as 30,000.

Example 2 Preparation of reagent combinations for different test items

In this example, a combination of reagents used in the experiment shown in Table 2 was prepared.

Table 2

Among them, the labeling reagents in Experiments 1 to 4 were prepared according to the method in "Preparation of Labeling Reagents".

The preparation steps of each solid phase reagent in experiments 1 to 4 are as follows:

First, prepare magnetic microspheres coated with Toxoplasma antigen, magnetic microspheres coated with rubella virus antigen, magnetic microspheres coated with cytomegalovirus antigen, and coated magnetic beads according to "Preparation of magnetic bead coating". Magnetic microspheres with herpes simplex virus type 1 antigen, herpes simplex virus type 2 antigen (1:1), magnetic microspheres coated with parvovirus B19 antigen, magnetic microspheres and coatings coated with herpes zoster virus antigen Concentration of magnetic beads with magnetic microspheres with Coxsackie virus antigen. Choose 50mM Tris pH7.4 buffer solution to dilute the above magnetic microspheres respectively, keep the concentration of the labeling reagent at about 1ug/mL, adjust the concentration of the magnetic microsphere coating, adjust the luminescence threshold to 30,000, allow ±2% deviation, The results are shown in Table 3.

Table 3 Determination of the concentration of each magnetic bead coating in experiments 1 to 4

As shown in Table 3, magnetic microspheres coated with toxoplasma antigen, magnetic microspheres coated with rubella virus antigen, magnetic microspheres coated with cytomegalovirus antigen, and herpes simplex virus type 1/2 The magnetic bead concentrations of the antigen magnetic microspheres, the magnetic microspheres coated with parvovirus B19 antigen, the magnetic microspheres coated with the herpes zoster virus antigen, and the magnetic microspheres coated with the Coxsackie virus antigen are respectively: 0.15mg/mL, 0.15mg/mL, 0.15mg/mL, 0.12mg/mL, 0.2mg/mL, 0.15mg/mL, 0.12mg/mL.

According to the concentration of each magnetic bead coating determined in Table 3, the corresponding magnetic bead coatings were mixed according to the items in Experiments 1 to 4, and the solid phase reagents in Experiments 1 to 4 were prepared respectively.

Example 3 Test using the reagent combination in experiments 1 to 4

Select samples with clear clinical diagnosis results, a total of 3315 cases. Among them, 1952 were negative samples and 1363 were positive samples. Among the positive samples, there were 970 positive samples of any one of Toxoplasma gondii IgM antibodies, rubella virus IgM antibodies, cytomegalovirus IgM antibodies, and herpes simplex virus type 1/2 IgM antibodies. There were 120 samples with positive parvovirus B19IgM antibody, 138 samples with positive IgM antibody against herpes zoster virus, and 135 samples with positive IgM antibody against Coxsackie virus.

Using the reagent combinations of Experiments 1 to 4 prepared in Example 2, the above-mentioned samples were tested using the immunoanalyzer of the present invention according to the method in the "detection step" above.

In the embodiment of the present invention, COI (Cutoff index) is the ratio of the chemiluminescence signal value (RLU) of the measured sample to the threshold value (Cutoff value). For qualitative detection methods, the threshold value (cutoff value) is the judgment that the test result is positive Or negative cutoff value. In the embodiment of the present invention, whether the sample contains a test substance, the COI value of the test result of the sample needs to be compared with a reference value (reference value 1.10). If it is greater than or equal to 1.10, it means one or more test substances in the sample. Species are positive; if it is less than 0.90, it means that the test substances in the sample are all negative. The COI is between 0.90-1.10, and the result is a gray zone (indeterminate).

The positive and negative results of the test were compared with the clinical diagnosis results, and the coincidence rate was calculated. The results are shown in Table 4 below.

Table 4. Sensitivity and specificity of experiments 1～4

As shown in Table 4, the overall coincidence rate of experiment 1 (detection of Toxoplasma antibody IgM+rubella virus antibody IgM+cytomegalovirus antibody IgM+herpes simplex virus type 1/2) was 99.49%; experiment 2 (detection of Toxoplasma gondii antibody IgM+rubella virus antibody IgM+ The overall coincidence rate of cytomegalovirus antibody IgM + herpes simplex virus type 1/2 + parvovirus B19IgM antibody) is 99.51%; experiment 3 (detection of parvovirus B19IgM + herpes zoster virus IgM antibody + Coxsackie virus IgM antibody) The rate was 99.74%; Experiment 4 (detection of Toxoplasma gondii antibody IgM+rubella virus antibody IgM+cytomegalovirus antibody IgM+herpes simplex virus type 1/2 IgM antibody+parvovirus B19IgM antibody+herpes zoster virus IgM antibody+coxsackie virus IgM antibody The overall coincidence rate of) is 99.52%. This can meet the needs for rapid and effective Torch screening.

Claims (19)

1. A kit including:

   The capture mixture, which is mixed with at least two antigens selected from the following items coated on a solid carrier: Toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, and parvovirus B19 antigen , Coxsackie virus antigen and herpes zoster virus antigen; and

   Labeled anti-human IgM antibody.
2. The kit according to claim 1, wherein the at least two antigens are coated on the same solid-phase carrier or separately coated on different solid-phase carriers.
3. The kit according to claim 1 or 2, wherein the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed so that in the same reaction system When each antigen coated on the solid phase carrier and the anti-human IgM antibody are used to detect the internal reference separately, the corresponding luminescence threshold is basically the same.
4. The kit according to claim 3, wherein the luminescence threshold is determined by an ROC curve.
5. The kit according to any one of claims 1 to 4, wherein the capture mixture comprises Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus 1/2 coated on a solid phase carrier Type antigen.
6. The kit according to any one of claims 1 to 4, wherein the capture mixture comprises toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus 1/2 Type antigen and parvovirus B19 antigen.
7. The kit according to any one of claims 1 to 4, wherein the capture mixture comprises parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen coated on a solid phase carrier.
8. The kit according to any one of claims 1 to 4, wherein the capture mixture comprises toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus 1/2 Type antigen, parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen.
9. The kit according to any one of claims 1 to 8, further comprising:

   The specification states that when at least two antigens on the capture mixture and the labeled anti-human IgM antibody are used to detect the corresponding pathogen, the ratio of the mixed detection value to the luminescence threshold value is greater than or equal to 1.1. It is a positive result.
10. The kit according to claim 9, wherein the instructions further state that when a positive result is determined, it is recommended that the pathogen corresponding to each of the at least two antigens be further examined.
11. An immunoassay method includes the following steps:

    The sample to be tested is coated with at least two of the toxoplasma antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen, and herpes zoster virus antigen. Mixing and incubating a solid-phase carrier of three antigens for a period of time, so that the at least two antigens coated on the solid-phase carrier can bind to the IgM antibodies corresponding to the at least two antigens in the sample to be tested;

    Washing the mixture of the sample to be tested and the solid phase carrier to remove unbound substances;

    Add the labeled anti-human IgM antibody to the washed mixture and incubate, so that the labeled anti-human IgM antibody can bind to the at least two antigens on the solid phase carrier The corresponding IgM antibody binds to form a complex;

    Cleaning the complex to remove unbound substances;

    A luminescent substrate is added to the washed complex to detect the mixed detection value of IgM antibodies corresponding to the at least two antigens in the sample to be tested.
12. The method according to claim 11, wherein the concentration of the labeled anti-human IgM antibody and the concentration of each antigen coated on the solid-phase carrier are designed such that each is used in the same reaction system. When the antigens coated on the solid phase carrier and the anti-human IgM antibody individually detect the internal reference, the corresponding luminescence thresholds are basically the same.
13. The method according to claim 11 or 12, wherein the at least two antigens coated on a solid phase carrier are added to the test sample to be mixed and incubated, or added in a pre-mixed form Mix and incubate the sample to be tested.
14. The method according to any one of claims 11 to 13, wherein the at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen and herpes simplex virus type 1/2 antigen; or

    The at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen and parvovirus B19 antigen; or

    The at least two antigens are parvovirus B19 antigen, Coxsackie virus antigen and herpes zoster virus antigen; or

    The at least two antigens are Toxoplasma gondii antigen, rubella virus antigen, cytomegalovirus antigen, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen and herpes zoster virus antigen.
15. The method according to any one of claims 11 to 14, wherein a positive result is determined when the ratio of the mixed detection value to the threshold value is greater than or equal to 1.1.
16. The method according to claim 15, wherein, when the result is judged as a positive result, the method further comprises the step of individually detecting the pathogen corresponding to each of the at least two antigens.
17. An immune analyzer that can selectively detect different types of IgM antibodies in blood samples, including:

    The sample device has a sample storage component and a sample dispensing component, the sample storage component is used to store the sample to be tested, and the sample dispensing component is used to suck the sample to be tested and discharge it into the reaction cup to be added;

    The reagent device has a reagent storage component and a reagent dispensing component. The reagent storage component is used to store a reagent kit. The reagent kit includes a solid-phase reagent and a labeling reagent. The solid-phase reagent includes a coating with Toxoplasma gondii antigen and rubella. A solid phase component of at least two antigens among viral antigens, cytomegalovirus antigens, herpes simplex virus type 1/2 antigen, parvovirus B19 antigen, coxsackie virus antigen and herpes zoster virus antigen, and the labeling reagent comprises An anti-human IgM antibody with a label, the reagent dispensing part is used for sucking the solid phase reagent and the labeling reagent in the kit stored on the reagent storage part and discharging them into the reaction cup where the reagent is to be added;

    The luminescent substrate dispensing device is connected to the container storing the luminescent substrate and is used to inject the luminescent substrate into the reaction cup where the luminescent substrate is to be added;

    The reaction device has a plurality of placement positions for placing the reaction cup and is used for incubating the reaction solution in the reaction cup;

    The optical measurement component is used to perform optical measurement on the reaction solution after the incubation to obtain the detection result of the sample to be tested;

    The control device is electrically connected to the sample device, the reagent device, the luminescent substrate dispensing device and the light measuring component, and is configured to:

    Receiving a test instruction, where the test instruction includes the type of IgM antibody to be tested;

    In response to the test instruction:

    Controlling the sample dispensing component to add the sample to be tested in the sample storage component to the reaction cup on the reaction device;

    Control the reagent dispensing component to add the solid phase component corresponding to the type of IgM antibody to be tested into the reaction cup on the reaction device, so that the sample to be tested and the solid phase component are in the Mixing in the reaction cup and incubating for a period of time, so that the antigen coated on the solid phase component can bind to the test IgM antibody in the test sample;

    The reagent dispensing component is controlled to further add a labeling reagent to the reaction cup, so that the labeling reagent is mixed with the mixture in the reaction cup and incubated for a period of time, so that the labeling reagent can be combined with the solid phase component Binding to the IgM antibody to be tested;

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

    The detection result is obtained according to the ratio of the luminescence value measured in the light measuring component to the luminescence threshold value.
18. The immunoassay analyzer according to claim 17, wherein the at least two antigens coated on the solid phase component are present in the kit in the form of separate packaging.
19. The immune analyzer according to claim 17, wherein the kit is the kit according to any one of claims 1 to 10.

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