Chemiluminescence immunoassay for quantitative detection of antibody against porcine reproductive and respiratory syndrome virus

Abstract

The present application relates to a kind of porcine reproductive and respiratory syndrome antibody chemiluminescence immunoassay quantitative detection method, for the immune quantitative detection of porcine reproductive and respiratory syndrome antibody in ex vivo pig serum sample.The method prepares GP5-M capture phase, N protein capture phase, GP5-M background capture phase and N protein background capture phase;Ex vivo pig serum sample is mixed with sample diluent containing anti-pig rheumatoid factor antibody and pig immunoglobulin Fc fragment blocking protein, respectively incubated with each capture phase, and corresponding chemiluminescence labeled antigen is added;Collect each channel chemiluminescence signal, deduct corresponding background channel signal to obtain channel correction signal, and convert into GP5-M channel antibody value and N protein channel antibody value;When the ratio falls into the ratio interval determined by double positive calibrant, output quantitative result, not fall into when free antigen blocking retest is outputted.

Description

Technical Field

[0001] This invention relates to the field of viral antibody immunoquantitative detection technology, and in particular to a chemiluminescent immunoquantitative detection method for porcine reproductive and respiratory syndrome (PRRS) antibodies. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is a viral disease in pigs caused by infection with the porcine reproductive and respiratory syndrome virus (PRRSV). PRRS antibody detection is commonly used to evaluate the serological response status of pig herds after infection or immunization. Immunoassay based on antigen-antibody specific binding is a frequently used technique in PRRS antibody detection. Compared to conventional enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay typically captures the antibody on a solid-phase carrier and uses the chemiluminescent signal to quantify the antibody concentration, making it suitable for automated detection and quantitative analysis.

[0003] In the prior art, immunomagnetic microparticle chemiluminescence immunoassay kits for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies and their usage methods have been disclosed. For example, patent application CN108872602A discloses an immunomagnetic microparticle chemiluminescence immunoassay kit for quantitative detection of PRS antibodies. The kit includes magnetic microparticle reagents, enzyme-labeled antibodies, substrate solutions, washing solutions, standards, and diluents. The magnetic microparticle reagents are composed of magnetic bead suspensions conjugated with recombinant PRS virus capsid proteins and magnetic bead suspensions conjugated with recombinant PRS virus envelope glycoproteins in a specific ratio. In use, the magnetic microparticles coated with recombinant viral antigens capture PRS antibodies in the sample. A luminescent signal is then generated by the enzyme-labeled antibody and the chemiluminescent substrate, and the antibody titer in the test sample is calculated based on a standard curve established using the standards.

[0004] The above method can achieve chemiluminescent quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies. However, when actually detecting isolated porcine serum samples, the following technical problems may still exist: On the one hand, in addition to the target antibody, porcine serum samples may also contain rheumatoid factor-like interfering substances, immunoglobulin Fc fragment-related binding components, and other serum components that can non-specifically bind to solid-phase carriers, expression tags, or labeled antigens. Under the high-sensitivity reading conditions of chemiluminescence detection, such non-specific bindings can easily form background luminescence signals. On the other hand, existing magnetic particle chemiluminescence detection usually combines the reaction results corresponding to different viral antigens into a single luminescence result, making it difficult to distinguish the specific signals formed by different viral antigen reactions and the background signals caused by serum matrix, expression tags, or solid-phase carriers.

[0005] Furthermore, porcine reproductive and respiratory syndrome (PRRS) antibody test samples may exhibit differences in responses to different viral antigens under varying infection stages, immune backgrounds, and individual serum matrix conditions. When a single overall luminescence value is abnormally elevated or the contributions of different antigen responses are inconsistent, existing testing procedures typically rely on repeat testing, dilution testing, or quality control samples to determine the validity of the test batch. However, it is difficult to distinguish between specific antibody responses and non-specific background responses within the same testing procedure, and it is also difficult to conduct targeted verification of inconsistent quantitative results.

[0006] Therefore, the main technical problem that still needs to be solved in the existing chemiluminescent immunoassay methods for detecting porcine reproductive and respiratory syndrome (PRRS) antibodies is: how to distinguish between specific antibody reactions related to different antigens of PRRS virus and non-specific background signals caused by serum matrix or expression tags during the chemiluminescent immunoassay of isolated porcine serum samples, and how to perform targeted verification and correction when the quantitative results of different antigen reactions are inconsistent. Summary of the Invention

[0007] To overcome the aforementioned technical deficiencies, this invention discloses a chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies. This method can distinguish between PRRS virus-specific antibody reactions and non-specific background signals within the same detection process, and can perform antigen-blocking retesting when quantitative results from different antigen reactions are inconsistent. The method includes the following steps: GP5-M capture phase, N protein capture phase, GP5-M background capture phase and N protein background capture phase were prepared. The GP5-M capture phase was a magnetic solid-phase carrier with directional immobilization of porcine reproductive and respiratory syndrome virus (PRRSV) GP5-M conformational recombinant antigen. The N protein capture phase was a magnetic solid-phase carrier with immobilized PRRSV N protein recombinant antigen pre-adsorbed with porcine negative serum. Both the GP5-M background capture phase and the N protein background capture phase were magnetic solid-phase carriers with immobilized tag proteins without PRRSV antigenic epitopes. The isolated porcine serum sample was mixed with a sample diluent to obtain the test reaction solution. The sample diluent contained anti-porcine rheumatoid factor antibody and porcine immunoglobulin Fc fragment blocking protein. The test reaction solution was incubated with the GP5-M capture phase, the N protein capture phase, the GP5-M background capture phase and the N protein background capture phase, respectively, and then magnetically separated and washed. Chemiluminescently labeled GP5-M conformational recombinant antigen was added to the reaction systems corresponding to the GP5-M capture phase and the GP5-M background capture phase, and chemiluminescently labeled N protein recombinant antigen was added to the reaction systems corresponding to the N protein capture phase and the N protein background capture phase. After incubation, the mixture was magnetically separated and washed. Chemiluminescence signals of GP5-M channel, N protein channel, GP5-M background channel, and N protein background channel were collected separately. The GP5-M channel chemiluminescence signal was subtracted from the GP5-M background channel chemiluminescence signal to obtain the GP5-M channel correction signal, and the N protein channel chemiluminescence signal was subtracted from the N protein background channel chemiluminescence signal to obtain the N protein channel correction signal. Based on the calibration curve established using porcine reproductive and respiratory syndrome (PRRS) antibody calibrators, the GP5-M channel correction signal was converted into the GP5-M channel antibody value, and the N protein channel correction signal was converted into the N protein channel antibody value. When the ratio of the GP5-M channel antibody value to the N protein channel antibody value falls within the ratio range determined by the double-positive calibrator, the quantitative result of porcine reproductive and respiratory syndrome (PRRS) antibody determined by the GP5-M channel antibody value and the N protein channel antibody value is output; when the ratio does not fall within the ratio range, the test reaction solution is subjected to antigen blocking retesting using free GP5-M conformational recombinant antigen and free N protein recombinant antigen, and the quantitative result of PRRS antibody is output according to the channel antibody value that reaches the corresponding blocking decrease limit.

[0008] Preferably, the sample diluent also contains porcine negative serum protein pre-coated magnetic microparticles. After the isolated porcine serum sample is mixed with the sample diluent, it is first adsorbed by the porcine negative serum protein pre-coated magnetic microparticles, and then the supernatant is obtained by magnetic separation as the test reaction solution.

[0009] Preferably, the porcine negative serum protein pre-coated magnetic microparticles are formed by immobilizing total protein from porcine reproductive and respiratory syndrome (PRRS) antibody-negative porcine serum onto the surface of carboxylated magnetic microparticles. Before immobilization, the total protein from porcine PRRS antibody-negative porcine serum is treated with adsorption of GP5-M conformational recombinant antigen and N protein recombinant antigen, respectively.

[0010] Preferably, the GP5-M conformational recombinant antigen comprises a GP5 protein extracellular antigen fragment, a flexible linker peptide, an M protein hydrophilic antigen fragment, and a directional fixation tag connected in sequence. The GP5-M conformational recombinant antigen is linked to a magnetic solid-phase carrier through the directional fixation tag.

[0011] Preferably, the GP5 protein extracellular antigen fragment is located at the end of the GP5-M conformation recombinant antigen away from the orientation fixation tag, and the M protein hydrophilic antigen fragment is located between the GP5 protein extracellular antigen fragment and the orientation fixation tag.

[0012] Preferably, the chemiluminescent labeling site of the chemiluminescently labeled GP5-M conformational recombinant antigen is located at an end different from that of the directional immobilization tag, and the chemiluminescently labeled GP5-M conformational recombinant antigen and the GP5-M conformational recombinant antigen in the GP5-M capture phase have the same extracellular antigen fragment of GP5 protein and the same hydrophilic antigen fragment of M protein.

[0013] Preferably, before the N protein recombinant antigen is immobilized on the magnetic solid-phase support, it is pre-adsorbed sequentially with porcine reproductive and respiratory syndrome (PRRS) antibody-negative porcine serum, an N protein background capture phase, and a tag protein without PRRS virus antigenic epitopes.

[0014] Preferably, the chemiluminescently labeled N protein recombinant antigen and the N protein recombinant antigen in the N protein capture phase have the same amino acid sequence, and both are pre-adsorbed with porcine negative serum under conditions where they do not share the same pre-adsorption mixture.

[0015] Preferably, the GP5-M background capture phase and the GP5-M capture phase are prepared using the same magnetic solid support, the same coupling reaction system, and the same blocking reaction system; the N protein background capture phase and the N protein capture phase are prepared using the same magnetic solid support, the same coupling reaction system, and the same blocking reaction system.

[0016] Preferably, the calibration curves include a GP5-M channel calibration curve and an N protein channel calibration curve. The GP5-M channel calibration curve is established using a porcine reproductive and respiratory syndrome (PRRS) antibody calibrator with a known GP5-M response antibody value, and the N protein channel calibration curve is established using a porcine PRRS antibody calibrator with a known N protein response antibody value.

[0017] Preferably, the double-positive calibrators include at least three antibody levels, each double-positive calibrator having both GP5-M reaction antibody and N protein reaction antibody values, and the ratio range is formed by the ratio of the GP5-M channel antibody value to the N protein channel antibody value of each double-positive calibrator.

[0018] Preferably, the antigen blocking retest includes a first blocking retest and a second blocking retest. The first blocking retest involves pre-incubating the test reaction solution with free GP5-M conformational recombinant antigen and then entering the GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase, respectively. The second blocking retest involves pre-incubating the test reaction solution with free N protein recombinant antigen and then entering the GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase, respectively.

[0019] Preferably, the blocking descent limit includes the blocking descent limit for the GP5-M channel and the blocking descent limit for the N protein channel; the formula for calculating the channel correction signal descent rate is: ,in, Indicates the descent rate of the channel correction signal. This indicates the channel calibration signal before antigen blocking and retesting. The channel correction signal indicates the result of antigen blocking retesting. The GP5-M channel blocking descent limit is determined by the descent rate of the channel correction signal after the first blocking retest of the GP5-M blocking positive control and the GP5-M non-specific interference control. The N protein channel blocking descent limit is determined by the descent rate of the channel correction signal after the second blocking retest of the N protein blocking positive control and the N protein non-specific interference control.

[0020] Preferably, when the GP5-M channel correction signal decline rate after the first closed retest reaches the GP5-M channel closed decline limit, and the N protein channel correction signal decline rate after the second closed retest reaches the N protein channel closed decline limit, the GP5-M channel antibody value corresponding to the first closed retest and the N protein channel antibody value corresponding to the second closed retest are used to determine the quantitative result of porcine reproductive and respiratory syndrome (PRRS) antibody; when only one channel correction signal decline rate reaches the corresponding closed decline limit, the channel antibody value reaching the corresponding closed decline limit is used to determine the quantitative result of PRRS antibody; when neither channel correction signal decline rate reaches the corresponding closed decline limit, a verification failure status is output.

[0021] Preferably, the test reaction solutions are allocated from the same batch to four parallel reaction systems containing a GP5-M capture phase, an N protein capture phase, a GP5-M background capture phase, and an N protein background capture phase. The chemiluminescence signals of the GP5-M channel, the N protein channel, the GP5-M background channel, and the N protein background channel are associated with the same detection batch number and output.

[0022] Compared with existing technologies, the above technical solution has the following advantages: (i) This invention sets up a GP5-M capture phase, an N protein capture phase, a GP5-M background capture phase, and an N protein background capture phase, so that isolated pig serum samples can generate GP5-M channel chemiluminescence signals, N protein channel chemiluminescence signals, and corresponding background channel chemiluminescence signals in the same detection process. By subtracting the corresponding background channel signals to obtain channel correction signals, it is possible to distinguish the specific antibody reactions related to different antigens of porcine reproductive and respiratory syndrome virus (PRRSV) from non-specific background signals caused by serum matrix, expression tags, or solid-phase carriers, thereby reducing the influence of non-specific background signals on quantitative results.

[0023] (II) This invention uses GP5-M conformation recombinant antigen and N protein recombinant antigen to construct dual antigen detection channels respectively, and judges based on the ratio range of antibody value of GP5-M channel to antibody value of N protein channel. This makes the detection results no longer dependent on the overall luminescence value of a single antigen channel or mixed antigen, and can identify isolated pig serum samples with inconsistent contributions of different antigen reactions, thereby improving the discriminability of chemiluminescent immunoassay results of porcine reproductive and respiratory syndrome (PRRS) antibody.

[0024] (III) When the ratio of antibody values ​​in the two channels does not fall within the ratio range determined by the double-positive calibrator, the present invention further uses free GP5-M conformation recombinant antigen and free N protein recombinant antigen to perform antigen blocking retesting, and outputs the quantitative result of porcine reproductive and respiratory syndrome (PRRS) antibody based on the antibody value of the channel that reaches the corresponding blocking decrease limit. This can perform targeted verification and correction of suspected non-specific interference samples and samples with a dominant response to specific antigens, reducing the risk of direct output of false positive or abnormally high value results.

[0025] (iv) The sample diluent of the present invention contains anti-porcine rheumatoid factor antibody and porcine immunoglobulin Fc fragment blocking protein, and can be further combined with porcine negative serum protein pre-coated magnetic microparticles to pre-treat isolated porcine serum samples, which can reduce the influence of rheumatoid factor-like interfering substances, Fc fragment related binding components and other serum matrix components on chemiluminescent immunoreaction in porcine serum samples, and reduce background luminescence signal from the sample processing stage.

[0026] (v) In this invention, the GP5-M capture phase, N protein capture phase, GP5-M background capture phase and N protein background capture phase are respectively pre-placed in the four reaction wells of the same detection cartridge, and are distributed to each reaction well in parallel by the same test reaction solution for detection. This allows the four detection channels to complete the reaction under the same sample source and the same test batch conditions, which facilitates automated detection, well position traceability and result verification, and improves the consistency and repeatability of the chemiluminescent immunoassay quantitative detection process for porcine reproductive and respiratory syndrome (PRRS) antibodies. Attached Figure Description

[0027] Figure 1 The overall flowchart of the chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies provided in this embodiment of the invention; Figure 2 This is a schematic diagram of the GP5-M conformational recombinant antigen oriented and immobilized on a magnetic solid support according to an embodiment of the present invention; Figure 3 This is a schematic diagram illustrating the detection principle of background subtraction corresponding to the GP5-M channel and N protein channel provided in an embodiment of the present invention. Figure 4 This is a schematic diagram showing the ratio range and sample distribution of the antibody levels of the GP5-M channel and the N protein channel provided in this embodiment of the invention. Figure 5 This is a schematic diagram comparing the changes in channel correction signals before and after the closed retest of free antigen, provided in an embodiment of the present invention. Detailed Implementation

[0028] The embodiments of the present invention will be further described below with reference to the accompanying drawings and specific examples. It should be understood that the following examples are for illustrating the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Without departing from the technical concept of the present invention, those skilled in the art can make adaptive adjustments to the specific reagent sources, incubation time, reaction temperature, luminescent labeling system, and automated detection equipment.

[0029] like Figure 1 As shown, this embodiment provides a chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies. This method is used for the chemiluminescent immunoassay of PRRS antibodies in isolated porcine serum samples. The method is based on the immunospecific binding between PRRS virus antigen and PRRS antibody. Through parallel reactions of the GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase, chemiluminescent signals are obtained for the GP5-M channel, N protein channel, GP5-M background channel, and N protein background channel, respectively. The quantification of PRRS antibodies in isolated porcine serum samples is achieved by subtracting the corresponding background channel signals, determining the ratio of antibody values ​​between the two channels, and performing antigen blocking retesting.

[0030] In this embodiment, the GP5-M capture phase is a magnetic solid-phase carrier with recombinant GP5-M conformation antigen of porcine reproductive and respiratory syndrome virus (PRRSV) directionally immobilized. The N protein capture phase is a magnetic solid-phase carrier with recombinant N protein antigen of PRRSV immobilized after pre-adsorption treatment with porcine negative serum. Both the GP5-M background capture phase and the N protein background capture phase are magnetic solid-phase carriers with tag proteins immobilized without PRRSV antigenic epitopes. The GP5-M background capture phase is used to obtain the chemiluminescence signal of the background channel corresponding to the GP5-M channel, and the N protein background capture phase is used to obtain the chemiluminescence signal of the background channel corresponding to the N protein channel.

[0031] See Figure 2 The GP5-M conformation recombinant antigen comprises, in sequence, a GP5 protein extracellular antigen fragment, a flexible linker peptide, an M protein hydrophilic antigen fragment, and a directional fixation tag. The GP5 protein extracellular antigen fragment is positioned at the end of the GP5-M conformation recombinant antigen furthest from the directional fixation tag, while the M protein hydrophilic antigen fragment is located between the GP5 protein extracellular antigen fragment and the directional fixation tag. This structure maintains a relatively fixed linking order between the GP5 protein extracellular antigen fragment and the M protein hydrophilic antigen fragment on the surface of a magnetic solid support.

[0032] In this embodiment, the amino acid sequence of the GP5 protein extracellular antigen fragment is shown in SEQ ID NO:1: SWSFADGNGNSSTYQYIYNLTICELNGTTWLSD The amino acid sequence of the hydrophilic antigen fragment of the M protein is shown in SEQ ID NO:2: MGSLDQLCQNCIFHNT The amino acid sequence of the flexible linker peptide is shown in SEQ ID NO:3: GGGGSGGGGSGGGGS The amino acid sequence of the GP5-M conformation recombinant antigen is shown in SEQ ID NO:4: SWSFADGNGNSSTYQYIYNLTICELNGTTWLSDGGGGSGGGGSGGGGSMGSLDQLCQNCIFHNTHHHHHH The amino acid sequence of the N protein recombinant antigen is shown in SEQ ID NO:5: MPNNNGKQQQKKKGGDNAAQKQKKQQGNNGQVDNNVVNPASVQQVKNWPQKIAEIVHDFQRGVVSANITGISNAAAAYCQLVSNGRVNTVVCGRSLKLGNVHWLQRDPEVEEFVDEVLRSNKS The amino acid sequence of the targeted fixed tag is shown in SEQ ID NO:6: HHHHHH The GP5-M conformation recombinant antigen shown in SEQ ID NO:4 is formed by sequentially linking the GP5 protein extracellular antigen fragment shown in SEQ ID NO:1, the flexible linker peptide shown in SEQ ID NO:3, the M protein hydrophilic antigen fragment shown in SEQ ID NO:2, and the directional fixation tag shown in SEQ ID NO:6. In this embodiment, the directional fixation tag shown in SEQ ID NO:6 is a hexahistidine tag. To avoid shifting the detection target to the nucleic acid amplification or virus culture process, this embodiment only uses the expressed and purified recombinant antigen in the immunoassay reaction, without involving primers, probes, amplification, or virus culture quantification steps.

[0033] In this embodiment, the GP5-M conformational recombinant antigen was prepared using a recombinant expression method. The coding sequences for the extracellular antigen fragment of the GP5 protein, the flexible linker peptide, the hydrophilic antigen fragment of the M protein, and the directional fixed tag were ligated into an expression vector and transformed into host cells for expression. The host cells can be E. coli expression systems, yeast expression systems, insect cell expression systems, or mammalian cell expression systems. For detection systems requiring the preservation of conformational epitopes, insect cell expression systems or mammalian cell expression systems are preferred. In this embodiment, the GP5-M conformational recombinant antigen was expressed using an insect cell expression system. After purification by affinity chromatography and molecular sieve chromatography, a GP5-M conformational recombinant antigen solution with a purity of not less than 90% was obtained.

[0034] In this embodiment, the directional fixation tag uses a hexahistine tag, and the surface of the magnetic solid-phase carrier is pre-modified with nickel ion chelating groups. The GP5-M conformational recombinant antigen is linked to the magnetic solid-phase carrier via the directional fixation tag. In other embodiments, the directional fixation tag can also be a biotinylated tag, a Strep-tag tag, or other tags capable of connecting to affinity conjugates on the surface of the magnetic solid-phase carrier. When the directional fixation tag uses a biotinylated tag, streptavidin can be immobilized on the surface of the magnetic solid-phase carrier, and the GP5-M conformational recombinant antigen is directionally linked to the magnetic solid-phase carrier through biotin-streptavidin interaction.

[0035] The chemiluminescently labeled GP5-M conformational recombinant antigen shares the same extracellular antigen fragment of GP5 protein and the same hydrophilic antigen fragment of M protein as the GP5-M conformational recombinant antigen in the GP5-M capture phase. The chemiluminescent labeling site of the chemiluminescently labeled GP5-M conformational recombinant antigen is located at an end different from the oriented fixation tag. Specifically, chemiluminescent labeling can be achieved using acridine ester labeling, isoluminol labeling, or alkaline phosphatase labeling. In this embodiment, an acridine ester labeling system is used. The activated acridine ester is coupled to the labelable amino group at the end of the GP5-M conformational recombinant antigen furthest from the oriented fixation tag. After labeling, the free acridine ester is removed by a desalting column to obtain the chemiluminescently labeled GP5-M conformational recombinant antigen.

[0036] In this embodiment, the N protein recombinant antigen was expressed using an *E. coli* expression system and obtained after affinity chromatography, ion exchange chromatography, and desalting. Before being immobilized on a magnetic solid-phase support, the N protein recombinant antigen was pre-adsorbed sequentially with porcine reproductive and respiratory syndrome (PRRS) antibody-negative swine serum, an N protein background capture phase, and a tag protein without PRRS virus antigenic epitopes. Specifically, the N protein recombinant antigen solution was first mixed with PRRS antibody-negative swine serum at a volume ratio of 10:1 and incubated at 37°C for 1 hour; then, the N protein background capture phase was added, and the mixture was incubated at 25°C by rotation for 30 minutes followed by magnetic separation, and the supernatant was collected; then, a tag protein without PRRS virus antigenic epitopes was added to the supernatant, and the mixture was incubated at 25°C for 30 minutes, followed by ultrafiltration or chromatography to remove the free tag protein, resulting in the N protein recombinant antigen pre-adsorbed with swine negative serum. The chemiluminescently labeled N protein recombinant antigen and the N protein recombinant antigen in the N protein capture phase have the same amino acid sequence, and both have undergone independent pre-adsorption treatment with swine negative serum. Independent pre-adsorption treatment means that the N protein recombinant antigen used to prepare the N protein capture phase and the N protein recombinant antigen used to prepare the chemiluminescent label are pre-adsorbed separately, rather than sharing the same pre-adsorption mixture.

[0037] In the preparation of the GP5-M capture phase, a magnetic solid-phase support with nickel ion chelating groups modified on its surface was washed three times with phosphate buffer (pH 7.4). Then, a GP5-M conformational recombinant antigen solution was added, and the mixture was incubated at 25°C for 1 hour, allowing the GP5-M conformational recombinant antigen to be attached to the surface of the magnetic solid-phase support via a directional immobilization tag. After incubation, magnetic separation was performed, the supernatant was discarded, and the mixture was blocked for 30 minutes with a blocking solution containing casein and porcine negative serum albumin. The phase was then resuspended in a preservation solution to obtain the GP5-M capture phase. The preservation solution may include phosphate buffer, sucrose, casein, preservatives, and nonionic surfactants.

[0038] In the preparation of the N protein capture phase, a carboxylated magnetic solid-phase support was placed in 2-(N-morpholine)ethanesulfonic acid buffer. Carbodiimide and N-hydroxysuccinimide were added to activate the carboxyl groups. After activation, N protein recombinant antigen pre-adsorbed with porcine negative serum was added, and the mixture was incubated at 25°C for 2 hours to couple the N protein recombinant antigen to the surface of the magnetic solid-phase support. After coupling, magnetic separation was performed, and the mixture was blocked for 30 minutes with a blocking solution containing casein and porcine negative serum protein. The mixture was then resuspended in a preservation solution to obtain the N protein capture phase.

[0039] The GP5-M background capture phase and the N protein background capture phase were prepared using the same magnetic solid-phase support, the same coupling reaction system, and the same blocking reaction system as the corresponding antigen capture phase. The difference between the GP5-M background capture phase and the GP5-M capture phase is that the GP5-M background capture phase immobilizes a tag protein without a porcine reproductive and respiratory syndrome virus (PRRSV) antigenic epitope, rather than the GP5-M conformational recombinant antigen. Similarly, the difference between the N protein background capture phase and the N protein capture phase is that the N protein background capture phase immobilizes a tag protein without a PRRSV antigenic epitope, rather than the N protein recombinant antigen. This preparation method ensures consistency between the background capture phase and the corresponding antigen capture phase in terms of the magnetic solid-phase support, coupling system, and blocking system, facilitating the acquisition of a non-specific background signal matching the corresponding channel during detection.

[0040] The sample diluent is used to process isolated porcine serum samples. In this embodiment, the sample diluent includes phosphate buffer (pH 7.4), anti-porcine rheumatoid factor antibody, porcine immunoglobulin Fc fragment blocking protein, casein, polysorbate-20, preservatives, and pre-coated magnetic microparticles of porcine negative serum protein. The working concentration of the anti-porcine rheumatoid factor antibody is 0.05 mg / mL to 0.50 mg / mL, the working concentration of the porcine immunoglobulin Fc fragment blocking protein is 0.10 mg / mL to 1.00 mg / mL, the mass-volume fraction of casein is 0.1% to 1.0%, and the volume fraction of polysorbate-20 is 0.02% to 0.10%. In one specific embodiment, the working concentration of the anti-porcine rheumatoid factor antibody is 0.20 mg / mL, the working concentration of the porcine immunoglobulin Fc fragment blocking protein is 0.50 mg / mL, the mass-volume fraction of casein is 0.5%, and the volume fraction of polysorbate-20 is 0.05%.

[0041] Porcine negative serum protein pre-coated magnetic microparticles were formed by immobilizing total protein from porcine reproductive and respiratory syndrome (PRRS) antibody-negative porcine serum onto the surface of carboxylated magnetic microparticles. Prior to immobilization, the total protein from PRRS antibody-negative porcine serum was treated with GP5-M conformational recombinant antigen and N protein recombinant antigen, respectively. Specifically, after centrifugation and collection of the supernatant from PRRS antibody-negative porcine serum, GP5-M conformational recombinant antigen and N protein recombinant antigen were added, and the mixture was incubated at 25°C for 1 hour. Components bound to the recombinant antigens were then removed by antigen-coupled microparticle adsorption or chromatography to obtain the treated PRRS antibody-negative porcine serum protein. Subsequently, the treated PRRS antibody-negative porcine serum protein was coupled to the surface of carboxylated magnetic microparticles using carbodiimide and N-hydroxysuccinimide, blocked, and resuspended in sample diluent to obtain a sample diluent containing porcine negative serum protein pre-coated magnetic microparticles.

[0042] During testing, the isolated porcine serum sample is mixed with the sample diluent, preferably at a volume ratio of 1:10. The mixture is then incubated at 25°C for 5 minutes, allowing the anti-porcine rheumatoid factor antibody, porcine immunoglobulin Fc fragment blocking protein, and porcine negative serum protein pre-coated with magnetic microparticles in the sample diluent to treat interfering components in the sample. After incubation, magnetic separation is performed, and the supernatant is used as the test reaction solution. For low-interference samples, the porcine negative serum protein pre-coated magnetic microparticle adsorption step can be omitted, and the isolated porcine serum sample can be directly mixed with the sample diluent as the test reaction solution.

[0043] See Figure 3The test reaction solution was added to four reaction wells pre-filled with GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase. In this embodiment, the four reaction wells were located in the same detection cartridge, and the GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase were pre-filled in the four reaction wells of the same detection cartridge. The test reaction solution was aspirated using the same sampling needle and distributed to the four reaction wells, ensuring that the four reaction systems originated from the same ex vivo porcine serum sample and the same batch of test reaction solution.

[0044] After the test reaction solution entered the four reaction wells, it was incubated at 37°C for 10 minutes to allow the porcine reproductive and respiratory syndrome (PRRS) antibody to immunobind with the corresponding capture phase. After incubation, magnetic separation and washing were performed three times. Subsequently, chemiluminescently labeled GP5-M conformational recombinant antigen was added to the reaction systems corresponding to the GP5-M capture phase and GP5-M background capture, and chemiluminescently labeled N protein recombinant antigen was added to the reaction systems corresponding to the N protein capture phase and N protein background capture. The mixture was incubated at 37°C for 8 minutes. After incubation, magnetic separation and washing were performed three times, followed by the addition of trigger solution corresponding to the chemiluminescent labeling system, and chemiluminescent signals were collected.

[0045] In this embodiment, the chemiluminescence signal includes the GP5-M channel chemiluminescence signal, the N protein channel chemiluminescence signal, the GP5-M background channel chemiluminescence signal, and the N protein background channel chemiluminescence signal. For background subtraction, the GP5-M channel correction signal and the N protein channel correction signal are obtained using the following formulas:

[0046]

[0047] in, This indicates the GP5-M channel correction signal. This indicates the chemiluminescence signal of the GP5-M channel. This indicates the chemiluminescence signal in the GP5-M background channel; This indicates the N protein channel correction signal. This indicates the chemiluminescent signal of the N protein channel. This represents the chemiluminescence signal of the N protein background channel. The above symbols are used only in computational expressions; the full technical names will be used to represent the corresponding detection indicators in subsequent results descriptions.

[0048] Porcine reproductive and respiratory syndrome (PRRS) antibody calibrators include GP5-M reaction antibody calibrators, N protein reaction antibody calibrators, and double-positive calibrators. The GP5-M channel calibration curve is established using PRRS antibody calibrators with GP5-M reaction antibody values, and the N protein channel calibration curve is established using PRRS antibody calibrators with N protein reaction antibody values. The calibration curves can be fitted using a four-parameter logistic regression model. The four-parameter logistic regression model is expressed by the following formula:

[0049] in, Indicates the channel correction signal. This indicates the antibody level for the corresponding channel. This represents the lower asymptotic value of the calibration curve. This represents the high-end asymptotic value of the calibration curve. This indicates the antibody level corresponding to the midpoint of the calibration curve. This represents the slope parameter of the calibration curve. The parameter values ​​were obtained by fitting the GP5-M channel calibration curve and the N protein channel calibration curve, respectively, and were used to convert the GP5-M channel correction signal into the GP5-M channel antibody value and the N protein channel correction signal into the N protein channel antibody value, respectively.

[0050] The double-positive calibrators include those with at least three antibody levels, each with both GP5-M and N protein antibody values. In this embodiment, low-level, medium-level, and high-level double-positive calibrators are provided. Each double-positive calibrator is detected in both the GP5-M and N protein channels, and the ratio of the GP5-M channel antibody value to the N protein channel antibody value is calculated. The ratio is expressed using the following formula:

[0051] in, This represents the ratio of the antibody level in the GP5-M channel to the antibody level in the N protein channel. This indicates the antibody level in the GP5-M channel. This indicates the antibody level in the N protein channel. The ratios of low-level, medium-level, and high-level double-positive calibrators are used to form ratio ranges.

[0052] In this embodiment, to determine the ratio range, 10 samples each of low-level, medium-level, and high-level double-positive calibrators were selected, and three test batches were performed, with two replicate tests per batch, resulting in 180 sets of dual-channel antibody value data. Statistical results showed that the ratio distribution for low-level double-positive calibrators ranged from 0.78 to 0.91, for medium-level double-positive calibrators from 0.86 to 1.04, and for high-level double-positive calibrators from 0.96 to 1.16. Considering the ratio distributions of the three levels of double-positive calibrators and the intra-batch allowable deviation, the ratio range was determined to be 0.70 to 1.25. This ratio range is used to determine whether the GP5-M channel antibody value and the N protein channel antibody value in isolated porcine serum samples are within the same range. See also... Figure 4 Samples within the ratio range can have their porcine reproductive and respiratory syndrome (PRRS) antibody quantification results determined directly based on the antibody levels in both channels; samples outside the ratio range will proceed to the antigen-blocked retesting process.

[0053] When the ratio of the GP5-M channel antibody value to the N protein channel antibody value falls within the ratio range determined by the double-positive calibrator, the quantitative result of porcine reproductive and respiratory syndrome (PRRS) antibody determined by the GP5-M channel antibody value and the N protein channel antibody value is output. In this embodiment, the arithmetic mean of the GP5-M channel antibody value and the N protein channel antibody value can be used as the quantitative result of PRRS antibody, or a weighted average can be determined based on the channel stability of the double-positive calibrator. When using the weighted average, the calculation formula is as follows:

[0054] in, This indicates the quantitative results of porcine reproductive and respiratory syndrome (PRRS) antibody testing. This indicates the antibody level in the GP5-M channel. This indicates the antibody level in the N protein channel. This represents the GP5-M channel weighting coefficient. This represents the weighting coefficient of the N protein channel, and In this embodiment, the weighting coefficients for both the GP5-M channel and the N protein channel are 0.50. In other embodiments, the weighting coefficients can be determined based on the intra-batch precision of the double-positive calibrator, with channels having higher intra-batch precision having higher weighting coefficients.

[0055] When the ratio of the GP5-M channel antibody level to the N protein channel antibody level does not fall within the specified range, antigen blocking retesting is performed using free GP5-M conformational recombinant antigen and free N protein recombinant antigen, respectively. Antigen blocking retesting includes a first blocking retest and a second blocking retest. The first blocking retest involves pre-incubating the test reaction solution with free GP5-M conformational recombinant antigen, followed by entering the GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase, respectively. The second blocking retest involves pre-incubating the test reaction solution with free N protein recombinant antigen, followed by entering the GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase, respectively. The free GP5-M conformational recombinant antigen and free N protein recombinant antigen pre-bind to their corresponding specific antibodies in the test reaction solution, thereby causing a decrease in the subsequent correction signal of the corresponding channel. See also... Figure 5 For true positive abnormal samples, after blocking with the corresponding free antigen, the decrease in the corresponding channel correction signal reaches the blocking decrease limit; for false positive interference samples, after blocking with the free antigen, the decrease in the corresponding channel correction signal does not reach the blocking decrease limit.

[0056] The channel correction signal descent rate is expressed by the following formula:

[0057] in, Indicates the descent rate of the channel correction signal. This indicates the channel calibration signal before antigen blocking and retesting. This represents the channel correction signal after antigen blocking retesting. The GP5-M channel blocking descent limit is determined by the decrease rate of the channel correction signal after the first blocking retest of the GP5-M blocked positive control and the GP5-M non-specific interference control. The N protein channel blocking descent limit is determined by the decrease rate of the channel correction signal after the second blocking retest of the N protein blocked positive control and the N protein non-specific interference control. Specifically, the decrease rate of the channel correction signal of the blocked positive control and the non-specific interference control is first detected separately, and then the boundary of the decrease rate that can distinguish between the blocked positive control and the non-specific interference control is selected as the corresponding blocking descent limit.

[0058] In this embodiment, to determine the blocking descent limit, 30 GP5-M blocking positive control samples, 30 GP5-M non-specific interference control samples, 30 N protein blocking positive control samples, and 30 N protein non-specific interference control samples were selected and subjected to a first blocking retest and a second blocking retest, respectively. The channel correction signal descent rate of the GP5-M blocking positive control samples after the first blocking retest was 61.8% to 87.6%, with an average descent rate of 74.9%; the channel correction signal descent rate of the GP5-M non-specific interference control samples after the first blocking retest was 4.8% to 29.3%, with an average descent rate of 16.7%. The channel correction signal descent rate of the N protein blocking positive control samples after the second blocking retest was 63.5% to 89.1%, with an average descent rate of 76.8%; the channel correction signal descent rate of the N protein non-specific interference control samples after the second blocking retest was 3.9% to 27.8%, with an average descent rate of 15.6%. Based on the aforementioned quality control sample decline rate distribution, the GP5-M channel closure decline limit and the N protein channel closure decline limit are both set at 50%. In other embodiments, the closure decline limit can be adjusted based on the statistical results of different batches of quality control samples.

[0059] When the GP5-M channel correction signal decrease rate after the first closed retest reaches the GP5-M channel closed decrease limit, and the N protein channel correction signal decrease rate after the second closed retest reaches the N protein channel closed decrease limit, the quantitative result of porcine reproductive and respiratory syndrome (PRRS) antibody is determined using the GP5-M channel antibody value corresponding to the first closed retest and the N protein channel antibody value corresponding to the second closed retest. When only one channel correction signal decrease rate reaches the corresponding closed decrease limit, the quantitative result of PRRS antibody is determined using the antibody value of the channel that reaches the corresponding closed decrease limit. When neither channel correction signal decrease rate reaches the corresponding closed decrease limit, a verification failure status is output, indicating that the isolated swine serum sample has non-specific background interference or is not suitable for outputting quantitative results according to the current batch calibration curve.

[0060] In this embodiment, the detection cartridge includes four reaction wells, each pre-set with a GP5-M capture phase, an N protein capture phase, a GP5-M background capture phase, and an N protein background capture phase, respectively. After the fully automated chemiluminescence immunoassay analyzer draws the reaction solution from the sample cup, it sequentially dispenses the solution into the four reaction wells using the same dispensing needle. The analyzer records the chemiluminescence signals of the GP5-M channel, the N protein channel, the GP5-M background channel, and the N protein background channel, and outputs these signals in association with the same detection cartridge number. The detection cartridge number can also be stored in association with the sample number, calibration curve number, quality control status, and detection time to facilitate traceability of the detection well location and batch during retesting.

[0061] To verify the role of the background capture phase, this embodiment uses a conventional method of mixed detection with N protein magnetic beads and GP5 protein magnetic beads as a comparative example, and a detection method with background capture phase subtraction corresponding to this embodiment as an example. A total of 120 isolated porcine serum samples were selected for this embodiment, including 40 samples with negative label cross-reactivity, 40 samples with positive porcine reproductive and respiratory syndrome (PRRS) antibodies, 20 ordinary negative samples, and 20 borderline samples. Each sample was tested using both the comparative mixed detection method and the background subtraction detection method of this embodiment, with each method repeated twice, and the average value was taken as the result. For ease of explanation, Table 1 lists the statistical results for each type of sample.

[0062] Table 1 Comparison of Background Capture Phase Subtraction Effects

[0063] As shown in Table 1, for label cross-reaction negative samples, 31 samples were judged positive in the comparative proportion pooled detection method, while only 2 samples were judged positive in the background subtraction detection method of this embodiment. This indicates that the corresponding background capture phase can reduce false positive outputs caused by non-specific binding related to the expression tag or solid-phase carrier. For porcine reproductive and respiratory syndrome (PRRS) antibody positive samples, the background subtraction detection method of this embodiment still maintained 40 positive results, indicating that the corresponding background subtraction did not eliminate the specific immune response signal of PRRS antibodies. For ordinary negative samples and borderline samples, the background subtraction detection method of this embodiment can reduce the influence of background signals on quantitative judgment, making the detection results more suitable for subsequent ratio judgment and antigen blocking retesting processes.

[0064] To verify the effectiveness of sample diluents and porcine negative serum protein pre-coated magnetic microparticles in reducing interference with the porcine serum matrix, this embodiment used conventional PBS-BSA diluent as a control and a sample diluent containing anti-porcine rheumatoid factor antibody, porcine immunoglobulin Fc fragment blocking protein, and porcine negative serum protein pre-coated magnetic microparticles as an example. A total of 150 ex vivo porcine serum samples were selected for this embodiment, including 50 samples of porcine serum with high rheumatoid factor-like interference, 30 samples of lipemic porcine serum, 30 samples of hemolytic porcine serum, 20 samples of porcine serum with high immunoglobulin background, and 20 samples of ordinary negative porcine serum. Each sample was treated with both conventional PBS-BSA diluent and the sample diluent of this embodiment before testing. Each treatment method was repeated twice, and the average value was taken as the test result.

[0065] Table 2 Comparison of blocking effects of sample dilutions

[0066] As shown in Table 2, compared with the conventional PBS-BSA diluent, the sample diluent of this embodiment can reduce the chemiluminescence signal of the background channel in porcine serum with high rheumatoid factor-like interference, lipemic porcine serum, hemolytic porcine serum, and porcine serum with high immunoglobulin background, and also reduce the channel correction signal remaining after background subtraction. These results indicate that anti-porcine rheumatoid factor antibody, porcine immunoglobulin Fc fragment blocking protein, and porcine negative serum protein pre-coated magnetic microparticles can cooperate with background capture to reduce porcine serum matrix interference during the sample processing stage.

[0067] To verify the role of ratio judgment and antigen blocking retesting, this embodiment selected routine true positive samples, false positive interference samples, true positive samples from special infection stages, GP5-M reaction dominant samples, dual-channel abnormal true positive samples, and ordinary negative samples for testing. A total of 240 clinical ex vivo swine serum samples were selected in this embodiment, including 60 routine true positive samples, 40 false positive interference samples, 40 true positive samples from special infection stages, 30 GP5-M reaction dominant samples, 40 dual-channel abnormal true positive samples, and 30 ordinary negative samples. The ratio range was set from 0.70 to 1.25, and the blocking thresholds for both the GP5-M channel and the N protein channel were 50%. Each sample underwent initial testing; for samples whose ratios did not fall within the ratio range, a first blocking retest and a second blocking retest were performed.

[0068] Table 3. Ratio Determination and Clinical Validation Table for Free Antigen Blocking Retest

[0069] As shown in Table 3, most routine true positive samples fell within the ratio range, allowing for direct output of dual-channel quantitative results. Among false positive interference samples, although most showed abnormal antibody levels in certain channels, they failed to reach the corresponding blocking threshold after retesting with free antigen blocking, thus being identified as failing the retest. In true positive samples from specific infection phases, most reached the N protein channel blocking threshold after a second retest, allowing for output using the N protein channel antibody level. In GP5-M dominant reaction samples, most reached the GP5-M channel blocking threshold after a first retest, allowing for output using the GP5-M channel antibody level. These results indicate that ratio judgment and antigen blocking retesting can provide targeted retesting and correction for samples with inconsistent quantitative results from different antigen reactions.

[0070] To further verify the overall effectiveness of this embodiment compared to existing mixed antigen magnetic particle chemiluminescence detection methods, this embodiment summarizes and analyzes the above 240 clinical isolated porcine serum samples, using previous viral antibody confirmation results, retest results, and positive / negative reference results as a comprehensive reference. The results are shown in Table 4.

[0071] Table 4. Comparison of overall testing performance between this embodiment and the comparative example.

[0072] As shown in Table 4, background subtraction alone can reduce the number of false positives; further combining it with ratio judgment can identify more abnormal samples that require retesting; adding antigen blocking retesting on top of ratio judgment can convert some abnormal samples into output quantitative results and reduce the number of false positives and false negatives. The above data shows that the background subtraction, dual-channel ratio judgment, and antigen blocking retesting in this embodiment are not isolated and parallel, but form a continuous detection logic from background processing, anomaly identification to retesting output.

[0073] To verify the repeatability of the test, this embodiment selected 20 low-value positive samples, 20 medium-value positive samples, 20 high-value positive samples, and 20 negative samples, totaling 80 in vitro pig serum samples. These samples were repeatedly tested in three different batches, with each sample tested twice per batch. The results are shown in Table 5.

[0074] Table 5. Results of Repeatability Validation

[0075] As shown in Table 5, the intra-batch and inter-batch coefficients of variation in low-value positive samples, medium-value positive samples, high-value positive samples, and negative samples in this embodiment are all within acceptable ranges, indicating that the four-channel parallel reaction, corresponding background subtraction, dual-channel antibody value conversion, and antigen blocking retesting process have good repeatability.

[0076] In one optional embodiment, the GP5-M capture phase, N protein capture phase, GP5-M background capture phase, and N protein background capture phase are not pre-placed in the detection cartridge, but are stored in the reagent compartment as magnetic microparticle suspensions. These are then aspirated and added to the corresponding reaction wells by a fully automated chemiluminescence immunoassay analyzer according to a preset program. This embodiment can also generate chemiluminescence signals for the GP5-M channel, N protein channel, GP5-M background channel, and N protein background channel, and perform corresponding background subtraction, ratio determination, and antigen blocking retesting.

[0077] In another alternative embodiment, the directional immobilization tag for the GP5-M conformational recombinant antigen employs a biotinylated tag, and streptavidin is immobilized on the surface of a magnetic solid-phase support. The GP5-M conformational recombinant antigen is directionally linked to the magnetic solid-phase support via biotin-streptavidin interaction. This method, compared to the hexahistine tag and nickel ion chelate group linkage methods, can achieve directional immobilization of the GP5-M conformational recombinant antigen on the surface of the magnetic solid-phase support. In other embodiments, the chemiluminescent labeling system can also employ isoluminol or alkaline phosphatase labeling systems, as long as they can generate a chemiluminescent signal that can be read by a chemiluminescent immunoassay analyzer.

[0078] This embodiment uses isolated porcine serum samples for detection. The detection is based on the specific immune binding reaction between porcine reproductive and respiratory syndrome (PRRS) virus antigen and PRRS antibody. The output is a quantitative result of PRRS antibody. This method does not involve nucleic acid amplification, primers, probes, virus culture, or live animal disease diagnosis steps. It belongs to the chemiluminescent immunoassay quantitative detection technology for viral antibodies and can stably correspond to the technical field of viral immunoassay.

[0079] It should be noted that the embodiments of the present invention have better implementability and are not intended to limit the present invention in any way. Any person skilled in the art may use the above-disclosed technical content to change or modify it into equivalent effective embodiments. However, any modifications or equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. A chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies, characterized in that, Includes the following steps: A GP5-M capture phase, an N protein capture phase, a GP5-M background capture phase, and an N protein background capture phase were prepared. The GP5-M capture phase was a magnetic solid-phase carrier in which the GP5-M conformational recombinant antigen of porcine reproductive and respiratory syndrome virus (PRRSV) was directionally immobilized. The N protein capture phase was a magnetic solid-phase carrier in which the recombinant N protein antigen of PRRSV was immobilized after pre-adsorption treatment with porcine negative serum. Both the GP5-M background capture phase and the N protein background capture phase were magnetic solid-phase carriers in which tag proteins without PRRSV antigenic epitopes were immobilized. An isolated porcine serum sample was mixed with a sample diluent to obtain a test reaction solution. The sample diluent contained anti-porcine rheumatoid factor antibody and porcine immunoglobulin Fc fragment blocking protein. The test reaction solution was incubated with the GP5-M capture phase, the N protein capture phase, the GP5-M background capture phase, and the N protein background capture phase, respectively, followed by magnetic separation and washing; Chemiluminescently labeled GP5-M conformational recombinant antigen was added to the reaction system corresponding to the GP5-M capture phase and the GP5-M background capture phase. Chemiluminescently labeled N protein recombinant antigen was added to the reaction system corresponding to the N protein capture phase and the N protein background capture phase. After incubation, the mixture was magnetically separated and washed. Chemiluminescence signals of the GP5-M channel, N protein channel, GP5-M background channel, and N protein background channel were collected separately. The GP5-M channel chemiluminescence signal was subtracted from the GP5-M background channel chemiluminescence signal to obtain the GP5-M channel correction signal, and the N protein channel chemiluminescence signal was subtracted from the N protein background channel chemiluminescence signal to obtain the N protein channel correction signal. Based on the calibration curve established using the porcine reproductive and respiratory syndrome (PRRS) antibody calibrator, the GP5-M channel correction signal was converted into the GP5-M channel antibody value, and the N protein channel correction signal was converted into the N protein channel antibody value. When the ratio of the GP5-M channel antibody value to the N protein channel antibody value falls within the ratio range determined by the double-positive calibrator, the quantitative result of porcine reproductive and respiratory syndrome (PRRS) antibody determined by the GP5-M channel antibody value and the N protein channel antibody value is output; when the ratio does not fall within the ratio range, the test reaction solution is subjected to antigen blocking retesting using free GP5-M conformational recombinant antigen and free N protein recombinant antigen, respectively, and the quantitative result of PRRS antibody is output according to the channel antibody value that reaches the corresponding blocking decrease limit.

2. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 1, characterized in that, The sample diluent also contains porcine negative serum protein pre-coated magnetic microparticles. After the isolated porcine serum sample is mixed with the sample diluent, it is first adsorbed by the porcine negative serum protein pre-coated magnetic microparticles, and then the supernatant is obtained by magnetic separation as the test reaction solution.

3. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 2, characterized in that, The porcine negative serum protein pre-coated magnetic microparticles are formed by immobilizing total protein from porcine reproductive and respiratory syndrome (PRRS) antibody-negative porcine serum onto the surface of carboxylated magnetic microparticles. The total protein from porcine PRRS antibody-negative porcine serum is treated with adsorption of the GP5-M conformational recombinant antigen and the N protein recombinant antigen before immobilization.

4. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 1, characterized in that, The GP5-M conformational recombinant antigen comprises a GP5 protein extracellular antigen fragment, a flexible linker peptide, an M protein hydrophilic antigen fragment, and a directional fixation tag connected in sequence. The GP5-M conformational recombinant antigen is connected to the magnetic solid-phase carrier through the directional fixation tag.

5. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 4, characterized in that, The GP5 protein extracellular antigen fragment is disposed at the end of the GP5-M conformation recombinant antigen away from the orientation fixation tag, and the M protein hydrophilic antigen fragment is located between the GP5 protein extracellular antigen fragment and the orientation fixation tag.

6. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 4, characterized in that, The chemiluminescent labeling site of the chemiluminescently labeled GP5-M conformational recombinant antigen is located at an end different from that of the directional immobilization tag, and the chemiluminescently labeled GP5-M conformational recombinant antigen has the same extracellular antigen fragment of GP5 protein and the same hydrophilic antigen fragment of M protein as the GP5-M conformational recombinant antigen in the GP5-M capture phase.

7. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 1, characterized in that, Before being immobilized on the magnetic solid-phase support, the N protein recombinant antigen is pre-adsorbed sequentially with porcine reproductive and respiratory syndrome (PRRS) antibody-negative porcine serum, the N protein background capture phase, and a tag protein without PRRS virus antigenic epitopes.

8. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 7, characterized in that, The chemiluminescently labeled N protein recombinant antigen and the N protein recombinant antigen in the N protein capture phase have the same amino acid sequence, and both are pre-adsorbed with porcine negative serum under conditions where they do not share the same pre-adsorption mixture.

9. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 1, characterized in that, The GP5-M background capture phase and the GP5-M capture phase are prepared using the same magnetic solid support, the same coupling reaction system, and the same blocking reaction system; the N protein background capture phase and the N protein capture phase are prepared using the same magnetic solid support, the same coupling reaction system, and the same blocking reaction system.

10. The chemiluminescent immunoassay method for quantitative detection of porcine reproductive and respiratory syndrome (PRRS) antibodies according to claim 1, characterized in that, The calibration curves include a GP5-M channel calibration curve and an N protein channel calibration curve. The GP5-M channel calibration curve is established using a porcine reproductive and respiratory syndrome (PRRS) antibody calibrator with a known GP5-M response antibody value, and the N protein channel calibration curve is established using a porcine PRRS antibody calibrator with a known N protein response antibody value.

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