Anti-sal1 protein monoclonal antibody, detection kit and application thereof in early pregnancy diagnosis of sows

Abstract

The application discloses an anti-SAL1 protein monoclonal antibody, a detection kit and application of the monoclonal antibody in early pregnancy diagnosis of sows. The monoclonal antibody comprises a monoclonal antibody 1A3, a monoclonal antibody 2G11 and a monoclonal antibody 5H5; the monoclonal antibody 1A3 comprises a 1A3 heavy chain variable region and a 1A3 light chain variable region, the monoclonal antibody 2G11 comprises a 2G11 heavy chain variable region and a 2G11 light chain variable region, and the monoclonal antibody 5H5 comprises a 5H5 heavy chain variable region and a 5H5 light chain variable region. The antigen of the polyclonal antibody pAb-SAL1 is SAL1 protein, and the polyclonal antibody is prepared after animals are immunized by the antigen. The application establishes a single antibody-single antibody sandwich ELISA detection method and a single antibody-polyclonal antibody sandwich ELISA detection method, and the two methods show excellent sensitivity, precision and accuracy.

Description

Technical Field

[0001] This invention relates to the field of antibody technology, specifically to an anti-SAL1 protein monoclonal antibody, a detection kit, and its application in early pregnancy diagnosis in sows. Background Technology

[0002] Early pregnancy diagnosis in sows is a key technological step in shortening non-productive days (NPD), improving sow reproductive efficiency, and enhancing the economic benefits of pig farming. Non-productive days refer to the number of days a sow is neither pregnant nor lactating; each additional day results in losses of feed, labor, pen depreciation, utilities, and opportunity costs. The main cause of prolonged NPD is the failure to promptly identify unsuccessful pregnancies after mating, leading to sows remaining non-pregnant until the next estrus cycle (approximately 21 days later) before re-mating. Therefore, developing an early, accurate, and convenient pregnancy diagnosis technology that can detect pregnancy before the first estrus cycle after mating (earlier than traditional ultrasound detection) is crucial.

[0003] I. Currently, commonly used clinical methods for diagnosing pregnancy mainly include: ①B-ultrasound detection method: The detection window is usually 24 to 30 days after mating. Although it is intuitive and reliable, the detection time is too late and requires a high level of experience from the operator, which cannot meet the needs of rapid on-site screening in large-scale pig farms.

[0004] ② Hormone detection method: Although ELISA detection based on hormones such as progesterone and estradiol sulfate can achieve laboratory diagnosis, the detection time is too late or the accuracy needs to be improved, and the sampling process may cause stress to sows.

[0005] ③ Exogenous hormone-induced estrus method: Pregnancy status is determined by observing the estrus response after injecting exogenous hormones. This method has a significant impact on the physiological state of sows and may affect subsequent reproductive performance.

[0006] In recent years, point-of-care testing (POCT) technology based on specific biomarkers has become a research hotspot in veterinary clinical diagnosis due to its advantages of being non-invasive, rapid, and easy to operate. Non-invasive or minimally invasive samples such as saliva, urine, and vaginal secretions are ideal sources of samples for large-scale screening because they are simple to collect, stress-free, and easy to repeat. However, specific biomarkers and related testing products for early pregnancy diagnosis in sows are still very scarce. The limitations and shortcomings of existing technologies are as follows: ① Existing detection technologies generally have the problem of a late detection window, making it impossible to achieve early diagnosis 15-18 days after mating (before the first estrus period); ② Existing detection methods rely heavily on specialized equipment and operational experience, making them difficult to meet the actual needs of rapid on-site screening in large-scale pig farms; ③ The accuracy of existing hormone-based immunoassay methods needs to be improved, and the sample collection process may cause stress to sows; ④ Currently, there are no high-affinity, high-specificity antibodies against sow SAL1 protein and their corresponding detection products, which limits the clinical translational application of SAL1 biomarkers; ⑤ Existing studies lack a systematic analysis of the conformational epitope characteristics of the SAL1 protein, resulting in a lack of structural biology basis for antibody pairing strategies, which affects the sensitivity and specificity of the detection system. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an anti-SAL1 protein monoclonal antibody, a detection kit, and its application in early pregnancy diagnosis in sows. This invention achieves quantitative detection of SAL1 protein in sow saliva samples 15 days after mating by preparing a high-affinity antibody, analyzing its conformational epitope region that recognizes SAL1, and establishing a sandwich ELISA detection system with non-overlapping epitope pairing, thereby enabling early assessment of the sow's pregnancy status.

[0008] To achieve the above objectives, the technical solution designed by the present invention is as follows: This invention provides a monoclonal antibody against SAL1 protein, the amino acid sequence of which is shown in SEQ ID NO: 2; The monoclonal antibodies include monoclonal antibody 1A3, monoclonal antibody 2G11 and monoclonal antibody 5H5; The monoclonal antibody 1A3 includes a 1A3 heavy chain variable region and a 1A3 light chain variable region, and the 1A3 heavy chain variable region includes three heavy chain complementarity-determining regions 1A3-CDR-VH, which are as follows: 1A3-CDR1-VH: GYTFTEYT; 1A3-CDR2-VH: INPNGVT; 1A3-CDR3-VH: ARSEVNPFTY; The 1A3 light chain variable region includes three light chain complementarity determination regions 1A3-CDR-VL, which are: 1A3-CDR1-VL: QTIVHSNGNTF; 1A3-CDR2-VL: KVS; 1A3-CDR3-VL: FQGSHVPPT; The monoclonal antibody 2G11 includes a 2G11 heavy chain variable region and a 2G11 light chain variable region, and the 2G11 heavy chain variable region includes three heavy chain complementarity-determining regions 2G11-CDR-VH, which are as follows: 2G11-CDR1-VH: GYTFTSYW; 2G11-CDR2-VH: INPSNGRT; 2G11-CDR3-VH: TRGLSDY; The 2G11 light chain variable region includes three light chain complementarity determination regions 2G11-CDR-VL, which are: 2G11-CDR1-VL: QDINSY; 2G11-CDR2-VL: RAN; 2G11-CDR3-VL: LQYDEFPYT; The monoclonal antibody 5H5 includes a 5H5 heavy chain variable region and a 5H5 light chain variable region, and the 5H5 heavy chain variable region includes three heavy chain complementarity-determining regions 5H5-CDR-VH, which are as follows: 5H5-CDR1-VH: GYTFTSYW; 5H5-CDR2-VH:INPSNGRT; 5H5-CDR3-VH: TRGLSDY; The 5H5 light chain variable region includes three light chain complementarity-determining regions, 5H5-CDR-VL, which are: 5H5-CDR1-VL: QDINSY; 5H5-CDR2-VL: RAN; 5H5-CDR3-VL:LQYDEFPYT.

[0009] Furthermore, in the monoclonal antibody 1A3, the amino acid sequence of the heavy chain variable region of 1A3 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region of 1A3 is shown in SEQ ID NO: 4; The monoclonal antibody 2G11 has the following amino acid sequence: heavy chain variable region as shown in SEQ ID NO: 5, and light chain variable region as shown in SEQ ID NO: 6. The monoclonal antibody 5H5 has the following amino acid sequence: heavy chain variable region as shown in SEQ ID NO: 7, and light chain variable region as shown in SEQ ID NO: 8.

[0010] This invention also provides a method for preparing the polyclonal antibody pAb-SAL1, comprising the following steps: i. Use the SAL1 protein, whose amino acid sequence is shown in SEQ ID NO: 2, as an antigen to immunize animals and collect animal serum; ii. Animal serum was purified by antigen-conjugated agarose affinity chromatography and then dialyzed to obtain the purified polyclonal antibody pAb-SAL1.

[0011] The present invention also provides a polyclonal antibody pAb-SAL1 prepared by the preparation method described above.

[0012] The present invention also provides the application of an antibody in the preparation of a kit for detecting early pregnancy status in sows, the antibody comprising monoclonal antibody 1A3, monoclonal antibody 2G11, monoclonal antibody 5H5 and the polyclonal antibody pAb-SAL1.

[0013] The present invention also provides a kit for detecting early pregnancy status in sows, the kit comprising any one of a monoclonal antibody-monoclonal antibody sandwich ELISA kit and a monoclonal antibody-polyclonal antibody sandwich ELISA kit; The monoclonal antibody-monoclonal antibody sandwich ELISA kit includes monoclonal antibody 2G11 and HRP-labeled monoclonal antibody 1A3; The monoclonal antibody-polyclonal antibody sandwich ELISA kit includes the monoclonal antibody 5H5 and the HRP-labeled polyclonal antibody pAb-SAL1; The early pregnancy period is defined as 12 to 18 days after artificial insemination.

[0014] Furthermore, the early pregnancy is defined as the 15th day after artificial insemination.

[0015] The present invention also provides a method for ELISA detection using the aforementioned kit, comprising the following steps: (1) The capture antibody was diluted and added to the wells of an ELISA plate for incubation; the capture antibody was monoclonal antibody 2G11 or monoclonal antibody 5H5; (2) After adding blocking solution to the wells of the ELISA plate, block at 2-4℃; (3) Add the sample to be tested to the well of the ELISA plate and incubate; (4) Add detection antibody to the well of the ELISA plate and incubate; when the capture antibody is monoclonal antibody 2G11, the detection antibody is HRP-labeled monoclonal antibody 1A3; when the capture antibody is monoclonal antibody 5H5, the detection antibody is HRP-labeled polyclonal antibody pAb-SAL1. (5) Add TMB substrate chromogenic solution to the wells of the microplate and perform chromogenic development in the dark; (6) Add stop solution to the wells of the ELISA plate and measure the absorbance at 450 nm wavelength using an ELISA reader.

[0016] The present invention also provides the application of the kit described herein in the non-diagnostic detection of early pregnancy status in sows.

[0017] The present invention also provides a computer program product related to the early pregnancy status of sows, the computer program product being used to perform detection of the early pregnancy status of sows, the early pregnancy being the 12th to 18th day after artificial insemination; Includes the following steps: ① Collect saliva samples from the sows to be tested, and centrifuge to collect the supernatant; ② The collected supernatant was analyzed by ELISA using the kit described above to obtain the absorbance at a wavelength of 450 nm; ③ Calculate the concentration of SAL1 protein in the saliva sample based on absorbance; ④ Make a judgment based on the SAL1 protein concentration: When the kit is a monoclonal antibody-monoclonal antibody sandwich ELISA kit, if the SAL1 protein concentration is greater than 1.136 μg / mL, the sow to be tested is pregnant; otherwise, the sow to be tested is non-pregnant. When the kit is a monoclonal antibody-polyclonal antibody sandwich ELISA kit, if the SAL1 protein concentration is greater than 0.907 μg / mL, the sow to be tested is pregnant; otherwise, the sow to be tested is non-pregnant.

[0018] Furthermore, the ELISA detection method is as follows: I. Dilute the capture antibody and add it to the wells of an ELISA plate for incubation; the capture antibody is monoclonal antibody 2G11 or monoclonal antibody 5H5; II. After adding blocking buffer to the wells of the ELISA plate, block at 2-4℃; III. Add the collected supernatant to the wells of the ELISA plate and incubate. IV. Add the detection antibody to the wells of the ELISA plate and incubate; when the capture antibody is monoclonal antibody 2G11, the detection antibody is HRP-labeled monoclonal antibody 1A3; when the capture antibody is monoclonal antibody 5H5, the detection antibody is HRP-labeled polyclonal antibody pAb-SAL1. V. Add TMB substrate chromogenic solution to the wells of the ELISA plate and perform chromogenic development in the dark; VI. Add stop solution to the wells of the ELISA plate and measure the absorbance at 450 nm using an ELISA reader.

[0019] The beneficial effects of this invention are: 1. This invention first clearly demonstrates the application value of SAL1 protein as a biomarker for early pregnancy diagnosis in sows: Through parallel analysis of saliva 4D-DIA and vaginal secretion 4D Label-free proteomics, this invention identified that SAL1 is closely related to pregnancy status in both body fluids, establishing the cross-body fluid stability of SAL1 as a pregnancy diagnostic biomarker and laying the foundation for its clinical detection application.

[0020] 2. First-ever resolution of the conformational epitope features of the SAL1 protein: This invention systematically resolved the conformational epitope regions of SAL1 recognized by monoclonal antibodies 1A3 and 2G11 using hydrogen-deuterium exchange mass spectrometry (HDX-MS). It clarified that monoclonal antibody 1A3 recognizes peptides 28-44 and 73-84, while monoclonal antibody 2G11 recognizes peptides 28-44 and 105-120. This discovery reveals the molecular mechanism by which antibody binding induces conformational rearrangement of the antigen, providing a structural biology basis for antibody pairing.

[0021] 3. Preparation of high-affinity monoclonal antibodies: The equilibrium dissociation constants (KD) of monoclonal antibody 1A3 and monoclonal antibody 2G11 with the SAL1 antigen, determined by surface plasmon resonance (SPR) technology, were 5.12 × 10⁻⁶. -9 M and 1.74×10 -9 M, both reaching nanomolar levels of extremely high efficiency. Crucially, the dissociation rate constants (Kd) of the two antibodies are 9.57 × 10⁻⁶. -6 s -1 and 5.27×10 -6 s -1 It exhibits typical "extremely slow dissociation" kinetic characteristics, ensuring the high stability of the antigen-antibody complex during multiple washing processes and significantly improving the detection signal-to-noise ratio.

[0022] 4. A high-sensitivity sandwich ELISA detection method was established: Based on a non-overlapping conformational epitope pairing strategy, this invention established a monoclonal antibody-monoclonal antibody sandwich ELISA method (2G11 / HRP-1A3) and a monoclonal antibody-polyclonal antibody sandwich ELISA method (5H5 / HRP-pAb-SAL1). The detection limits of the two methods reached 0.248 ng / mL and 0.226 ng / mL, respectively, with coefficients of variation below 8%, and spiked recoveries ranging from 86.15% to 115.32%, demonstrating excellent sensitivity, precision, and accuracy.

[0023] 5. Early pregnancy diagnosis 15 days after mating: The detection method established in this invention can detect sow saliva samples 15 days after mating (24-30 days earlier than the traditional B-ultrasound detection window), realizing early pregnancy diagnosis before the first estrus period, and providing technical support for timely return to estrus management and shortening non-productive days.

[0024] 6. Excellent diagnostic efficacy, non-invasive sampling, and simple operation: This invention was validated with 100 clinical saliva samples. A combined analysis of two ELISA detection methods using a Logistic binary logistic regression model yielded an AUC value of 0.918. This invention uses saliva samples, collected non-invasively, causing no stress to sows and facilitating repeated sampling and large-scale screening. The ELISA method is simple to operate, suitable for batch testing in laboratories, and provides a precise quantitative detection tool for large-scale pig farms.

[0025] 7. Laying the Foundation for POCT Product Development: The antibody and conformational epitope information of this invention provides core technical support for the subsequent development of rapid on-site detection products such as immunochromatographic test strips, and has significant clinical translational value and application prospects. This invention is the first high-affinity antibody targeting sow SAL1 protein and its sandwich ELISA detection method, filling a core technological gap in this field and having important practical significance for improving the refined management and economic benefits of the pig farming industry. Attached Figure Description

[0026] Figure 1 A volcano plot to illustrate the differential protein expression between the pregnant and non-pregnant groups; Figure 2 Cluster heatmap to show the expression patterns of differentially expressed proteins between the pregnant and non-pregnant groups; In the figure, each row represents a protein, each column represents a sample, and the color from blue to red indicates the expression level from low to high. Figure 3 Bar chart for functional enrichment analysis of Gene Ontology (GO); Figure 4 Bubble chart for KEGG pathway enrichment analysis; Figure 5 This is a diagram showing the predicted transmembrane domains of the SAL1 protein. Figure 6 This is a diagram illustrating the hydrophilicity and hydrophobicity of the SAL1 protein. Figure 7 This is a diagram showing the predicted signal peptide of the SAL1 protein. Figure 8 This is a diagram showing the immunogenicity analysis of the SAL1 protein. Figure 9 This diagram shows the complete gene synthesis, prokaryotic expression, purification, and identification of the SAL1 protein. In the figure, A is the PCR amplification product of the SAL1 gene detected by agarose gel electrophoresis, lanes 1-8: amplification products of different clones; B is the recombinant plasmid DNA sequencing identification; C is the SDS-PAGE analysis of the induced expression of recombinant SAL1 protein, lane 1: cells induced by cold shock (empty vector) with pET-28a(+) expression vector, lane 2: cells of recombinant strain without cold shock induction, lane 3: cells of recombinant strain after cold shock induction, lane 4: supernatant after cell cold shock induction and lysis, lane 5: precipitate after cell cold shock induction and lysis; D is the SDS-PAGE analysis of the solubility of recombinant protein, lane 1: supernatant after cell cold shock induction and lysis, lane 2: containing 30 The elution buffer after elution with 250 mM imidazole wash buffer; lanes 3-4: elution buffer after elution with 250 mM imidazole elution buffer; E is the result of SDS-PAGE analysis of the purification process of recombinant protein, lane 1: purified protein; lane 2: 0.5 mg / mL BSA; F is the result of Western Blot identification of purified protein, lane 1: purified protein; lane 2: multi-tag protein; Figure 10 A schematic diagram of the preparation process for SAL1 protein monoclonal antibody; Figure 11 The image shows the identification results of monoclonal and polyclonal antibodies against the SAL1 protein. In the figure, A shows the titer results of ELISA on mouse tail blood; B-D show the results of Western blot analysis of the binding specificity of monoclonal antibodies 1A3 (B), 2G11 (C), and 5H5 (D), respectively. Lane 1: Cold shock induced expression of purified recombinant SAL1 protein; Lane 2: Recombinant strain without cold shock induction (negative control); E-F show the results of WB (E) and SDS-PAGE (F) verification of the light and heavy chains of monoclonal antibodies, respectively. Lanes 1-3: 1A3, 2G11, and 5H5; G shows the results of SDS-PAGE verification of polyclonal antibodies against SAL1 protein. Lane 1: Light and heavy chains of SAL1 polyclonal antibody. Figure 12 This is a graph showing the results of monoclonal antibody subtype identification; Figure 13 The image shows the results of ascites titer determination before purification. Figure 14 The image shows the results of potency determination after purification of ascites fluid; Figure 15 The graph shows the titer results of HRP-labeled monoclonal and polyclonal antibodies. Figure 16 The results of SPR analysis of the binding kinetics of monoclonal antibodies 1A3 and 2G11 with SAL1 are shown in the figure. Figure 17The graph shows the Tm1 values ​​(330 / 350 nm) for monoclonal and polyclonal antibodies. Figure 18 Figure showing the results of HDX-MS sequence coverage analysis; Figure 19 Line graph showing the deuteration uptake kinetics of differentially expressed peptides; Figure 20 Histogram of deuterated uptake of differentially expressed peptides (ΔHDX>0.54 Da, P<0.01); Figure 21 Mapping differentially expressed peptides to the three-dimensional structure of the SAL1 protein; Figure 22 The figure shows the results of optimizing the conditions for ELISA with monoclonal antibody-monoclonal antibody sandwich. In the figure, A is the result of optimized conditions for coating and capturing antibodies; B is the result of optimized conditions for antigen incubation; C is the result of optimized working conditions for detection antibodies; and D is the standard curve (4PL fitted curve) of the monoclonal antibody-monoclonal antibody sandwich ELISA kit. Figure 23 The figure shows the results of ELISA condition optimization for monoclonal antibody-polyclonal antibody sandwich ELISA. In the figure, A is the result of optimized conditions for coating and capturing antibodies; B is the result of optimized conditions for antigen incubation; C is the result of optimized working conditions for detection antibodies; and D is the standard curve (4PL fitted curve) of the monoclonal antibody-polyclonal antibody sandwich ELISA kit. Figure 24 This is a graph showing the receiver operating characteristic (ROC) curve analysis results for the ELISA detection method. In the figure, A is the ROC of the monoclonal antibody-monoclonal antibody sandwich ELISA method; B is the ROC of the monoclonal antibody-polyclonal antibody sandwich ELISA method; C is the ROC of the two ELISA methods analyzed by the combined analysis of the logistic binary logistic regression model. Figure 25 Figure 1 shows the structural model of the 1A3-SAL1-2G11 ternary complex constructed based on experimental constraints and the results of interaction analysis. In the figure, A is a three-dimensional structural band diagram of the ternary complex; B is a surface contact area analysis diagram of the binding interface (red area); and C is an analytical diagram of atomic-level non-covalent interactions at key recognition sites. Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0028] Example 1: Proteomic analysis of 4D-Label-free vaginal secretions from sows 1. Collection and processing of experimental samples Multiparous Landrace-Large White crossbred sows with normal estrus, good reproductive performance, uniform body size, and consistent body condition were selected and artificially inseminated. The day of insemination was recorded as day 0. On day 15 post-insemination, a sterile cotton swab was gently inserted 8-10 cm into the sow's vagina and slowly rotated along the vaginal wall 2-3 times to fully absorb vaginal secretions. After collection, the swab tip was cut off, placed in a sterile cryovial, and immediately immersed in liquid nitrogen for freezing. Within 2 hours, it was transferred to a -80°C ultra-low temperature freezer for long-term storage. Pregnancy was confirmed by ultrasound examination on days 28 and 55 post-insemination. Based on the ultrasound results, vaginal secretion samples collected on day 15 post-insemination were divided into a pregnant group and a non-pregnant group, with three biological replicates in each group.

[0029] 2. 4D Label-free quantitative proteomics analysis The above samples were subjected to protein extraction, reductive alkylation, and trypsin digestion, respectively. The obtained peptides were desalted and then subjected to 4D label-free quantitative proteomics data acquisition using a tandem liquid chromatography system. Chromatographic separation was performed using a C18 analytical column (75 μm × 25 cm) with gradient elution for 120 min. Mass spectrometry data acquisition was performed in data-independent mode.

[0030] The raw mass spectrometry data were retrieved by searching a database, specifically the porcine UniProt reference proteome database (https: / / www.uniprot.org / proteomes / ). Search parameters were set as follows: trypsin restriction enzyme, maximum allowed missed cleavage sites ≤ 2, fixed modification of cysteine ​​(Carbamidomethyl), variable modification of methionine (Oxidation), and false discovery rate (FDR) of <1% for both protein and peptide levels.

[0031] 3. Screening of differentially expressed proteins Based on the above mass spectrometry quantitative results, a total of 3682 proteins were identified and quantified. Using the fold change and statistical significance of the difference between the pregnant and non-pregnant groups as screening criteria, a screening threshold of |log2FC|>2 and P<0.05 was set. Using this criterion, 47 differentially expressed proteins were screened from the 3682 quantified proteins.

[0032] 4. Functional enrichment analysis of differentially expressed proteins To clarify the biological functions and metabolic pathways involved in differentially expressed proteins, Gene Ontology (GO) functional enrichment analysis and KEGG pathway enrichment analysis were performed on the aforementioned 47 differentially expressed proteins. GO enrichment analysis covered three dimensions: biological processes, cellular components, and molecular functions, while KEGG pathway enrichment analysis focused on metabolic and signal transduction pathways. A p-value < 0.05 was used as a significance threshold to screen for significantly enriched GO entries and KEGG pathways. The analysis results (specific pathway names for significantly enriched differentially expressed proteins, such as "significant enrichment of differentially expressed proteins in the RNA polymerase pathway, protein digestion and absorption pathway, and Notch signaling pathway") suggest that local metabolic activity in the reproductive tract during the critical window of embryo implantation underwent reprogramming.

[0033] 5. Determination of candidate biomarker SAL1 To identify the most promising candidate biomarkers for early pregnancy diagnosis from 47 differentially expressed proteins, a comprehensive evaluation was conducted based on the following criteria: (1) significant fold change, i.e., ranking high among all differentially expressed proteins in |log2FC|; (2) secreted protein or detectable across body fluids, suitable for non-invasive sampling; (3) potentially associated with reproductive physiological processes; and (4) good immunogenicity, suitable for subsequent antibody preparation and establishment of immunological detection methods.

[0034] Based on the above criteria, the results are as follows: Figures 1-8 As shown, salivary lipocalin 1 (UniProt ID: A0A8D0HYQ4) was selected as a candidate biomarker. In the proteomics results, SAL1 was significantly downregulated in vaginal secretions of the pregnant group compared to the non-pregnant group, with differential expression parameters: FC=0.015, log2FC=-6.08, P=0.034. The downregulation magnitude was among the highest of all differentially expressed proteins. Figure 1 Meanwhile, previous 4D-DIA proteomics studies of salivary fluid showed that SAL1 was significantly upregulated in the saliva of pregnant sows. This cross-humoral differential expression pattern further confirmed the high correlation between SAL1 and the pregnancy status of sows, and verified its detectability in multiple body fluids such as saliva and vaginal secretions. Figure 2 ).

[0035] GO Figure 3 ) and KEGG ( Figure 4 Enrichment analysis showed significant enrichment in the RNA polymerase pathway and the Notch signaling pathway. Furthermore, bioinformatics analysis (transmembrane domain prediction, signal peptide prediction, hydrophilicity / hydrophobicity analysis, and immunogenicity analysis) revealed that the SAL1 protein lacks a transmembrane domain. Figure 5 ), has hydrophilicity ( Figure 6 ), and the presence of signal peptides ( Figure 7Good immunogenicity () Figure 8 It possesses the typical characteristics of secretory proteins and the feasibility of preparing specific antibodies.

[0036] In summary, this embodiment successfully screened and identified SAL1 protein from sow vaginal secretions as a candidate biomarker for early pregnancy diagnosis 15 days after mating by combining 4D label-free quantitative proteomics technology with bioinformatics analysis. This lays the material foundation for subsequent preparation of recombinant proteins, development of specific antibodies, and establishment of immunological detection methods.

[0037] Example 2: Preparation of recombinant SAL1 protein 1. Gene synthesis (1) The full-length SAL1 gene was synthesized by overlap extension PCR. Twelve oligonucleotide fragments with a length of 60-70 bp and an overlap region of 20 bp were designed and synthesized (primer sequences are shown in Table 1). The 12 oligonucleotides were mixed in equal molar amounts to obtain mixed oligonucleotides. Using the mixed oligonucleotides as templates, a first round of PCR was performed using high-fidelity DNA polymerase to assemble the fragments into the full-length gene.

[0038] (2) Subsequently, using the first-round PCR amplification product as a template, a second round of nested PCR was performed using specific primers NJ0141378-1_1 and NJ0141378-1_12 located at both ends of the gene to amplify and enrich the full-length fragment. The PCR product was verified by agarose gel electrophoresis and then excised to obtain a 576 bp SAL1 gene fragment. The nucleotide sequence of the SAL1 protein is shown in SEQ ID NO: 1.

[0039] Table 1. Oligonucleotide sequences of SAL1 gene synthesis 2. Construction of expression carrier The pET-28a(+) expression vector was linearized and purified by double digestion with Nco I and Xho I restriction endonucleases. A one-step cloning method was used to ligate the purified SAL1 gene fragment into the linearized vector. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB agar plates containing 50 μg / mL kanamycin, and incubated overnight at 37°C. Single colonies were picked for preliminary screening by colony PCR; the results are shown below. Figure 9 As shown in Figure A, positive clones were inoculated into LB liquid medium and cultured. After plasmid extraction, DNA sequencing was performed using T7 terminator primers. The results are as follows: Figure 9 As shown in B, the recombinant plasmid pET-28a(+)-SAL1 with the correct sequence was obtained after sequence alignment.

[0040] 3. Induced expression and purification (1) The recombinant plasmid pET-28a(+)-SAL1 was transformed into Escherichia coli BL21(DE3) competent cells to obtain a recombinant strain. The recombinant strain was then induced to produce heterologous proteins by cold shock at 15°C combined with 0.2 mM IPTG to obtain cold-shock induced cells. After the cells were disrupted by sonication, the supernatant and precipitate were obtained by centrifugation and analyzed by SDS-PAGE. The results are as follows: Figure 9 As shown in Figure C, a distinct specific induction band appears at 20.04 kDa, consistent with the theoretical molecular weight of the SAL1 fusion protein, which is mainly present in the supernatant fraction in a soluble form.

[0041] (2) Soluble proteins in the supernatant were purified using Ni-NTA agarose affinity chromatography. The sample was loaded at a flow rate of 0.5 mL / min and equilibrated. It was then washed and eluted sequentially with binding buffer, wash buffer containing 30 mM imidazole, and elution buffer containing 250 mM imidazole. The eluent was collected and dialyzed overnight with PBS to obtain the purified protein. The results were verified by 12% SDS-PAGE electrophoresis. Figure 9 D and Figure 9 As shown in E, the appearance of a single clear band at the expected molecular weight indicates that the target protein has been successfully purified with a purity of over 85%.

[0042] (3) The purified protein was validated by Western blot, using anti-His tag monoclonal antibody as the primary antibody, goat anti-mouse IgG as the secondary antibody, and multi-tag protein as the control group. The results are as follows: Figure 9 As shown in F, the SAL1 protein can specifically bind to the anti-His tag antibody, confirming the successful acquisition of high-purity soluble recombinant SAL1 protein. The amino acid sequence of the recombinant SAL1 protein is shown in SEQ ID NO: 2.

[0043] Example 3: Preparation and Identification of Monoclonal Antibodies like Figure 10 As shown, it includes the following steps: 1. Mouse immunization 50 μg of purified recombinant SAL1 protein was mixed with QuickAntibody-Mouse 5W adjuvant at a 1:1 volume ratio and administered subcutaneously to female BALB / c mice (6-8 weeks old) in a volume of 100 μL. After two rounds of immunization, serum was collected using an indirect ELISA method for titer assessment. Results are as follows: Figure 11 As shown in Figure A, the antibody titer in mouse serum reached 1:409600.

[0044] 2. Hybridoma cell preparation Mice with high serum antibody titers were selected and immunized intraperitoneally with 50 μg of purified recombinant SAL1 protein. Three days after immunization, mouse spleen cells were isolated and fused with SP2 / 0 myeloma cells in the presence of polyethylene glycol (PEG). After screening in HAT medium, hybridoma cells were screened for antigen-specific monoclonal antibodies using an indirect ELISA method. Positive hybridoma cells underwent three rounds of restrictive dilution subcloning to obtain three hybridoma cell lines that stably secrete SAL1-specific monoclonal antibodies, named hybridoma cell 1A3, hybridoma cell 2G11, and hybridoma cell 5H5, respectively.

[0045] 3. Production and purification of monoclonal antibodies Selected hybridoma cells were injected intraperitoneally into female BALB / c mice (8-10 weeks old) pretreated with an ascites-specific adjuvant to induce ascites formation. After collecting the ascites, monoclonal antibodies were isolated and purified from the ascites using recombinant protein A / G affinity chromatography. Monoclonal antibodies 1A3, 2G11, and 5H5 were obtained using hybridoma cells 1A3, 2G11, and 5H5, respectively.

[0046] The integrity and purity of the heavy and light chains of the monoclonal antibody were detected by SDS-PAGE, and the results are as follows: Figure 11 E and Figure 11 As shown in Figure F, monoclonal antibodies 1A3, 2G11, and 5H5 all exhibited clear 55 kDa heavy chain and 25 kDa light chain bands, with a purity exceeding 90%. Antibody concentrations were determined using a NanoDrop 2000: 2.8 mg / mL for monoclonal antibody 1A3, 2.3 mg / mL for monoclonal antibody 2G11, and 2.6 mg / mL for monoclonal antibody 5H5.

[0047] 4. Monoclonal antibody identification (1) Monoclonal antibody isotype detection was performed using a commercially available mouse antibody typing kit. The results are as follows: Figure 12 As shown, the heavy chains of monoclonal antibodies 1A3, 2G11, and 5H5 all belong to the IgG1 subclass, and their light chains are all Kappa (κ) chains.

[0048] (2) Antibody titer was assessed by indirect ELISA, and the results are as follows: Figure 13 and Figure 14 As shown, the titers of monoclonal antibody 1A3, monoclonal antibody 2G11, and monoclonal antibody 5H5 before and after purification all reached 1:409600.

[0049] (3) Western blot was used to verify the specificity of the monoclonal antibody, and the results are as follows: Figure 11 B~ Figure 11 As shown in D, monoclonal antibodies 1A3, 2G11, and 5H5 can all specifically bind to the recombinant SAL1 protein, demonstrating that monoclonal antibodies 1A3, 2G11, and 5H5 have high specificity.

[0050] 5. Monoclonal antibody sequencing (1) Monoclonal antibody 1A3 includes a 1A3 heavy chain variable region and a 1A3 light chain variable region: ① The 1A3 heavy chain variable region includes 3 heavy chain complementarity-determining regions (1A3-CDR-VH) and 4 heavy chain backbone regions (1A3-FR-VH), which are as follows: 1A3-FR1-VH:EVQLQQSGPELVKPGASVKTSCKTS; 1A3-CDR1-VH: GYTFTEYT; 1A3-FR2-VH:IHWVKQSHGKSLEWIGG; 1A3-CDR2-VH: INPNGVT; 1A3-FR3-VH: SYNQKFKGMATLTVDKSSSTAYMELRRSLTSEDSAVYYC; 1A3-CDR3-VH: ARSEVNPFTY; 1A3-FR4-VH: WGQGTLVTVSA; The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 1A3 is shown in SEQ ID NO: 3.

[0051] ②The 1A3 light chain variable region includes 3 light chain complement determination regions (1A3-CDR-VL) and 4 light chain backbone regions (1A3-FR-VL), which are as follows: 1A3-FR1-VL: DVLMTQTPLSLPVSLGDQASISCRSS; 1A3-CDR1-VL: QTIVHSNGNTF; 1A3-FR2-VL:LEWYLQKPGQSPKLLIY; 1A3-CDR2-VL: KVS; 1A3-FR3-VL: NRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYC; 1A3-CDR3-VL: FQGSHVPPT; 1A3-FR4-VL: FGGGTKLEIK; The amino acid sequence of the variable region of the light chain of monoclonal antibody 1A3 is shown in SEQ ID NO: 4.

[0052] (2) The monoclonal antibody 2G11 includes the 2G11 heavy chain variable region and the 2G11 light chain variable region: ① The 2G11 heavy chain variable region includes 3 heavy chain complement determination regions (2G11-CDR-VH) and 4 heavy chain backbone regions (2G11-FR-VH), which are as follows: 2G11-FR1-VH:QVQLQQPGSELVKPGASVKLSCKAS; 2G11-CDR1-VH: GYTFTSYW; 2G11-FR2-VH:NHWVKQRPGQGLEWIGE; 2G11-CDR2-VH: INPSNGRT; 2G11-FR3-VH: KYNEKFKSKAILTVDKSSSTAYMQLSSLTSEDSAVYYC; 2G11-CDR3-VH: TRGLSDY; 2G11-FR4-VH:WGQGTTLTVSS; The amino acid sequence of the variable region of the heavy chain of monoclonal antibody 2G11 is shown in SEQ ID NO: 5.

[0053] ②The 2G11 light chain variable region includes 3 light chain complementarity determination regions (2G11-CDR-VL) and 4 light chain backbone regions (2G11-FR-VL), which are as follows: 2G11-FR1-VL:DIKMTQSPSSMYASLGERVTFTCKAS; 2G11-CDR1-VL: QDINSY; 2G11-FR1-VL:LSWFQQKPGKSPKTLIY; 2G11-CDR2-VL: RAN; 2G11-FR3-VL: RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC; 2G11-CDR3-VL: LQYDEFPYT; 2G11-FR4-VL: FGGGTKLEIK; The amino acid sequence of the variable region of the light chain of monoclonal antibody 2G11 is shown in SEQ ID NO: 6.

[0054] (3) Monoclonal antibody 5H5 includes the 5H5 heavy chain variable region and the 5H5 light chain variable region: ① The 5H5 heavy chain variable region includes 3 heavy chain complement determination regions (5H5-CDR-VH) and 4 heavy chain backbone regions (5H5-FR-VH), which are as follows: 5H5-FR1-VH:QVQLQQPGSELVKPGASVKLSCKAS; 5H5-CDR1-VH: GYTFTSYW; 5H5-FR2-VH:NHWVKQRPGQGLEWIGE; 5H5-CDR2-VH:INPSNGRT; 5H5-FR3-VH: KYNEKFKTKAILTVDKSSSTAYMQLSSLTSEDSAVYYC; 5H5-CDR3-VH: TRGLSDY; 5H5-FR4-VH:WGQGTTLTVSS; The amino acid sequence of the variable region of the heavy chain of the monoclonal antibody 5H5 is shown in SEQ ID NO: 7.

[0055] ②The 5H5 light chain variable region includes 3 light chain complementarity determination regions (5H5-CDR-VL) and 4 light chain backbone regions (5H5-FR-VL), which are as follows: 5H5-FR1-VL:DIKMTQSPSSMYASLGERVTFTCKAS; 5H5-CDR1-VL: QDINSY; 5H5-FR1-VL:LSWFQQKPGKSPKTLIY; 5H5-CDR2-VL: RAN; 5H5-FR3-VL: RLVDGVPSRFSGSGSGQDYSLTISSLEYEDMGIYYC; 5H5-CDR3-VL: LQYDEFPYT; 5H5-FR4-VL:FGGGTKLEIK; The amino acid sequence of the variable region of the light chain of monoclonal antibody 5H5 is shown in SEQ ID NO: 8.

[0056] Example 4: Preparation of polyclonal antibodies 1. Animal immunization New Zealand white rabbits (2-2.5 kg) were immunized with SAL1 protein as the antigen, 400 μg subcutaneously every 2-3 weeks. Blood samples were collected for testing, and the titer of the antiserum was determined by indirect ELISA. When the titer was greater than 1:50,000, blood was collected for final preparation of antiserum, which was then purified.

[0057] 2. Antibody purification An antigen affinity purification chromatography column was prepared by coupling SAL1 protein with agarose medium. The obtained antiserum was mixed with an equal volume of PBS and slowly loaded onto the column. After the antibody and protein were bound, the column was eluted with glycine elution buffer to obtain the desired purified antibody. The purified antibody was then immediately dialyzed overnight (10-12 h) in PBS at 4°C to obtain the polyclonal antibody pAb-SAL1, which was then subjected to purity, concentration and titer determination.

[0058] 3. Antibody identification The titer of the purified antibody was detected by ELISA, and the concentration of the obtained antibody was determined using a BCA concentration assay kit. The purity of the purified antibody was observed by SDS-PAGE electrophoresis and Coomassie brilliant blue staining.

[0059] The purified polyclonal antibody pAb-SAL1 was analyzed by SDS-PAGE, and the results are as follows: Figure 11 As shown in G, the polyclonal antibody pAb-SAL1 exhibits two clear protein bands under reducing conditions, with molecular weights of 55 kDa and 25 kDa, respectively, consistent with the theoretical molecular weights of the heavy and light chains of the monoclonal antibody. The purity of the polyclonal antibody is over 85%, and the concentration of the polyclonal antibody pAb-SAL1 determined by the BCA method is 1.4 mg / mL.

[0060] Example 5: Surface Plasmon Resonance (SPR) Analysis The interaction kinetics between monoclonal antibodies 1A3 and 2G11 and recombinant SAL1 protein were systematically characterized using surface plasmon resonance (SPR) technology on a Biacore 8K system.

[0061] SAL1 protein was immobilized on the surface of a CM5 sensor chip as a ligand using an amino-coupled method. The monoclonal antibody to be tested was diluted as an analyte in 1×PBS-P+ running buffer (pH 7.4) containing 5% (v / v) DMSO. Different concentrations of monoclonal antibody solutions were sequentially injected at a flow rate of 30 μL / min, and the response signal changes during the binding phase (60 s) and dissociation phase (120 s) were monitored in real time. The chip was regenerated after each cycle using 10 mM glycine-hydrochloric acid buffer (pH 2.0). All kinetic curves were globally fitted using a 1:1 Langmuir binding model in Biacore Insight software.

[0062] The results are as follows Figure 16 As shown, the binding affinity (KD) of monoclonal antibody 1A3 to the SAL1 antigen is 5.12 × 10⁻⁶. -9 M, the binding rate constant Ka is 1.87 × 10 3 M -1 s -1 The dissociation rate constant Kd is 9.57 × 10⁻⁶. -6 s -1 The binding affinity (KD) of monoclonal antibody 2G11 to the SAL1 antigen was 1.74 × 10⁻⁶. -9 M, the binding rate constant Ka is 3.03 × 10 3 M -1 s -1 The dissociation rate constant Kd is 5.27 × 10⁻⁶. -6 s -1 Both monoclonal antibodies exhibited good affinity characteristics, especially extremely slow dissociation kinetics, which ensured the high stability of the antigen-antibody complex.

[0063] Example 6: Epitope resolution by hydrogen-deuterium exchange mass spectrometry (HDX-MS) 1. Sample preparation and labeling SAL1 protein was mixed with excess monoclonal antibodies 1A3 and 2G11 in buffer to form complexes; a separate SAL1 protein was used as a control. The complex samples were mixed with deuteration buffer (90% D2O, 1×PBS) and subjected to hydrogen-deuterium exchange labeling reactions at room temperature for 3 minutes and 30 minutes, respectively. After labeling, the reaction was terminated by rapidly adding pre-cooled low-pH quenching buffer.

[0064] 2. Mass spectrometry data acquisition and analysis (1) The quenched samples were immediately processed using an online enzymatic digestion system. The samples were first rapidly digested by passing through a pre-cooled immobilized pepsin digestion column, followed by online desalting using a C18 desalting column. The resulting peptides were separated by a Hypersil GOLDC18 analytical column, and data were acquired on a SCIEX X500B QTOF mass spectrometer equipped with an electrospray ionization source.

[0065] (2) Mass spectrometry data were first matched against a database using Biologics Explorer software to obtain high-coverage SAL1 peptide identification results. Subsequently, Mass Spec Studio software was used to perform precise quantitative analysis of the deuteration level of the identified peptides. The Student's t-test was used to compare the deuteration level of the corresponding peptides at the same labeling time when SAL1 was present alone and when it was bound to different monoclonal antibodies. The significance threshold was set as a deuteration difference ΔHDX > 0.54 Da and P < 0.01.

[0066] 3. Results The results are as follows Figure 18 As shown, the sequence coverage of SAL1 protein in the monoclonal antibody 1A3 binding group was 89.20%, with a total of 22 quantifiable peptides identified; the sequence coverage in the monoclonal antibody 2G11 binding group was 90.34%, with a total of 27 quantifiable peptides identified.

[0067] Comparing the differences in deuteration levels, the results are as follows: Figures 19-21 As shown, monoclonal antibody 1A3 recognizes peptide regions 28-44 and 73-84 of the SAL1 protein. After binding with monoclonal antibody 1A3, the above peptides show a significant increase in deuteration uptake, indicating that monoclonal antibody binding induces conformational relaxation or increased solvent accessibility in the local region of the antigen. Monoclonal antibody 2G11 recognizes peptide regions 28-44, 105-120 and their adjacent regions (including 28-42, 108-120, 108-119, and 110-120) of the SAL1 protein. After binding with monoclonal antibody 2G11, the above peptides show a significant increase in deuteration uptake. When SAL1 protein binds to monoclonal antibody 2G11, the deuteration rate of peptides 28-44 in SAL1 protein alone is 61.63%-61.71%, while after binding to monoclonal antibody 2G11, the deuteration rate of this peptide increases to 74.70%-78.69%, indicating that the conformation of this region changes or solvent accessibility increases after antibody binding. These results suggest that monoclonal antibodies 1A3 and 2G11 recognize non-overlapping conformational epitopes of SAL1. The epitopes of monoclonal antibody 1A3 are concentrated in the N-terminus and core domain of SAL1, while the epitopes of monoclonal antibody 2G11 extend to the C-terminal region, providing a structural biology basis for the establishment of a dual-antibody sandwich ELISA detection system.

[0068] Example 7: Antibody Structure and Tm1 Analysis 1. To further elucidate the molecular recognition mechanism between SAL1 protein and monoclonal antibodies 1A3 and 2G11 at the structural level, computer-aided molecular docking simulations were conducted. The three-dimensional structure of the SAL1 (1GM6) protein was obtained from the PDB database (https: / / www.rcsb.org / ). Based on the obtained heavy chain and light chain variable regions of the monoclonal antibodies, Fab segment models of monoclonal antibodies 1A3 and 2G11 were constructed using homology modeling. Constrained docking was performed using HDOCKlitev 1.1 software. Epitopes of 1A3 (Residues 28–44, 73–84) and epitopes of monoclonal antibody 2G11 (Residues 28–44, 105–120) determined by HDX-MS were set as active residues to guide sampling. Based on binary docking, a ternary complex model of 1A3-SAL1-2G11 was constructed, and the binding probability was assessed using a confidence score. The binding free energy of the complex was calculated using the MM / GBSA module of the HawkDock server. Finally, non-covalent interactions such as hydrogen bonds and salt bridges at the antigen-antibody interface were analyzed using the PLIP platform, and visualized using PyMOL.

[0069] The results are as follows Figure 25As shown, preliminary binary docking results confirm that, under given experimental constraints, both monoclonal antibodies 1A3 and 2G11 can form high-confidence complex models with SAL1, and the predicted binding interface perfectly matches the HDX-MS high-protection zone. Based on the spatial orientation consistency of the binary model, the results of the further constructed ternary complex model show that SAL1 can simultaneously bind with monoclonal antibodies 1A3 and 2G11 to form a stable sandwich structure. The model with the lowest binding energy has a confidence score as high as 0.9834, and the docking score is -354.33. The binding free energy of the ternary complex calculated by MM / GBSA is -48.74 kcal / mol, indicating that this "sandwich" mode has extremely high thermodynamic spontaneity and stability. Spatial hindrance analysis shows that although both monoclonal antibodies involve the residue 28-44 region, monoclonal antibodies 1A3 and 2G11 enter from different spatial orientations of the antigen, and the surface contact area (red) shows that they do not conflict in physical occupancy. This spatial complementarity explains why the presence of shared epitopes did not interfere with the detection of the double-antibody sandwich assay. Atomic-level interaction analysis revealed that the antigen-antibody interface is maintained by a complex network of non-covalent bonds. THR-29 of the 2G11 heavy chain variable region forms a key hydrogen bond (2.9–3.0 Å) with ASN-65 of SAL1, while ASP-76 of SAL1 forms a strong salt bridge with ARG-64 of the 2G11 heavy chain variable region. Monoclonal antibody 1A3 forms a hydrogen bond with ASN-112 of SAL1 via TYR-88, and a salt bridge is formed between LYS-14 and ASP-80. The synergistic effect of these high-energy chemical bonds constitutes the molecular basis for the sensor's high affinity and extremely slow dissociation rate.

[0070] 2. The Tm1 values ​​of monoclonal antibody 1A3, monoclonal antibody 2G11, monoclonal antibody 5H5 and polyclonal antibody pAb-SAL1 were analyzed.

[0071] The results are as follows Figure 17 As shown, the Tm1 value of monoclonal antibody 1A3 was 69.82±0.34℃, the Tm1 value of monoclonal antibody 2G11 was 73.79±6.75℃, the Tm1 value of monoclonal antibody 5H5 was 70.19±0.30℃, and the Tm1 value of polyclonal antibody pAb-SAL1 was 78.31±7.97℃. The relatively large standard deviations of monoclonal antibody 2G11 and polyclonal antibody pAb-SAL1 suggest that they may exist in a conformational intermediate state during thermal denaturation. These results indicate that the monoclonal antibodies 1A3, 2G11, and 5H5 prepared in this invention all possess good thermal stability and are suitable for establishing subsequent immunological detection methods.

[0072] Example 8: Optimization of Antibody Pairing Screening and Sandwich ELISA Method Conditions 1. Antibody HRP labeling Using a commercial HRP labeling kit (Wuhan Sanying Biotechnology Co., Ltd. CAT NO. PK20001), horseradish peroxidase was used to label monoclonal antibodies 1A3, 2G11, 5H5, and polyclonal antibody pAb-SAL1 with HRP, resulting in HRP-labeled antibodies HRP-1A3, HRP-2G11, HRP-5H5, and HRP-pAb-SAL1, respectively. The specific steps are as follows: (1) Remove the HRP labeling kit from -20℃ and equilibrate at room temperature for 30 minutes to allow the reaction start solution and reaction stop solution to fully thaw and mix.

[0073] (2) Add 1 μL of reaction start solution to every 10 μL of antibody to be labeled, and use a pipette to repeatedly blow and mix several times to ensure thorough mixing and avoid generating bubbles.

[0074] (3) Open the cap of the horseradish peroxidase tube, add the activated antibody directly into the tube, and incubate at 37°C and 300 RPM for 1 h with shaking.

[0075] (4) Add the reaction termination solution to the horseradish peroxidase reaction tube (bottle) at a ratio of 1 μL of reaction termination solution to 10 μL of antibody, mix thoroughly, and let stand at room temperature for 1 h.

[0076] (5) After termination, add an equal volume of product protection solution, mix thoroughly, and store at -20℃.

[0077] The titer of the HRP-labeled antibody was determined, and the results are as follows: Figure 15 As shown, the potency of HRP-1A3 reached 1:409600, the potency of HRP-2G11 and HRP-5H5 reached 1:204800, and the potency of HRP-pAb-SAL1 reached 1:102400.

[0078] 2. Antibody pairing screening In the antibody pair screening process, monoclonal antibodies 1A3, 2G11, 5H5 and polyclonal antibody pAb-SAL1 were used as capture antibodies to coat 96-well plates, and HRP-labeled antibody was used as the detection antibody. The 2 μg / mL antigen SAL1 protein was detected by sandwich ELISA.

[0079] ① Coating: Dilute the capture antibody to 1 μg / mL with coating buffer, add it to the wells of the ELISA plate, and incubate at 37°C for 2 hours; ② Sealing: Add 5% skim milk powder and seal at 37℃ for 1 hour; ③ Sample addition: Add 2 μg / mL of antigen SAL1 protein and incubate at 37℃ for 1 hour; ④ Add detection antibody: Add detection antibody at a ratio of 1:10000 and incubate at 37°C for 1 hour; ⑤ Development: Add TMB substrate development solution and develop at 37°C in the dark for 15 minutes; ⑥ Termination and detection: Add stop solution and measure absorbance at 450 nm wavelength (OD450nm) using an ELISA reader.

[0080] Testing revealed that the combination of capture antibody 2G11 and detection antibody HRP-1A3 achieved the highest signal-to-noise ratio (SNR) (OD450nm 3.476, background 0.058, S / N = 59.931); the combination of capture antibody 5H5 and detection antibody HRP-pAb-SAL1 also achieved a relatively high SNR (OD450nm 2.885, background 0.074, S / N = 38.986). Two combinations were ultimately selected for further ELISA method optimization: ① capture antibody 2G11 and detection antibody HRP-1A3, named monoclonal antibody-monoclonal antibody sandwich (2G11 / HRP-1A3); ② capture antibody 5H5 and detection antibody HRP-pAb-SAL1, named monoclonal antibody-polyclonal antibody sandwich (5H5 / HRP-pAb-SAL1).

[0081] 3. Optimization of sandwich ELISA conditions The detection conditions for the two combinations mentioned above were systematically optimized using the chessboard titration method.

[0082] (1) Optimization results of combination one (2G11 / HRP-1A3): The results are as follows Figure 22 A~ Figure 22 As shown in C, the optimal concentration of the capture antibody for 2G11 / HRP-1A3 coating is 1 μg / mL; the optimal coating conditions are: incubation at 37°C for 4 hours; the optimal blocking conditions are: blocking with 3% skim milk powder at 37°C for 1 hour; the optimal antigen incubation time is 120 minutes; the optimal detection antibody dilution is 1:10000; and the optimal detection antibody incubation time is 120 minutes.

[0083] (2) Optimization results of combination two (5H5 / HRP-pAb-SAL1): The results are as follows Figure 23 A~ Figure 23 As shown in C, the optimal concentration of the capture antibody for coating 5H5 / HRP-pAb-SAL1 is 2 μg / mL; the optimal coating conditions are: incubation at 37°C for 1 hour; the optimal blocking conditions are: blocking with 1% BSA at 37°C for 2 hours; the optimal antigen incubation time is 120 minutes; the optimal detection antibody dilution is 1:6000; and the optimal detection antibody incubation time is 90 minutes.

[0084] Example 9: A kit for detecting early pregnancy status in sows The kit for detecting early pregnancy status in sows in this embodiment includes either a monoclonal antibody-monoclonal antibody sandwich ELISA kit or a monoclonal antibody-polyclonal antibody sandwich ELISA kit. The monoclonal antibody-monoclonal antibody sandwich ELISA kit includes monoclonal antibody 2G11 (as a capture antibody) and HRP-labeled monoclonal antibody HRP-1A3 (as a detection antibody). The monoclonal antibody-polyclonal antibody sandwich ELISA kit includes the monoclonal antibody 5H5 (as a capture antibody) and the HRP-labeled polyclonal antibody HRP-pAb-SAL1 (as a detection antibody).

[0085] Example 10: ELISA Detection Method 1. Monoclonal antibody-monoclonal antibody sandwich ELISA detection method ① Coating: Dilute the capture antibody (monoclonal antibody 2G11) with coating buffer to a working concentration of 1 μg / mL, add it to the wells of the ELISA plate, and incubate at 37°C for 4 hours; ② Sealing: Add 3% skim milk powder and seal at 37℃ for 1 hour; ③ Sample addition: Add the sample to be tested and incubate at 37℃ for 2 hours; ④ Add detection antibody: Add detection antibody (HRP-1A3) diluted 1:10000 and incubate at 37°C for 2 hours; ⑤ Development: Add TMB substrate development solution and develop at 37°C in the dark for 15 minutes; ⑥ Termination and detection: Add stop solution and measure absorbance at 450 nm wavelength (OD450nm) using an ELISA reader.

[0086] 2. Monoclonal antibody-polyclonal antibody sandwich ELISA detection method ① Coating: Dilute the capture antibody (monoclonal antibody 5H5) with coating buffer to a working concentration of 2 μg / mL, add it to the wells of the ELISA plate, and incubate at 37°C for 1 hour; ② Blocking: Add 1% BSA for blocking, and block at 37℃ for 2 hours; ③ Sample addition: Add the sample to be tested and incubate at 37℃ for 2 hours; ④ Add detection antibody: Add detection antibody (HRP-pAb-SAL) diluted 1:6000 and incubate at 37°C for 1.5 hours; ⑤ Development: Add TMB substrate development solution and develop at 37°C in the dark for 15 minutes; ⑥ Termination and detection: Add stop solution and measure absorbance at 450 nm wavelength (OD450nm) using an ELISA reader.

[0087] 3. Establishment of Standard Curve Recombinant SAL1 protein dilution buffer (1–2048 ng / mL as the test sample) was used for ELISA detection via steps 1 and 2, respectively. ELISA standard curves were generated and fitted using a four-parameter logistic regression (4PL) model. The results are as follows: Figure 22 D and Figure 23 As shown in D: The standard curve equation for the monoclonal antibody-monoclonal antibody sandwich ELISA detection method is: Y = (3.367 - 0.03331) / [1 + (X / 53.66)^0.7327] + 0.03331, R 2 =0.995; The standard curve equation for the monoclonal antibody-polyclonal antibody sandwich ELISA detection method is: Y = (3.119 - 0.02106) / [1 + (X / 25.16)^0.8597] + 0.02106, R 2 =0.996.

[0088] 4. Methodological Evaluation (1) Determination of detection limit and quantitation limit The limits of detection (LOD) and quantitation (LOQ) of the two ELISA methods were determined using blank PBS solutions (n=30). LOD was calculated as the average absorbance of the blank samples plus three times the standard deviation, and LOQ was calculated as the average absorbance of the blank samples plus ten times the standard deviation.

[0089] Table 2. LOD and LOQ determination in monoclonal antibody-monoclonal antibody sandwich ELISA assay. Table 3. LOD and LOQ determination in monoclonal antibody-polyclonal antibody sandwich ELISA methods. The results are shown in Tables 2 and 3. For the monoclonal antibody-monoclonal antibody sandwich ELISA method, the LOD was 0.248 ng / mL and the LOQ was 0.525 ng / mL. For the monoclonal antibody-polyclonal antibody sandwich ELISA method, the LOD was 0.226 ng / mL and the LOQ was 0.346 ng / mL.

[0090] (2) Precision determination The precision of the two ELISA detection methods was assessed by measuring the within-group and between-group coefficients of variation (CV%) of SAL1 protein samples with different concentrations.

[0091] Table 4. Determination of within-group coefficient of variation in monoclonal antibody-monoclonal antibody sandwich ELISA detection method Table 5. Determination of Coefficient of Variation Between Groups in Monoclonal Antibody-Monoclonal Antibody Sandwich ELISA Detection Method Table 6. Determination of within-group coefficient of variation in monoclonal antibody-polyclonal antibody sandwich ELISA detection method Table 7. Determination of Coefficient of Variation Between Groups in Monoclonal Antibody-Multiclonal Antibody Sandwich ELISA Detection Method The results are shown in Tables 4-7. For the monoclonal antibody-monoclonal antibody sandwich ELISA method, the intra-group coefficient of variation (COP) ranged from 0.50% to 7.40%, and the inter-group COP ranged from 0.30% to 4.50%. For the monoclonal antibody-polyclonal antibody sandwich ELISA method, the COP ranged from 0.30% to 2.10%, and the inter-group COP ranged from 0.60% to 4.10%. These results indicate that both methods exhibit good repeatability and stability.

[0092] (3) Determination of spiked recovery rate Recombinant SAL1 protein (50 ng / mL and 100 ng / mL) was added to the saliva matrix of pigs, and the spiked recovery rate was determined.

[0093] Table 8. Recovery rate determination of monoclonal antibody-monoclonal antibody sandwich ELISA detection method Table 9. Recovery rate determination of monoclonal antibody-polyclonal antibody sandwich ELISA method The results are shown in Tables 8 and 9. The recovery rate of the monoclonal antibody-monoclonal antibody sandwich ELISA method was 97.05%–115.32%, and the recovery rate of the monoclonal antibody-double antibody sandwich ELISA method was 86.15%–89.81%. The results indicate that both methods have good accuracy and matrix compatibility.

[0094] Example 11: ELISA detection method to verify the pregnancy status of sows 1. Sample collection (1) Select multiparous Landrace-Large White crossbred sows with normal estrus and good reproductive performance, and inseminate them using artificial insemination. The day of insemination is recorded as day 0 of insemination, and saliva samples are collected from the sows on day 15 after insemination. The pregnancy status of the sows is confirmed by ultrasound on day 28 and day 55.

[0095] (2) Saliva sample collection method: Use sterile gauze to collect saliva samples non-invasively from the sow's mouth. Insert the gauze into the sow's mouth and let her chew fully to ensure full absorption of saliva. Then squeeze the saliva into a 5 mL cryovial. Centrifuge at 12,000 rpm for 10 minutes at 4°C to remove debris. Collect the supernatant and store it at -80°C for later use.

[0096] A total of 100 saliva samples were collected, of which 80 were confirmed to be pregnant by ultrasound and 20 were not.

[0097] 2. Diagnostic efficacy assessment One hundred clinical saliva samples were tested using the kit from Example 9 and two ELISA detection methods established in Example 10. The OD450nm value was used as the detection variable and the actual pregnancy status was used as the state variable. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was calculated. The pregnancy status of sows was determined by combining the critical value determined by the ROC curve.

[0098] Table 10 Parameters of Logistic Binary Logistic Regression Model The results are as follows Figure 24 As shown in Table 10, the AUC value of the combined Logistic binary logistic regression model analysis for the monoclonal antibody-monoclonal antibody sandwich ELISA detection method and the monoclonal antibody-double antibody sandwich ELISA detection method was 0.918 (95% CI: 0.874~0.962).

[0099] The concentration of SAL1 protein in clinical saliva samples was measured, and the results are shown in Table 11.

[0100] Table 11 SAL1 protein concentration in clinical saliva samples Therefore, when the kit is a monoclonal antibody sandwich ELISA kit, if the SAL1 protein concentration is greater than 1.136 μg / mL, the sow being tested is pregnant; otherwise, the sow being tested is non-pregnant. The accuracy rate for pregnancy diagnosis is 85.1%, the sensitivity is 81.25%, and the specificity is 80%. When the kit is a monoclonal antibody-polyclonal antibody sandwich ELISA kit, the sows being tested are considered pregnant when the SAL1 protein concentration is greater than 0.907 μg / mL; otherwise, they are considered non-pregnant. The accuracy rate for pregnancy diagnosis is 81.3%, the sensitivity is 52.5%, and the specificity is 100%. The results showed that both the anti-monoclonal antibody sandwich ELISA method and the monoclonal antibody-double antibody sandwich ELISA method can be used for early pregnancy diagnosis in sows 15 days after mating. Among them, the monoclonal antibody-monoclonal antibody sandwich ELISA method has higher diagnostic accuracy.

[0101] Example 12 Based on the above embodiments, this embodiment provides a computer program product related to the early pregnancy status of sows, used to detect the early pregnancy status of sows, with early pregnancy occurring on the 15th day after artificial insemination. It includes the following steps: (1) Collect saliva samples from the sows to be tested, and centrifuge to collect the supernatant; (2) The collected supernatant was subjected to ELISA detection using the kit from Example 9 to obtain the absorbance at a wavelength of 450 nm. The specific process is as follows: I. Dilute the capture antibody and add it to the wells of the ELISA plate for incubation; II. After adding blocking buffer to the wells of the ELISA plate, block at 2-4℃; III. Add the collected supernatant to the wells of the ELISA plate and incubate. IV. Add the detection antibody to the wells of the ELISA plate and incubate. V. Add TMB substrate chromogenic solution to the wells of the ELISA plate and perform chromogenic development in the dark; VI. Add stop solution to the wells of the ELISA plate and measure the absorbance at 450 nm using an ELISA reader.

[0102] (3) Calculate the concentration of SAL1 protein in the saliva sample based on absorbance; (4) Make a judgment based on the SAL1 protein concentration: When the kit is a monoclonal antibody sandwich ELISA kit, if the SAL1 protein concentration is greater than 1.136 μg / mL, the sow to be tested is pregnant; otherwise, the sow to be tested is non-pregnant. When the kit is a monoclonal antibody-polyclonal antibody sandwich ELISA kit, the sow to be tested is considered pregnant if the SAL1 protein concentration is greater than 0.907 μg / mL, otherwise the sow to be tested is considered non-pregnant.

[0103] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An anti-SAL1 protein monoclonal antibody, characterized in that: The amino acid sequence of the SAL1 protein is shown in SEQ ID NO: 2; The monoclonal antibodies include monoclonal antibody 1A3, monoclonal antibody 2G11 and monoclonal antibody 5H5; The monoclonal antibody 1A3 includes a 1A3 heavy chain variable region and a 1A3 light chain variable region, and the 1A3 heavy chain variable region includes three heavy chain complementarity-determining regions 1A3-CDR-VH, which are as follows: 1A3-CDR1-VH: GYTFTEYT; 1A3-CDR2-VH: INPNGVT; 1A3-CDR3-VH: ARSEVNPFTY; The 1A3 light chain variable region includes three light chain complementarity determination regions 1A3-CDR-VL, which are: 1A3-CDR1-VL: QTIVHSNGNTF; 1A3-CDR2-VL: KVS; 1A3-CDR3-VL: FQGSHVPPT; The monoclonal antibody 2G11 includes a 2G11 heavy chain variable region and a 2G11 light chain variable region, and the 2G11 heavy chain variable region includes three heavy chain complementarity-determining regions 2G11-CDR-VH, which are as follows: 2G11-CDR1-VH: GYTFTSYW; 2G11-CDR2-VH: INPSNGRT; 2G11-CDR3-VH: TRGLSDY; The 2G11 light chain variable region includes three light chain complementarity determination regions 2G11-CDR-VL, which are: 2G11-CDR1-VL: QDINSY; 2G11-CDR2-VL: RAN; 2G11-CDR3-VL: LQYDEFPYT; The monoclonal antibody 5H5 includes a 5H5 heavy chain variable region and a 5H5 light chain variable region, and the 5H5 heavy chain variable region includes three heavy chain complementarity-determining regions 5H5-CDR-VH, which are as follows: 5H5-CDR1-VH: GYTFTSYW; 5H5-CDR2-VH:INPSNGRT; 5H5-CDR3-VH: TRGLSDY; The 5H5 light chain variable region includes three light chain complementarity-determining regions, 5H5-CDR-VL, which are: 5H5-CDR1-VL: QDINSY; 5H5-CDR2-VL: RAN; 5H5-CDR3-VL:LQYDEFPYT.

2. The antibody according to claim 1, characterized in that: In the monoclonal antibody 1A3, the amino acid sequence of the heavy chain variable region of 1A3 is shown in SEQ ID NO: 3, and the amino acid sequence of the light chain variable region of 1A3 is shown in SEQ ID NO: 4; The monoclonal antibody 2G11 has the following amino acid sequence: heavy chain variable region as shown in SEQ ID NO: 5, and light chain variable region as shown in SEQ ID NO:

6. The monoclonal antibody 5H5 has the following amino acid sequence: heavy chain variable region as shown in SEQ ID NO: 7, and light chain variable region as shown in SEQ ID NO:

8.

3. A method for preparing a polyclonal antibody pAb-SAL1, characterized in that: Includes the following steps: i. Use the SAL1 protein, whose amino acid sequence is shown in SEQ ID NO: 2, as an antigen to immunize animals and collect animal serum; ii. Animal serum was purified by antigen-conjugated agarose affinity chromatography and then dialyzed to obtain the purified polyclonal antibody pAb-SAL1.

4. A polyclonal antibody pAb-SAL1 prepared by the preparation method of claim 3.

5. The application of an antibody in the preparation of a kit for detecting early pregnancy status in sows, characterized in that: The antibodies include monoclonal antibodies 1A3, 2G11, and 5H5 as described in claim 1, and polyclonal antibody pAb-SAL1 as described in claim 4.

6. A kit for detecting early pregnancy status in sows, characterized in that: The kit includes either a monoclonal antibody-monoclonal antibody sandwich ELISA kit or a monoclonal antibody-polyclonal antibody sandwich ELISA kit. The monoclonal antibody-monoclonal antibody sandwich ELISA kit comprises the monoclonal antibody 2G11 as described in claim 1 and the HRP-labeled monoclonal antibody 1A3; The monoclonal antibody-polyclonal antibody sandwich ELISA kit comprises the monoclonal antibody 5H5 of claim 1 and the HRP-labeled polyclonal antibody pAb-SAL1 of claim 4; The early pregnancy period is defined as 12 to 18 days after artificial insemination.

7. A method for ELISA detection using the kit described in claim 6, characterized in that: Includes the following steps: (1) The capture antibody was diluted and added to the wells of an ELISA plate for incubation; the capture antibody was monoclonal antibody 2G11 or monoclonal antibody 5H5; (2) After adding blocking solution to the wells of the ELISA plate, block at 2-4℃; (3) Add the sample to be tested to the well of the ELISA plate and incubate; (4) Add detection antibody to the well of the ELISA plate and incubate; when the capture antibody is monoclonal antibody 2G11, the detection antibody is HRP-labeled monoclonal antibody 1A3; when the capture antibody is monoclonal antibody 5H5, the detection antibody is HRP-labeled polyclonal antibody pAb-SAL1. (5) Add TMB substrate chromogenic solution to the wells of the microplate and perform chromogenic development in the dark; (6) Add stop solution to the wells of the ELISA plate and measure the absorbance at 450 nm wavelength using an ELISA reader.

8. The use of the kit according to claim 6 in the non-diagnostic detection of early pregnancy status in sows.

9. A computer program product related to the early pregnancy status of sows, characterized in that: The computer program product is used to detect the early pregnancy status of sows, wherein the early pregnancy period is 12 to 18 days after artificial insemination; Includes the following steps: ① Collect saliva samples from the sows to be tested, and centrifuge to collect the supernatant; ② The collected supernatant was subjected to ELISA detection using the kit described in claim 6 to obtain the absorbance at a wavelength of 450 nm; ③ Calculate the concentration of SAL1 protein in the saliva sample based on absorbance; ④ Make a judgment based on the SAL1 protein concentration: When the kit is a monoclonal antibody-monoclonal antibody sandwich ELISA kit, if the SAL1 protein concentration is greater than 1.136 μg / mL, the sow to be tested is pregnant; otherwise, the sow to be tested is non-pregnant. When the kit is a monoclonal antibody-polyclonal antibody sandwich ELISA kit, if the SAL1 protein concentration is greater than 0.907 μg / mL, the sow to be tested is pregnant; otherwise, the sow to be tested is non-pregnant.

10. The computer program product according to claim 9, characterized in that: The ELISA detection method is as follows: I. Dilute the capture antibody and add it to the wells of an ELISA plate for incubation; the capture antibody is monoclonal antibody 2G11 or monoclonal antibody 5H5; II. After adding blocking buffer to the wells of the ELISA plate, block at 2-4℃; III. Add the collected supernatant to the wells of the ELISA plate and incubate. IV. Add the detection antibody to the wells of the ELISA plate and incubate. V. Add TMB substrate chromogenic solution to the wells of the ELISA plate and perform chromogenic development in the dark; when the capture antibody is monoclonal antibody 2G11, the detection antibody is HRP-labeled monoclonal antibody 1A3; when the capture antibody is monoclonal antibody 5H5, the detection antibody is HRP-labeled polyclonal antibody pAb-SAL1. VI. Add stop solution to the wells of the ELISA plate and measure the absorbance at 450 nm using an ELISA reader.

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