WO2023098215A1 - 妊娠相关糖蛋白的单克隆抗体及其在牛早期怀孕检测中的应用 - Google Patents

妊娠相关糖蛋白的单克隆抗体及其在牛早期怀孕检测中的应用 Download PDF

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WO2023098215A1
WO2023098215A1 PCT/CN2022/117833 CN2022117833W WO2023098215A1 WO 2023098215 A1 WO2023098215 A1 WO 2023098215A1 CN 2022117833 W CN2022117833 W CN 2022117833W WO 2023098215 A1 WO2023098215 A1 WO 2023098215A1
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monoclonal antibody
pregnancy
test strip
pag
test
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PCT/CN2022/117833
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English (en)
French (fr)
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付辉
黄永健
谷琳琳
王伟维
马涛
李细清
梁秋乙
杨琳琳
曾楚怡
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深圳市爱医生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to the field of antibodies, more specifically, the invention relates to a monoclonal antibody of pregnancy-related glycoprotein and its application in early pregnancy detection of cattle.
  • calving interval is an important indicator to measure the reproductive ability of dairy cows. Through early and accurate pregnancy detection of dairy cows, it can help the ranch to find empty cows and pregnant cows as soon as possible, re-breed the non-pregnant cows in time, and help the ranch maximize milk production and economic benefits by shortening the calving interval.
  • B-ultrasound and rectal palpation are common methods for judging pregnancy at present, but these two methods can only be carried out at least 35 days and 45 days after mating, and the accuracy of the method is closely related to the experience and skills of the operator. lead to early death of the embryo.
  • Early pregnancy-associated glycoprotein is currently a commonly used diagnostic indicator for early pregnancy in dairy cows.
  • the protein is produced by the trophoblast of the fetal placenta of dairy cows and secreted into the peripheral blood of the female animal. It can be detected in the blood of the female animal about 28 days after mating. Changes in the concentration of PAG can be used to judge the pregnancy status of dairy cows as early as possible.
  • the products on the market to identify the pregnancy status of dairy cows by detecting PAG are all enzyme-linked immunosorbent assay (ELISA) detection kits.
  • the ELISA method takes a long time to detect (more than 3 hours), and the operation is cumbersome. It requires a special test site, equipment and professional operators, and is not suitable for grassroots and small and medium-sized pastures. At present, there is no rapid detection product based on the principle of immunochromatography.
  • the key to any immunological detection method is the preparation of specific antibodies.
  • this field urgently needs more specific PAG antibodies with high sensitivity and good stability.
  • the invention provides a product for rapid detection of early pregnancy in cattle with simple operation, good stability and high accuracy.
  • the present invention provides a monoclonal antibody that specifically binds to pregnancy-related glycoproteins and a detection test paper card containing the monoclonal antibody, so as to quickly and accurately detect early pregnancy in cattle.
  • the present invention provides a hybridoma cell, which was preserved on October 12, 2021 at the Guangdong Provincial Microbial Bacteria located on the 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City. Species Collection Center, the collection number is GDMCC No: 61975.
  • the present invention provides a hybridoma cell, which was preserved on October 12, 2021 at the Guangdongzhou Microbial Culture Collection located on the 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City Center, the deposit number is GDMCC No: 61976.
  • the present invention provides a monoclonal antibody specifically binding to bovine pregnancy-associated glycoprotein (PAG), wherein the monoclonal antibody is produced by the hybridoma cell of the first aspect or the second aspect of the present invention.
  • PAG bovine pregnancy-associated glycoprotein
  • the present invention provides a combination of monoclonal antibodies specifically binding to bovine pregnancy-associated glycoprotein (PAG), wherein the combination of antibodies includes a monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61975 Antibodies and monoclonal antibodies produced by hybridoma cells with a deposit number of GDMCC No: 61976.
  • PAG bovine pregnancy-associated glycoprotein
  • the present invention provides a test strip for detecting bovine pregnancy-associated glycoprotein (PAG), the test strip comprising a sample pad, a binding pad, a coating film and an absorbent pad, on the binding pad Coated with a labeled monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61975, and coated with a monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61976 Antibodies serve as detection lines.
  • PAG bovine pregnancy-associated glycoprotein
  • the present invention provides a test paper card for detecting bovine pregnancy-associated glycoprotein (PAG), the test paper card comprising the test paper strip of the fifth aspect of the present invention, and an upper shell and a lower shell composed of buckles.
  • PAG bovine pregnancy-associated glycoprotein
  • the present invention provides the application of the hybridoma cells of the first aspect or the second aspect, the monoclonal antibody of the third aspect, or the monoclonal antibody combination of the fourth aspect of the present invention in detecting early pregnancy in cattle.
  • the monoclonal antibody of the present invention can specifically bind to pregnancy-associated glycoprotein, therefore, the monoclonal antibody of the present invention can be used to prepare detection test strips based on antibody antigen immune reaction, to provide simple operation, good stability, accuracy High rapid detection of early pregnancy products in cattle.
  • Fig. 1 shows the structural representation of colloidal gold detection test strip
  • Fig. 2 is a schematic structural view showing a fluorescent immunoassay test strip
  • Fig. 3 is the structural representation showing the shell of colloidal gold detection test paper card and fluorescence detection test paper card;
  • Fig. 4 is a schematic diagram of the result determination of the colloidal gold detection test paper card.
  • the purpose of the present invention is to provide a monoclonal antibody capable of specifically binding to pregnancy-related glycoproteins so as to prepare a detection test card using the monoclonal antibody for rapid and accurate detection of early pregnancy in cattle.
  • the present invention provides a hybridoma cell, which was preserved on October 12, 2021 at the Guangdong Provincial Microbial Bacteria located on the 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City. Species Collection Center, the collection number is GDMCC No: 61975.
  • the present invention provides a hybridoma cell, which was preserved on October 12, 2021 at the Guangdongzhou Microbial Culture Collection located on the 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City Center, the deposit number is GDMCC No: 61976.
  • the preparation method of the hybridoma cell of the present invention is as follows: first, immunize the mouse with bovine pregnancy-associated glycoprotein (PAG), then carry out cell fusion with the splenocyte of the mouse and the myeloma cell Sp2/0, through limiting dilution, cloning And after indirect ELISA screening, hybridoma cells capable of secreting monoclonal antibodies were obtained.
  • PAG bovine pregnancy-associated glycoprotein
  • the inventor used PAG shown in SEQ ID NO: 1 as an antigen to immunize mice multiple times, used the spleen cells of the mice for cell fusion to prepare hybridoma cells, and screened positive hybridoma cell clones by indirect ELISA detection method, Thus, monoclonal antibodies capable of specifically binding to pregnancy-associated glycoproteins were prepared.
  • the present invention provides a monoclonal antibody specifically binding to bovine pregnancy-associated glycoprotein (PAG), wherein the monoclonal antibody is produced by the hybridoma cell in the first or second aspect of the present invention.
  • PAG bovine pregnancy-associated glycoprotein
  • the term "pregnancy-associated glycoprotein (PAG)” is a type of protein synthesized and secreted by ruminant fetal placental trophoblast cells, which plays an important role in pregnancy.
  • PAG pregnancy-associated glycoprotein
  • the inventors use bovine pregnancy-associated protein (PAG) with an amino acid sequence as shown in SEQ ID NO: 1 to immunize mice.
  • PAG bovine pregnancy-associated protein
  • SEQ ID NO: 1 bovine pregnancy-associated protein
  • the source of the PAG is not limited in the present invention, it can be extracted from natural cells or recombinant cells, and can also be artificially synthesized.
  • SEQ ID NO: 1 The sequence of SEQ ID NO: 1 is as follows: MKWLGLLGLVALSECMVIIPVTQMKAIRETLWERNLLTNFSEEHPCSLSQNATNDQNIVYHPLRNYKDLVYIGTIAIGTPPQEFRVIFDTGSSDLWVPSIHCHSPSCLTHSLFNPRTSTTFKLLNNIIDLIYASGRIKGVLGQDIIQVMWKPEAAGPGSNCCLQNRCRRSW RCLSFRKAPNGCRSAWKDSVLGETMPLLTYKLFVANAGRGLGCGFAASLPMKSRRFIMVSRRENGSVLMFGGVDHSYHTGKLNWVPVSRTHYWQITIGRISMNGKLIACKRGCQAIMDTGTTFLLGPSRHIAKIQRLIRIRPFGSLQYTVPCNITSTLPPLIFTIKGIDYPVPAQAYIHKMRGQS.
  • Pregnancy-associated glycoprotein is a commonly used diagnostic indicator for early pregnancy in dairy cows. About 28 days after mating, the pregnancy status of dairy cows can be diagnosed as early as possible by detecting the changes in the concentration of PAG in the blood of the female animal. According to the existing research findings, the diagnostic rate of pregnancy in dairy cows 35 days after mating is 93.03% by detecting PAG, indicating that PAG can be used as a marker for the diagnosis and detection of bovine pregnancy.
  • any immunological detection method of PAG is specific antibody, so the monoclonal antibody provided by the present invention can be used to detect bovine early pregnancy by various immunoassay methods, and said detection method includes but not limited to immunochromatographic technique , enzyme-linked immunosorbent assay, western blot, flow cytometry, etc.
  • the inventors used the IsoStrip Mouse Monoclonal Antibody Isotyping Kit to identify the subtypes of the two monoclonal antibodies, and determined that the subtypes of the two monoclonal antibodies were both IgG2b.
  • the monoclonal antibody is of the IgG2b subtype.
  • the present invention provides a combination of monoclonal antibodies specifically binding to bovine pregnancy-associated glycoprotein (PAG), wherein the combination of antibodies includes a monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61975 Antibody (Antibody 1) and a monoclonal antibody (Antibody 2) produced by hybridoma cells with a deposit number of GDMCC No: 61976.
  • PAG bovine pregnancy-associated glycoprotein
  • the present invention provides a test strip for detecting bovine pregnancy-associated glycoprotein (PAG), the test strip comprising a sample pad, a binding pad, a coating film and an absorbent pad, on the binding pad Coated with a labeled monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61975, and coated with a monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61976 Antibodies serve as detection lines.
  • PAG bovine pregnancy-associated glycoprotein
  • the test strip is formed by adhering the sample pad, binding pad, coating film and water-absorbing pad to the PVC bottom plate in sequence and connecting them by overlapping (as shown in Figure 1 or Figure 2).
  • the coated membrane usually refers to a membrane formed by coating antibodies on a nitrocellulose membrane, and two kinds of antibodies are coated on the coated membrane, and the first antibody is a hybridoma with a preservation number of GDMCC No: 61976
  • the monoclonal antibody produced by the cell is used as the detection line T
  • the second antibody is an antibody against the monoclonal antibody used as the detection line, which can be a monoclonal antibody or a polyclonal antibody, such as a goat anti-mouse IgG polyclonal antibody, which is used as a qualitative Control line C.
  • the test strip may also include tools or reagents for immunological analysis, such as labeling substances that generate detectable signals, solubilizers, cleaning agents, and the like.
  • the test strip may be a colloidal gold detection test strip or a fluorescent immunoassay test strip.
  • Colloidal gold immunochromatography is a rapid detection method based on antigen-antibody immune reaction. Antigens or antibodies are marked on red colloidal gold particles, and the results can be interpreted by visual observation. Fluorescent microsphere immunochromatography technology is a rapid detection method based on antigen-antibody immune reaction. Antigen or antibody is marked on fluorescent microspheres, and the result is interpreted by a fluorescence reader. The basic principles of the two are the same, and the sample pretreatment and detection methods are basically the same. The only difference is the antigen or antibody marker and the interpretation method. It can be completed), good stability and high accuracy.
  • the monoclonal antibody coated on the binding pad is labeled with colloidal gold; in the case where the test strip is a fluorescent immunodetection test strip Next, the monoclonal antibody coated on the conjugate pad is fluorescently labeled.
  • the labeling concentration of the monoclonal antibody coated on the binding pad is 7 ⁇ g/ml-25 ⁇ g/ml, and the concentration of the monoclonal antibody coated on the coating membrane is 0.5 mg/ml-0.6 mg/ml.
  • the present invention provides a test paper card for detecting bovine pregnancy-associated glycoprotein (PAG), the test paper card comprising the test paper strip of the fifth aspect of the present invention, and an upper shell and a lower shell Composed buckle (as shown in Figure 3).
  • PAG bovine pregnancy-associated glycoprotein
  • the present invention provides the application of the hybridoma cells of the first aspect or the second aspect, the monoclonal antibody of the third aspect, or the monoclonal antibody combination of the fourth aspect of the present invention in detecting early pregnancy in cattle.
  • the present invention adopts the immunochromatographic technique based on the double-antibody sandwich method.
  • the serum or plasma sample of the dairy cow to be tested is applied to the sample pad of the test strip, and when the sample flows to the binding pad, if there is PAG in the serum or plasma, it will be labeled (such as by colloidal gold or fluorescent Labeled) PAG monoclonal antibody (labeled antibody) combined to form a labeled antibody-PAG antigen complex.
  • the previously formed labeled antibody-PAG antigen complex will further combine with the PAG monoclonal antibody (T line antibody) coated as the detection line T to form a complex of the three (labeled antibody -PAG-T-line antibody) to visualize T-line. If there is no PAG in the blood or the content is lower than the detection limit of the product, the T line will not show color.
  • Immunogen preparation and purification Through gene cloning, construct PAG recombinant prokaryotic expression vector to obtain engineering bacteria capable of stably expressing PAG. After centrifugation and sonication, the bacterial solution induced to express was purified using Sepharose FF prepacked column, and the purified PAG (SEQ ID NO: 1) was used as the immunogen.
  • Cell fusion mix spleen cells and myeloma cells Sp2/0 according to the ratio of cell number 1:5, add incomplete RPMI-1640 culture medium to 30ml, mix well, centrifuge at 1200r/m for 10 minutes, and put the Aspirate the supernatant and lightly tap the bottom of the tube on the palm of your hand to loosen the precipitated cells evenly.
  • Add distilled water at 37°C to the beaker place the centrifuge tube containing the mixed cells in a water bath, add 1 ml of 50% PEG preheated at 37°C within 90 seconds, rotate the centrifuge tube while adding drops, and then let it stand for 1 minute.
  • hybridoma cell lines After fusion, the culture medium is replaced every 2-4 days. After 7-8 days after fusion, the cells are observed, and the cell wells with surviving hybridoma cells are marked. When more than 1/5 of the bottom of the well is covered, take 100 ⁇ l of the cell supernatant as the primary antibody, use PAG as the antigen to coat the microtiter plate, and use indirect ELISA to test the cell supernatant, and select hybridomas with positive test results and high titers cell.
  • the hybridoma cells in the positive wells screened by indirect ELISA were subcloned according to the limiting dilution method, and after at least 3 subclones, cell lines secreting monoclonal antibodies were obtained.
  • Ascites preparation and antibody purification take 7-week-old BALB/c female mice, inject paraffin oil into the peritoneal cavity of the mice, 0.5m1/mouse, and inoculate the intraperitoneal hybridization in the logarithmic growth phase diluted with serum-free medium after 7 days Tumor cells (from step 4), after an interval of 7 days, observe the production of ascites in mice every day, collect ascites with a 16-gauge needle, centrifuge the ascites, and take the supernatant.
  • the antibody was purified by saturated ammonium sulfate precipitation method, and 10 monoclonal antibodies (No. 1-10) were obtained after purification.
  • Antibody affinity determination the purified 10 monoclonal antibodies were labeled with colloidal gold, and then sprayed on glass fibers to prepare gold-binding pads. Assemble the nitrocellulose membrane coated with PAG antigen and 10 kinds of gold-binding pads to form a test card, drop PBS into the sample hole of the test card, and wait for 10 minutes for observation. According to the color depth of the PAG antigen coating position, exclude antibodies with no color or light color, and select antibodies with higher color depth (No. 1, 2, 3, 5, 8 and 10) for the next step of pair screening .
  • the experimental results in Table 1 below show that the combination of antibody 1 labeled with gold (conjugation pad)-antibody 2 envelope (coating membrane) has the highest sensitivity in detecting PAG antigen, which is less than 0.5ng/ml, which meets the needs of product development.
  • the antibody 1 is a monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61975
  • the antibody 2 is a monoclonal antibody produced by a hybridoma cell with a deposit number of GDMCC No: 61976.
  • Antibody subtype identification IsoStrip Mouse Monoclonal Antibody Isotyping Kit was used to identify the subtype of the two monoclonal antibodies, both of which were IgG2b.
  • the colloidal gold immunochromatography technology based on the double-antibody sandwich method can be used to quickly identify pregnant cows and empty pregnant cows on the 28th day after breeding. If the cow is pregnant, the PAG in the blood reaches a high level. During the flow of the sample, the PAG combines with the colloidal gold-labeled PAG monoclonal antibody, and when it continues to flow to the T area of the detection line, it combines with the PAG antibody coated with the detection line T to form The complex of the three (colloidal gold-labeled antibody-PAG-T line coated antibody), the T line shows a red band, and the color depth of the T line is greater than that of the B line; if the cow is not pregnant, there is no PAG in the blood or the content is low If it is lower than the threshold, the T line will not develop color or the color depth will be smaller than that of the B line.
  • the color of quality control line C indicates that the product is effective, and the color of quality control line C does not show that the product is invalid.
  • colloidal gold binding pad put the colloidal gold solution on a magnetic stirrer, adjust the pH to about 7.5 with 0.2M K 2 CO 3 , add 7 ⁇ g/ml PAG monoclonal antibody 1 under stirring, and stir at room temperature for 30 minutes ; Add 10% bovine serum albumin (BSA) solution to a final concentration of 1%, stir at room temperature for 30 minutes; put the solution in a centrifuge at 4°C and 10,000r/min for 30 minutes, discard the supernatant, and use the same volume of gold complex solution for precipitation Resuspend and mix well.
  • BSA bovine serum albumin
  • Every 30ml of the resuspension is evenly coated on a glass cellulose film with a size of 25cm*30cm, placed on a clean screen window, and dried in a room temperature drying room with a humidity of 10%-30% for 10 hours before use.
  • coating film Dilute goat anti-mouse IgG polyclonal antibody and PAG monoclonal antibody 2 with coating solution to final concentrations of 0.3mg/ml and 0.5mg/ml respectively, and spray them in nitric acid according to the amount of 1 ⁇ l/cm On the corresponding position of the cellulose film, prepare C line and T line, put the film in a 37°C incubator and dry it for 20 hours, and set it aside.
  • sample pad treatment solution Prepare the sample pad treatment solution, apply each 30ml sample pad treatment solution evenly on the glass cellulose film with a size of 25cm*30cm, place it in a room temperature drying room with a humidity of 10%-30%, and dry it for 15 Hours - 18 hours, spare.
  • Test paper card assembly Cut the assembled large board into 3.85mm width with a strip cutter, put it into a plastic card case, pack the assembled card case together with desiccant into an aluminum foil bag, and seal it.
  • the test paper card is composed of a test paper strip and a buckle, and the buckle is composed of an upper shell and a lower shell (see Figure 3); Absorbent pad, colloidal gold binding pad, sample pad, colloidal gold binding pad coated with PAG monoclonal antibody-colloidal gold marker, nitrocellulose membrane coated with quality control line composed of goat anti-mouse IgG polyclonal antibody and Test line composed of PAG monoclonal antibody.
  • Processing of the sample to be tested collect bovine whole blood 28 days after mating, separate serum or plasma, and choose to stand at 4°C overnight for natural precipitation or place in a centrifuge at 2000rpm/min-3000rpm/min, centrifuge for 5 minutes-15 minutes , take the supernatant for later use.
  • Sample detection return the sample to be tested to room temperature, mix well and add 3 drops (about 90 ⁇ L) of serum or plasma (EDTA, heparin sodium) to the sample hole of the test card drop by drop with a sample dropper , wait for 20 minutes to observe the results, after more than 30 minutes the results are for reference only.
  • EDTA serum or plasma
  • the quality control line C develops color
  • the detection line T has the same color depth as the colorimetric line B. It is recommended to re-take blood and test again after 7 days.
  • Collect the bovine blood sample (1720 parts) of 28 days after mating from the pasture adopt the colloidal gold test paper card of the present invention and competing product ELISA kit to detect and calculate the colloidal gold test paper card of the present invention to the separated serum and plasma samples The coincidence rate with commercial ELISA kits.
  • Table 2 shows a schematic diagram of the calculation method of the coincidence rate between the two detection methods (specifically colloidal gold test strips or fluorescent test strips and commercial ELISA in this application).
  • Table 2 Schematic diagram of calculation of coincidence rate between test strip card of the present invention and commercialized ELISA kit
  • c is the number of samples whose detection result of the test paper card of the present invention is positive and the detection result of the commercialized ELISA kit is negative;
  • d is the number of samples whose detection results of the test paper card of the present invention and the commercialized ELISA kit are all negative.
  • Table 3 shows the coincidence rate between the colloidal gold test strip of the present invention and the commercialized ELISA kit.
  • colloidal gold test paper card of the present invention is compared with commercialization ELISA test kit, and sensitivity (being positive coincidence rate) has reached 98.99%, and specificity has reached 98.07% (being negative coincidence rate), The total compliance rate reached 98.66%.
  • the present invention adopts the fluorescent immunochromatography technology based on the double-antibody sandwich method, coats the fluorescent marker on the glass fiber, and coats the detection line (T line) and the quality control line (C line) of the nitrocellulose membrane respectively.
  • the corresponding antibody and polyclonal antibody were secondary antibodies. If the cow is pregnant, the PAG in the sample combines with the fluorescent marker to form a complex during the flow process, and combines with the antibody coated with the detection line T to generate a fluorescent signal.
  • the instrument collects the fluorescence signals on the test line (T line) and quality control line (C line), and converts the curve equation input into the instrument through the ID card, and the test interface displays a positive result.
  • coating film Dilute goat anti-mouse IgG polyclonal antibody and PAG monoclonal antibody 2 with coating solution to final concentrations of 0.6mg/ml and 0.6mg/ml respectively, and spray them in nitric acid according to the amount of 1 ⁇ l/cm On the corresponding position of the cellulose film, prepare C line and T line, put the film in a 37°C incubator and dry it for 20 hours, and set it aside.
  • sample pad treatment solution Prepare the sample pad treatment solution, evenly coat 30ml of the sample pad treatment solution on a glass cellulose membrane with a size of 25cm*30cm, place it in a room temperature drying room with a humidity of 10%-30%, and dry it for 15 Hours - 18 hours, spare.
  • Test paper card assembly Cut the assembled large board into 3.85mm width with a strip cutter, put it into a plastic card case, pack the assembled card case together with desiccant into an aluminum foil bag, and seal it.
  • test paper card composition the test paper card is composed of a test paper card and a buckle, and the buckle is composed of an upper shell and a lower shell; the test paper card is composed of a PVC bottom plate, a nitrocellulose membrane, an absorbent pad, a fluorescent binding pad, and a sample pad , the fluorescent binding pad is coated with PAG monoclonal antibody-fluorescent marker, and the nitrocellulose membrane is coated with a quality control line composed of goat anti-mouse IgG polyclonal antibody and a detection line composed of PAG monoclonal antibody.
  • test modes can be selected according to actual testing requirements.
  • Rapid test (suitable for testing multiple samples at the same time): return the sample to be tested to room temperature, mix well and pipette 100 ⁇ L of serum or plasma (EDTA, heparin sodium) to be tested into the sample hole of the test card , start the reaction, insert the test card into the instrument after 15 minutes (the direction of the sample hole is facing inward), select the quick test mode, click to start the test, after the test is completed, the test result will be displayed on the instrument interface, and the test result with a reaction time of more than 20 minutes is only for reference.
  • EDTA serum or plasma
  • Standard test (suitable for testing a single sample): Return the sample to be tested to room temperature, mix it evenly, and use a pipette to pipette 100 ⁇ L of serum or plasma (EDTA, heparin sodium) to be tested into the sample hole of the test card. Insert the card into the instrument (the direction of the sample hole faces inward), select the standard test mode, click to start the test, the instrument will automatically count for 15 minutes, and the test result will be displayed on the instrument interface after 15 minutes.
  • EDTA serum or plasma
  • Embryo death is a relatively common phenomenon in the early stages of pregnancy of dairy cows. In the early stages of embryonic death, pregnancy-associated glycoproteins will still circulate in the cows. Therefore, it is recommended that cows with weak positive samples be re-taken blood after 7-10 days. Re-examination, if the result is still weakly positive after the re-examination, it is judged to be an empty bosom.
  • the best labeled amount of PAG monoclonal antibody is 25 ⁇ g/ml; the coating solution is PB (0.02mol/L, pH 7.4)+2% trehalose solution; the best goat anti-mouse IgG polyclonal antibody for quality control line
  • the coating concentration is 0.6mg/ml, and the best coating concentration of the PAG monoclonal antibody for the preparation of the test line is 0.6mg/ml; the membrane drying time is selected to be dried at 37°C for 20 hours; the best sample volume is 100 ⁇ l; The optimal reaction time is 15 minutes.
  • the detection fluorescence detection card prepared under this condition has the characteristics of high sensitivity, strong specificity and good stability.
  • the fluorescent test paper card of the present invention has a sensitivity (i.e. positive coincidence rate) of 98.39% compared with commercial ELISA kits, and a specificity of 98.23% (i.e. negative coincidence rate).
  • the compliance rate reached 98.33%.
  • test strip of the present invention whether it is a colloidal gold test strip or a fluorescent test strip, has a detection result equivalent to that of the highly sensitive ELISA method, and the accuracy is very high.
  • the test strip of the present invention does not require special test sites, equipment and professional operators, and is therefore very suitable for grass roots and small and medium-sized pastures.
  • the colloidal gold test strip of the present invention can realize detection in a relatively short time, which is of great significance for use in pastures.

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Abstract

提供了妊娠相关糖蛋白的单克隆抗体及其在牛早期怀孕检测中的应用。由于该单克隆抗体可以特异性地结合妊娠相关糖蛋白，因此可以用于制备操作简单、稳定性好、准确性高的快速牛早期怀孕检测产品，从而提高畜牧业的经济效益。

Description

妊娠相关糖蛋白的单克隆抗体及其在牛早期怀孕检测中的应用 技术领域
本发明涉及抗体领域，更具体地，本发明涉及妊娠相关糖蛋白的单克隆抗体及其在牛早期怀孕检测中的应用。
背景技术
牛早期怀孕检测是牧场生产过程中的一项重要组成部分，产犊间隔是衡量奶牛繁育能力的重要指标。通过早期精准的奶牛怀孕检测，可以帮助牧场尽早的发现空怀牛和怀孕牛，对未怀孕的奶牛及时重新配种，通过缩短产犊间隔来帮助牧场实现产奶量和经济效益的最大化。
B超和直肠触诊是目前常见的妊娠判断方法，但该两种方法至少在配种后35天和45天才能进行，且方法的准确性与操作人员的经验和技能密切相关，操作不当还会导致胚胎的早期死亡。
早期妊娠相关糖蛋白(PAG)是目前常用的奶牛早期妊娠诊断指标，该蛋白由奶牛胎儿胎盘的滋养层产生，并分泌到母畜外周血液中，在配种后28天左右即可通过检测母畜血液中PAG的浓度变化，尽早对奶牛妊娠状态进行判断。目前市面上通过检测PAG鉴别奶牛妊娠状态的产品均为酶联免疫吸附试验(ELISA)检测试剂盒。ELISA方法检测时间长(3小时以上)、操作繁琐，需要专门试验场地、设备及专业操作人员，不适于基层、中小型牧场使用。目前，尚无基于免疫层析原理的快速检测产品。
任何免疫学检测方法的关键都是特异性抗体的制备，为了解决现有方法的缺陷，满足更广泛的检测需求，本领域亟需更多的灵敏度高、稳定性好的特异性PAG抗体，用于制备操作简单、稳定性好、准确性高的快速检测牛早期怀孕的产品。
发明内容
有鉴于此，本发明提供了特异性结合妊娠相关糖蛋白的单克隆抗体以及包含所述单克隆抗体的检测试纸卡，以快速准确地检测牛早期怀孕。
因此，在第一方面，本发明提供了一种杂交瘤细胞，所述杂交瘤细胞于2021年10月12日保藏在位于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心，保藏号为GDMCC No：61975。
在第二方面，本发明提供了一种杂交瘤细胞，所述杂交瘤细胞于2021年10月12日保藏在位于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心，保藏号为GDMCC No：61976。
在第三方面，本发明提供了一种特异性结合牛妊娠相关糖蛋白(PAG)的单克隆抗体，其中，所述单克隆抗体由本发明第一方面或第二方面的杂交瘤细胞产生。
在第四方面，本发明提供了一种特异性结合牛妊娠相关糖蛋白(PAG)的单克隆抗体组合，其中所述抗体组合包括由保藏号为GDMCC No：61975的杂交瘤细胞产生的单克隆抗体以及由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体。
在第五方面，本发明提供了一种用于检测牛妊娠相关糖蛋白(PAG)的试纸条，所述试纸条包括样品垫、结合垫、包被膜和吸水垫，在所述结合垫上包被有经标记的由保藏号为GDMCC No：61975的杂交瘤细胞产生的单克隆抗体，并且在所述包被膜上包被有由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体作为检测线。
在第六方面，本发明提供了一种用于检测牛妊娠相关糖蛋白(PAG)的试纸卡，所述试纸卡包括本发明第五方面的试纸条、以及由上壳体和下壳体组成的卡扣。
在第七方面，本发明提供了本发明第一方面或者第二方面的杂交瘤细胞、第三方面的单克隆抗体、或者第四方面的单克隆抗体组合在检测牛早期怀孕中的应用。
本发明的单克隆抗体可以特异性地结合妊娠相关糖蛋白，因此，本发明的单克隆抗体可以用于制备基于抗体抗原免疫反应的检测试纸条，以提供操作简单、稳定性好、准确性高的快速检测牛早期怀孕的产品。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的实施方案。
图1是示出胶体金检测试纸条的结构示意图；
图2是示出荧光免疫检测试纸条的结构示意图；
图3是示出胶体金检测试纸卡和荧光检测试纸卡的壳体的结构示意图；
图4是胶体金检测试纸卡的结果判定的示意图。
具体实施方式
下面将结合本发明的实施方案和附图，对本发明进行清楚、完整的描述。显然，所描述的实施方案仅仅是本发明的一部分实施方案，而不是全部的实施方案。基于本发明中的实施方案，本领域普通技术人员可以获得的所有其他实施方案，都属于本发明保护的范围。
如上所述，本发明旨在提供能够特异性结合妊娠相关糖蛋白的单克隆抗体从而制备利用所述单克隆抗体的检测试纸卡，用于快速准确地检测牛早期怀孕。
因此，在第一方面，本发明提供了一种杂交瘤细胞，所述杂交瘤细胞于2021年10月12日保藏在位于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心，保藏号为GDMCC No：61975。
在第二方面，本发明提供了一种杂交瘤细胞，所述杂交瘤细胞于2021年10月12日保藏在位于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心，保藏号为GDMCC No：61976。
本发明的杂交瘤细胞的制备方法如下：首先，用牛妊娠相关糖蛋白(PAG)免疫小鼠，然后将小鼠的脾细胞与骨髓瘤细胞Sp2/0进行细胞融合，经有限稀释、克隆化以及间接ELISA筛选后，获得能够分泌单克隆抗体的杂交瘤细胞。
本发明人用SEQ ID NO:1所示的PAG作为抗原多次免疫小鼠，将小鼠的脾细胞用于细胞融合制备杂交瘤细胞，并通过间接ELISA检测方法筛选出阳性杂交瘤细胞克隆，由此制备出了能够特异性结合妊娠相关糖蛋白的单克隆抗体。
在第三方面，本发明提供了一种特异性结合牛妊娠相关糖蛋白(PAG)的单克隆抗体，其中，所述单克隆抗体由本发明第一方面或者第二方面的杂交瘤细胞产生。
在本说明书中，术语“妊娠相关糖蛋白(Pregnancy-associated glycoprotein，PAG)”是由反刍动物胎儿胎盘滋养层细胞合成分泌的一类蛋白质，在妊娠过程中具有重要作用。在本发明中，发明人使用氨基酸序列如SEQ ID NO:1所示的牛妊娠相关蛋白(PAG)来对小鼠进行免疫。应理解，本发明对所述PAG的来源没有限定，其可以从天然细胞或重组细胞中提取，也可以通过人工合成。SEQ ID NO:1的序列如下：MKWLGLLGLVALSECMVIIPVTQMKAIRETLWERNLLTNFSEEHPCSLSQNATNDQNIVYHPLRNYKDLVYIGTIAIGTPPQEFRVIFDTGSSDLWVPSIHCHSPSCLTHSLFNPRTSTTFKLLNNIIDLIYASGRIKGVLGQDIIQVMWKPEAAGPGSNCCLQNRCRRSWRCLSFRKAPNGCRSAWKDSVLGETMPLLTYKLFVANAGRGLGCGFAASLPMKSRRFIMVSRRENGSVLMFGGVDHSYHTGKLNWVPVSRTHYWQITIGRISMNGKLIACKRGCQAIMDTGTTFLLGPSRHIAKIQRLIRIRPFGSLQYTVPCNITSTLPPLIFTIKGIDYPVPAQAYIHKMRGQS。
牛妊娠相关糖蛋白(PAG)是目前常用的奶牛早期妊娠诊断指标。在配种后28天左右即可通过检测母畜血液中PAG的浓度变化，尽早对奶牛妊娠状态进行诊断。根据现有研究发现，通过检测PAG对配种后35天的奶牛妊娠确诊率达到93.03％，表明PAG完全可以作为牛妊娠诊断检测的标志物。而任何免疫学检测PAG方法的关键都是特异性抗体，因此本发明提供的单克隆抗体可以用于通过多种免疫分析法来检测牛早期怀孕，所述检测方法包括但不限于免疫层析技术、酶联免疫吸附测定法、免疫印迹法、流式细胞术等。
在本发明中，发明人通过IsoStrip Mouse Monoclonal Antibody Isotyping Kit抗体亚类试剂盒对这两株单克隆抗体进行了亚型鉴定，确定这两株单克隆抗体亚型均为IgG2b。因此，在一个实施方案中，所述单克隆抗体为IgG2b亚型。
在第四方面，本发明提供了一种特异性结合牛妊娠相关糖蛋白(PAG)的单克隆抗体组合，其中所述抗体组合包括由保藏号为GDMCC No：61975的杂交瘤细胞产生的单克隆抗体(抗体1)以及由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体(抗体2)。本领域的技术人员已知，抗体组合对于抗原的敏感性受多种因素影响，包括但不限于抗体亲和力、空间位阻和结合位点等。
在第五方面，本发明提供了一种用于检测牛妊娠相关糖蛋白(PAG)的试纸条，所述试纸条包括样品垫、结合垫、包被膜和吸水垫，在所述结合垫上包被有经标记的由保藏号为GDMCC No：61975的杂交瘤细胞产生的单克隆抗体，并且在所述包被膜上包被有由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体作为检测线。
试纸条是通过将样品垫、结合垫、包被膜和吸水垫依次粘附在PVC底板上并采用搭接的方式连接而成的(如图1或图2所示)。在本发明中，包被膜通常是指在硝酸纤维素膜上包被抗体而形成的膜，包被膜上包被有两种抗体，第一种抗体为由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体，作为检测线T，第二种抗体为针对作为检测线的单克隆抗体的抗体，可以是单克隆抗体或者多克隆抗体，例如羊抗鼠IgG多克隆抗体，其作为质控线C。所述试纸条还可以包括用于免疫学分析的工具或试剂，例如产生可检测信号的标记物质、增溶剂和清洁剂等。
因此，在一个实施方案中，所述试纸条可以为胶体金检测试纸条或荧光免疫检测试纸条。
胶体金免疫层析技术是一种基于抗原抗体免疫反应的快速检测方法，将抗原或抗体标记在红色胶体金颗粒上，通过肉眼观察即可进行结果判读。荧光微球免疫层析技术是一种基于抗原抗体免疫反应的快速检测方法，将抗原或抗体标记在荧光微球上，通过荧光读数仪进行结果判读。两者基本原理一致，样本前处理以及检测方法也基本相同，仅是抗原或抗体标记物和判读方法上的区别，同样具备操作简单(不需要专业实验室人员操作)、检测快速(20分钟内即可完成)、稳定性好、准确性高的特点。
在所述试纸条为胶体金检测试纸条的情况下，所述包被在所述结合垫上的单克隆抗体经胶体金标记；在所述试纸条为荧光免疫检测试纸条的情况下，所述包被在所述结合垫上的单克隆抗体经荧光标记。
优选地，所述结合垫上包被的单克隆抗体标记浓度为7μg/ml-25μg/ml，所述包被膜上包被的单克隆抗体浓度为0.5mg/ml-0.6mg/ml。
在第六方面，本发明提供了一种用于检测牛妊娠相关糖蛋白(PAG)的试纸卡，所述试纸卡包括本发明第五方面的试纸条、以及由上壳体和下壳体组成的卡扣(如图3所示)。
在第七方面，本发明提供了本发明第一方面或者第二方面的杂交瘤细胞、第三方面的单克隆抗体、或者第四方面的单克隆抗体组合在检测牛早期怀孕中的应用。
在一个实施方案中，本发明采用以双抗体夹心法为基础的免疫层析技术。具体地，将待检奶牛的血清或血浆样品加样到试纸条的样品垫上，在样品流动到结合垫时，血清或者血浆中如果存在PAG，则会与经标记(例如经胶体金或荧光标记)的PAG单克隆抗体(标记抗体)结合形成标记抗体-PAG抗原复合物。在样品继续流动至检测线T区时，此前形成的标记抗体-PAG抗原复合物会进一步与包被作为检测线T的PAG单克隆抗体(T线抗体)结合形成三者的复合物(标记抗体-PAG-T线抗体)，使T线显色。若血液中不存在PAG或含量低于该产品检测限，则T线不显色。
实施例
实施例1抗妊娠相关糖蛋白单克隆抗体的制备
1.免疫原制备及纯化：通过基因克隆，构建PAG重组原核表达载体以获得能够稳定表达PAG的工程菌。将诱导表达的菌液进行离心、超声破碎后，采用琼脂糖凝胶FF预装柱进行纯化，将纯化获得的PAG(SEQ ID NO:1)作为免疫原。
2.免疫程序：将纯化的PAG抗原与弗氏完全佐剂按1:1的比例充分混匀；选取健康的八周龄BALB/c小鼠，在其背部皮下注射100μg 乳化后的PAG抗原；分别在首次免疫后第15、30天用弗氏不完全佐剂乳化等量蛋白，然后加强免疫两次，免疫部位及方法同首次免疫一致；融合前三天，通过小鼠尾静脉注射用生理盐水制备的100μg PAG抗原再次加强免疫一次。
3.细胞融合：将脾细胞与骨髓瘤细胞Sp2/0按细胞数量1:5比例混合均匀，补加不完全RPMI-1640培养液至30ml，充分混匀，1200r/m离心10分钟，将上清液吸净，在手掌上轻击管底，使沉淀细胞松散均匀。在烧杯中加入37℃蒸馏水，将含有混合细胞的离心管置于水浴中，在90秒内加入37℃预热的50％PEG l ml，边滴加边旋转离心管，随后静置1分钟，再在5分钟内加入37℃预热的不完全RPMI-1640培养液，先慢慢滴入，然后加快，不可将底部细胞冲起，稀释完PEG后1500r/min离心10分钟，倒掉上清。
将细胞沉淀重悬于20％胎牛血清的HAT选择培养液中，混合均匀，同时将制备好的饲养细胞按相同体积加入，再次混合均匀，用多道移液器加入96孔细胞培养板中，每孔200μl，在饱和湿度、5％CO 2、37℃的条件下培养。
4.杂交瘤细胞株筛选：融合后的细胞每隔2-4天更换一次培养基，融合后7-8天时，开始观察细胞，标出有存活的杂交瘤细胞的细胞孔，待杂交瘤细胞长满孔底1/5以上时，取细胞上清100μl作为一抗，用PAG作为抗原包被酶标板，用间接ELISA对细胞上清进行检验，选择检测结果阳性且效价高的杂交瘤细胞。将通过间接ELISA筛选出的阳性孔中的杂交瘤细胞按照有限稀释法进行亚克隆，经过至少3次亚克隆，获得分泌单克隆抗体的细胞株。
5.腹水制备及抗体纯化：取7周龄BALB/c雌性小鼠，用石蜡油注入小鼠腹腔，0.5m1/只，7天后腹腔接种用无血清培养基稀释的处于对数生长期的杂交瘤细胞(来自步骤4)，间隔7天后，每天观察小鼠腹水产生情况，用16号针头采集腹水，将腹水离心，取上清。采用饱和硫酸铵沉淀法对抗体进行纯化，纯化后得到10株单克隆抗体(编号1-10)。
6.抗体亲和力测定：将纯化后的10株单克隆抗体分别进行胶体 金标记，随后喷涂于玻璃纤维上制备金结合垫。将用PAG抗原包被的硝酸纤维素膜分别与10种金结合垫组装成试纸卡，滴加PBS于试纸卡的加样孔中，等待10分钟观察。根据PAG抗原包被位置的显色深度，排除无显色或显色较浅的抗体，选择显色深度较高的抗体(编号1、2、3、5、8和10)进行下一步配对筛选。
7.抗体配对组合的筛选：将编号为1、2、3、5、8和10的共6株抗体分别包被到硝酸纤维素膜上，并同时经胶体金标记后包被在玻璃纤维上制备金结合垫，将包含6株不同单克隆抗体的6种硝酸纤维素膜和6种金结合垫交叉组合制备试纸卡，通过正交试验筛选敏感性最高的组合。通过各组合试纸卡分别检测不同浓度的PAG抗原(10ng/ml、5ng/ml、2.5ng/ml、1ng/ml、0.5ng/ml)，选择敏感性最高的配对组合用于产品的研发。下表1的实验结果表明，抗体1标金(结合垫)-抗体2包膜(包被膜)的组合在检测PAG抗原时敏感性最高，低于0.5ng/ml，满足产品研发需求，其中，所述抗体1为由保藏号为GDMCC No：61975的杂交瘤细胞产生的单克隆抗体，所述抗体2为由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体。
表1.抗体配对组合的敏感性
Figure PCTCN2022117833-appb-000001
8.抗体亚类鉴定：用IsoStrip Mouse Monoclonal Antibody Isotyping Kit抗体亚类试剂盒对两株单抗进行亚型鉴定，两株单抗亚型均为IgG2b。
实施例2牛早孕(妊娠相关糖蛋白)胶体金检测试纸卡的制备和检测
本实施例采用以双抗体夹心法为基础的胶体金免疫层析技术，在配种后第28天即可快速鉴别怀孕牛和空怀牛。若奶牛怀孕，其血液中的PAG达到较高水平，在样品流动过程中PAG与胶体金标记的PAG单克隆抗体结合，继续流动至检测线T区时与检测线T包被的PAG抗体结合形成三者的复合物(胶体金标记抗体-PAG-T线包被抗体)，T线呈现红色条带，且T线显色深度大于B线；若奶牛未怀孕，血液中不存在PAG或含量低于阈值，则T线不显色或者显色深度小于B线。质控线C显色表示该产品有效，质控线C不显色，表示产品无效。具体的实验操作如下。
1.胶体金溶液的制备：取1个洁净的圆底烧瓶，加入双蒸水，置于磁力加热搅拌器上，加热搅拌直至沸腾，加入1％的氯金酸溶液至终浓度为万分之一，继续加热至沸腾；随后按照1∶1.6的比例加入1％柠檬酸三钠溶液，继续加热煮沸10分钟左右，直至溶液呈现透亮的红色。室温(15℃-25℃)冷却后用双蒸水定容至原计划制备的体积(补偿蒸发损失)，由此获得胶体金溶液。
2.胶体金结合垫的制备：将胶体金溶液置于磁力搅拌器上，用0.2M K 2CO 3调节pH至7.5左右，搅拌状态下加入7μg/ml的PAG单克隆抗体1，室温搅拌30分钟；加入10％牛血清白蛋白(BSA)溶液至终浓度为1％，室温搅拌30分钟；溶液置4℃、10000r/min离心机离心30分钟，弃上清，沉淀用相同体积的金复溶液重悬，混匀。每30ml重悬液均匀涂布于25cm*30cm规格的玻璃纤维素膜上，放置在干净的纱网窗上，置湿度为10％-30％的室温干燥房干燥10小时，备用。
3.包被膜的制备：分别用包被液将羊抗鼠IgG多克隆抗体和PAG单克隆抗体2稀释至终浓度0.3mg/ml和0.5mg/ml，按照1μl/cm的量分别喷于硝酸纤维素膜的对应位置上，制备C线和T线，将膜置于37℃温箱烘干20小时，备用。
4.样品垫的制备：配制样品垫处理液，每30ml样品垫处理液均匀涂布于25cm*30cm规格的玻璃纤维素膜上，放置在湿度为10％-30％的室温干燥房烘干15小时-18小时，备用。
5.大板组装：将处理好的金结合垫裁成0.6cm*30cm规格；将处理好的样品垫裁成3.0cm*30cm规格；硝酸纤维素膜规格为2.5cm*30cm；吸水垫规格为2.6cm*30cm；PVC底板规格为7.8cm*30cm；将各组分按顺序组装于PVC底板上，具体组装方式参见图1。
6.试纸卡组装：将组装好的大板用切条机裁成3.85mm宽度，装入塑料卡壳内，组装好的卡壳连同干燥剂一包装入铝箔袋，封口。
7.胶体金试纸卡组成：该试纸卡由试纸条和卡扣组成，卡扣由上壳体和下壳体组成(参见图3)；试纸条组成包括PVC底板、硝酸纤维素膜、吸水垫、胶体金结合垫、样品垫，胶体金结合垫上包被有PAG单克隆抗体-胶体金标记物，硝酸纤维素膜上包被有由羊抗鼠IgG多克隆抗体构成的质控线及PAG单克隆抗体构成的检测线。
8.检测方法：
(1)待检样品处理：采集配种后28天的牛全血分离血清或血浆，可选择4℃静置过夜自然析出或置于离心机2000rpm/min-3000rpm/min，离心5分钟-15分钟，取上清备用。
(2)试剂的准备：打开包装袋，取出检测卡，平放于桌面待用。
(3)样品检测：将待检样品恢复至室温，混合均匀后用样品滴管逐滴滴加3滴(90μL左右)待检血清或血浆(EDTA、肝素钠)于检测卡的加样孔中，等待20分钟观察结果，超过30分钟后结果仅供参考。
(4)结果判定(参见图4)
1)怀孕：质控线C显色，且检测线T显色深度大于比色线B。
2)空怀：质控线C显色，且检测线T不显色或检测线T显色深度小于比色线B。
3)可疑：质控线C显色，且检测线T显色深度与比色线B相当，建议7天后重新采血复测。
4)无效：质控线C处不出现红色条带，判为无效，说明检测失误或检测卡失效，建议重新检测。
9.本发明的胶体金试纸卡与现有ELISA产品的比较实验结果
从牧场采集配种后28天的牛血液样品(1720份)，同时采用本发明的胶体金试纸卡和竞品ELISA试剂盒对分离后的血清和血浆样品进行检测并计算本发明的胶体金试纸卡与商业化ELISA试剂盒的符合率。
表2示出两种检测方法(在本申请中具体为胶体金试纸条或荧光试纸条与商业化ELISA)之间符合率的计算方法示意图。
表2.本发明试纸卡与商业化ELISA试剂盒符合率计算示意图
Figure PCTCN2022117833-appb-000002
注：
“a”表示本发明试纸卡和商业化ELISA试剂盒的检测结果均为阳性的样品数量；
“b”为本发明试纸卡的检测结果为阴性、商业化ELISA试剂盒的检测结果为阳性的样品数量；
“c”为本发明试纸卡的检测结果为阳性、商业化ELISA试剂盒的检测结果为阴性的样品数量；
“d”为本发明试纸卡和商业化ELISA试剂盒的检测结果均为阴性的样品数量。
其中阳性符合率、阴性符合率以及总符合率的具体计算公式如下：
Figure PCTCN2022117833-appb-000003
Figure PCTCN2022117833-appb-000004
Figure PCTCN2022117833-appb-000005
表3示出了本发明胶体金试纸条与商业化ELISA试剂盒之间的符合率。从表3中可以看出，本发明的胶体金试纸卡与商业化ELISA试剂盒相比，敏感性(即阳性符合率)达到了98.99％，特异性达到了98.07％(即阴性符合率)，总符合率达到了98.66％。
表3.胶体金试纸卡与商业化ELISA试剂盒符合率的计算结果
Figure PCTCN2022117833-appb-000006
实施例3.牛早孕(妊娠相关糖蛋白)荧光检测试纸卡的制备和检测
本发明采用双抗体夹心法为基础的荧光免疫层析技术，在玻纤上包被荧光标记物，并在硝酸纤维素膜的检测线(T线)和质控线(C线)处分别包被相应抗体和多克隆抗体二抗。若奶牛怀孕，则样品中的PAG在流动过程中与荧光标记物结合形成复合物，并与检测线T包被的抗体结合产生荧光信号。当检测卡插入仪器检测时，仪器收集检测线(T线)和质控线(C线)上的荧光信号后，通过ID卡输入到仪器中的曲线方程换算，检测界面显示为阳性结果。
1.荧光微球溶液的制备：在500mL三口烧瓶中加入25.5mL(0.22mol)苯乙烯、3mL(0.03mol)甲基丙烯酸和220mL去离子水，在300r/min转速搅拌下升温至回流并恒温，稳定5分钟后，加入0.173g(0.64mmol)过硫酸钾引发聚合，反应2小时之后，多次离心、洗涤，即得白色羧基聚苯乙烯微球。称取单分散羧基聚苯乙烯种子微球0.1g于10mL 0.25％SDS水溶液中，超声5分钟，使微球分散均匀。将5mg Eu配合物溶于1mL CH 2Cl 2后，加入到种子微球的分散体系中，乳化机乳化，室温剧烈振摇3小时，30℃旋蒸，除去CH 2Cl 2，产物经离心分离后，用去离子水、乙醇进行多次离心洗涤(3000r/min，3分钟)，直至上清液呈无色，即得到相应颜色的荧光微球(荧光编码微球)。
2.荧光结合垫的制备：将荧光微球加入至1.5mL离心管中，用0.05M MES洗脱三次，再用100mg/ml的EDC和NHS进行活化，室温反应30分钟；再用0.05M MES洗脱三次，最后一次洗脱后弃上清，沉淀用1mL的0.05M MES重悬；加入25μg/mL的PAG单克隆抗体1， 室温反应120分钟；用0.05M MES洗脱三次，最后一次洗脱后弃上清，沉淀用1mL的封闭液重悬，室温反应60分钟；溶液置4℃，15000r/min离心机离心20分钟，弃上清，沉淀用1mL的的荧光复溶液重悬，而后用荧光复溶液稀释至30mL。每30mL重悬液均匀涂布于25cm*30cm规格的玻璃纤维素膜上，放置在干净的纱网窗上，置于湿度为10％-30％的室温干燥房干燥10小时，备用。
3.包被膜的制备：分别用包被液将羊抗鼠IgG多克隆抗体和PAG单克隆抗体2稀释至终浓度0.6mg/ml和0.6mg/ml，按照1μl/cm的量分别喷于硝酸纤维素膜的对应位置上，制备C线和T线，将膜置于37℃温箱烘干20小时，备用。
4.样品垫的制备：配制样品垫处理液，将30ml样品垫处理液均匀涂布于25cm*30cm规格的玻璃纤维素膜上，放置在湿度为10％-30％的室温干燥房烘干15小时-18小时，备用。
5.大板组装：将处理好的荧光结合垫裁成0.6cm*30cm规格；将处理好的样品垫裁成3.0cm*30cm规格；硝酸纤维素膜规格为2.5cm*30cm；吸水垫规格为2.6cm*30cm；PVC底板规格为7.8cm*30cm；将各组分按顺序组装于PVC底板上，具体组装方式参见图2。
6.试纸卡组装：将组装好的大板用切条机裁成3.85mm宽度，装入塑料卡壳内，组装好的卡壳连同干燥剂一包装入铝箔袋，封口。
7.试纸卡组成：该试纸卡由试纸卡和卡扣组成，卡扣由上壳体和下壳体组成；试纸卡组成包括PVC底板、硝酸纤维素膜、吸水垫、荧光结合垫、样品垫，荧光结合垫上包被有PAG单克隆抗体-荧光标记物，硝酸纤维素膜上包被有由羊抗鼠IgG多克隆抗体构成的质控线及PAG单克隆抗体构成的检测线。
8.检测方法：
(1)待检样品处理：采集配种后28天的牛全血分离血清，4℃静置过夜自然析出或置于离心机4000rpm-5000rpm，离心30秒-60秒(血清或血浆均可)。
(2)试剂的准备：打开包装袋，取出检测卡，平放于桌面待用。
(3)可根据实际检测需求，选择以下不同的测试模式。
1)快速测试(适用于同时检测多个样本)：将待检样品恢复至室温，混合均匀后用移液枪吸取100μL待检血清或血浆(EDTA、肝素钠)于检测卡的加样孔中，开始反应，15分钟后将检测卡插入仪器内(加样孔方向朝内)，选择快速测试模式，点击开始测试，完成测试后，仪器界面显示检测结果，反应时间超过20分钟的检测结果仅供参考。
2)标准测试(适用检测单个样本)：将待检样品恢复至室温，混合均匀后用移液枪吸取100μL待检血清或血浆(EDTA、肝素钠)于检测卡的加样孔中，将检测卡插入仪器内(加样孔方向朝内)，选择标准测试模式，点击开始测试，仪器自动计时15分钟，15分钟后仪器界面显示检测结果。
(4)结果判读
1)阴性：空怀
2)弱阳：建议复检
3)阳性：怀孕
4)无效：检测卡插入方向错误或未进行点样操作
弱阳性分析：在奶牛怀孕初期，胚胎死亡是较为常见的现象，在胚胎死亡的早期，妊娠相关糖蛋白仍会在奶牛体内循环，因此建议弱阳性样本的奶牛，7-10天后重新取血进行复检，若复检后仍为弱阳性结果，则判定为空怀牛。
9.工艺参数:
PAG单克隆抗体最佳标记量为25μg/ml；包被液选择PB(0.02mol/L，pH值7.4)+2％海藻糖溶液；制备质控线的羊抗鼠IgG多克隆抗体的最佳包被浓度为0.6mg/ml，制备检测线的PAG单克隆抗体最佳包被浓度为0.6mg/ml；膜烘干时间选择于37℃烘干20小时；最佳加样量为100μl；最佳反应时间为15分钟。用该条件制备的检测荧光检 测卡，具有灵敏度高、特异性强、稳定性好的特点。
10.本发明的荧光试纸卡与现有ELISA产品的比较实验结果
从牧场采集配种后28天的牛血液样品(2450份)，同时采用本发明的荧光试纸卡和竞品ELISA试剂盒对分离后的血清和血浆样品进行检测并计算本发明的荧光试纸卡与商业化ELISA试剂盒的符合率。
荧光试纸卡与商业化ELISA试剂盒符合率的计算方法参见实施例2第9部分。结果示于表4。
表4.荧光试纸卡与商业化ELISA试剂盒符合率的计算结果
Figure PCTCN2022117833-appb-000007
通过以上实验结果可以看出，本发明的荧光试纸卡与商业化ELISA试剂盒相比，敏感性(即阳性符合率)达到了98.39％，特异性达到了98.23％(即阴性符合率)，总符合率达到了98.33％。
可见，本发明的试纸条，不管是胶体金试纸条还是荧光试纸条，均具有与灵敏度很高的ELISA方法相当的检测结果，准确度很高。本发明的试纸条不需要专门的试验场地、设备和专业操作人员，因此非常适于基层以及中小型牧场使用。并且，作为一种快速检测手段，本发明的胶体金试纸条可以在相对短的时间里实现检测，这对于在牧场使用具有非常的意义。

Claims (10)

  1. 一种杂交瘤细胞，所述杂交瘤细胞于2021年10月12日保藏在位于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心，保藏号为GDMCC No：61975。
  2. 一种杂交瘤细胞，所述杂交瘤细胞于2021年10月12日保藏在位于广州市先烈中路100号大院59号楼5楼的广东省微生物菌种保藏中心，保藏号为GDMCC No：61976。
  3. 一种特异性结合牛妊娠相关糖蛋白(PAG)的单克隆抗体，其中，所述单克隆抗体由权利要求1或者权利要求2所述的杂交瘤细胞产生。
  4. 根据权利要求3所述的单克隆抗体，其中，所述单克隆抗体为IgG2b亚型。
  5. 一种特异性结合牛妊娠相关糖蛋白(PAG)的单克隆抗体组合，其中所述抗体组合包括由保藏号为GDMCC No：61975的杂交瘤细胞产生的单克隆抗体以及由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体。
  6. 一种用于检测牛妊娠相关糖蛋白(PAG)的试纸条，所述试纸条包括样品垫、结合垫、包被膜和吸水垫，在所述结合垫上包被有经标记的由保藏号为GDMCC No：61975的杂交瘤细胞产生的单克隆抗体，并且在所述包被膜上包被有由保藏号为GDMCC No：61976的杂交瘤细胞产生的单克隆抗体作为检测线。
  7. 根据权利要求6所述的试纸条，其中，所述试纸条为胶体金检测试纸条或荧光免疫检测试纸条，在所述试纸条为胶体金检测试纸条的情况下，所述包被在所述结合垫上的单克隆抗体经胶体金标记；在所述试纸条为荧光免疫检测试纸条的情况下，所述包被在所述结合垫上的单克隆抗体经荧光标记。
  8. 根据权利要求6-7中任一项所述的试纸条，其中，所述结合垫上包被的单克隆抗体标记浓度为7μg/ml-25μg/ml，所述包被膜上包被的单克隆抗体浓度为0.5mg/ml-0.6mg/ml。
  9. 一种用于检测牛妊娠相关糖蛋白(PAG)的试纸卡，所述试纸 卡包括权利要求6-8中任一项所述的试纸条、以及由上壳体和下壳体组成的卡扣。
  10. 根据权利要求1或者2所述的杂交瘤细胞、根据权利要求3或者4所述的单克隆抗体、或者根据权利要求5所述的单克隆抗体组合在检测牛早期怀孕中的应用。
PCT/CN2022/117833 2021-12-02 2022-09-08 妊娠相关糖蛋白的单克隆抗体及其在牛早期怀孕检测中的应用 WO2023098215A1 (zh)

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