A human growth hormone detection method based on high-specificity double antibody sandwich method

Abstract

The present invention discloses a method for detecting human growth hormone based on a high-specificity double antibody sandwich method, including animal immunization, crude serum purification, serum fine purification, biomarkers, and human growth hormone content determination steps. The present invention establishes a method for detecting human growth hormone content, greatly improves the accuracy and technical requirements of the detected content, and provides a new detection method for medicine.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and more specifically, to a human growth hormone detection method based on a high-specificity double-antibody sandwich method. Background Art

[0002] Human Growth Hormone (HGH) is a peptide hormone secreted by the anterior pituitary gland of the human body. It is composed of 191 amino acids and can promote the growth of bones, internal organs and the whole body, promote protein synthesis, affect fat and mineral metabolism, and play a key role in human growth and development.

[0003] In general, growth hormone is the abbreviation of human growth hormone for injection (formerly known as recombinant human growth hormone for injection). It is produced through genetic recombination technology and is completely consistent with human pituitary growth hormone in terms of amino acid content, sequence and protein structure. In the field of pediatrics, the use of growth hormone for replacement therapy can significantly promote children's height growth. At the same time, growth hormone also plays an important role in the fields of reproduction, burns and anti-aging, and has been widely used in clinical practice.

[0004] Growth hormone has received continuous attention due to its high degree of abuse and difficulty in detection. At present, the growth hormone detection methods recognized by WADA include direct detection and indirect detection. The direct detection method uses an immunoassay kit to directly detect the 22kDa subtype of growth hormone and the non-22kDa subtype of growth hormone, and makes a judgment based on the change in the ratio of different subtypes of growth hormone exceeding a certain threshold; due to the cross-reaction of antigens and antibodies, the accuracy needs to be further improved. The indirect detection method is to detect two biomarkers IGF-I and P-III-NP that increase in the human body after growth hormone intake, using mass spectrometry or immunoassay methods to detect, substitute the corresponding values ​​into the formula to calculate, and judge growth hormone positive if it exceeds the threshold; this method has a long detection window period, a high threshold, and a certain missed detection rate. Therefore, it is necessary to develop a highly specific antigen detection method for direct detection of human growth hormone to improve the accuracy of the detection method. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a human growth hormone detection method based on a high-specificity double antibody sandwich method. The human growth hormone protein is used as an immunogen to prepare rabbit and guinea pig anti-human growth hormone polyclonal antibodies; the polyclonal antibodies are crudely purified by ammonium sulfate and then refined by affinity chromatography to obtain highly specific antibodies; an ELISA plate is coated with a rabbit / guinea pig anti-protease polyclonal antibody, human growth hormone in the sample is combined with the coated antibody, TMB is used as a color development system, and the OD450nm value is measured at a wavelength of OD450nm; the method is simple, short cycle, high sensitivity, good characteristics, economical and affordable, etc., and provides an effective detection means for human growth hormone research.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] A method for detecting human growth hormone based on a high-specificity double antibody sandwich method, comprising the following steps:

[0008] S1 Animal immunization: human growth hormone antigen is mixed with Freund's complete adjuvant to prepare antigen liquid A, and human growth hormone antigen is mixed with Freund's incomplete adjuvant to prepare antigen liquid A and antigen liquid B. Antigen liquid A is injected for primary animal immunization, and antigen liquid B is injected for secondary and tertiary animal immunization to obtain animal serum;

[0009] S2 crude serum purification: crude purification of animal serum after three immunizations with protein A or G to obtain crude purified animal serum;

[0010] S3 serum purification: the crude purified animal serum is subjected to two-step chromatography purification, affinity chromatography 1 and affinity chromatography 2, to obtain affinity chromatography 2 animal collection fluid;

[0011] S4 biomarker: biotin-label the collected fluid from affinity chromatography 2 animals to obtain animal serum affinity chromatography 2 labeled antibodies;

[0012] S5 Determination of human growth hormone content: The ELISA plate was coated with the affinity chromatography 2 animal collection fluid. After blocking and washing, the animal serum affinity chromatography 2 labeled antibody was used as the detection antibody and TMB was used as the color development system to determine the human growth hormone content.

[0013] Furthermore, in S1 animal immunization, the three immunization operations are specifically as follows:

[0014] (a) Primary immunization: Inject antigen solution A into animals at a dose of 0.2 ml per animal;

[0015] (b) Secondary immunization: 14 days after the initial immunization, blood was collected from the animals, and the serum was separated by centrifugation and frozen; then, a secondary immunization was performed, and the animals were injected with antigen solution B at a dose of 0.2 ml / animal;

[0016] (c) Three immunizations: 7 days after the second immunization, blood was collected from the animals, and the serum was separated by centrifugation and frozen; then, three immunizations were performed, and the animals were injected with antigen solution B at a dose of 0.2 ml / animal.

[0017] Furthermore, in S1 animal immunization, human growth hormone was diluted with 0.9% sterile sodium chloride to obtain an antigen dilution with a concentration of 120ug / ml. The antigen dilution was mixed with Freund's complete adjuvant in a volume ratio of 2:1 and then ground to obtain an antigen solution A with a final injection concentration of 80ug / ml. The antigen dilution was mixed with Freund's incomplete adjuvant in a volume ratio of 2:1 and then ground to obtain an antigen solution B with a final injection concentration of 80ug / ml.

[0018] Furthermore, in step S1, the antibody titer is monitored during the immunization process. If the antibody titer monitoring one week after three immunizations meets the use requirements, whole blood is collected to obtain animal serum.

[0019] Furthermore, in step S2, crude purification of serum includes the steps of primary precipitation with ammonium sulfate, secondary purification with ammonium sulfate, re-dissolution of precipitation, and chromatographic desalting.

[0020] Further, in step S3, the specific steps of affinity chromatography 1 are: in a protein purifier, first equilibrate the chromatography column with a crude pure equilibration buffer for 10CV, at a flow rate of 5 mL / min, load the sample after the UV absorption baseline is stable, and load the sample at a flow rate of 5 mL / min. After loading, equilibrate with buffer A for 10CV, and collect the UV280nm>15mAU flow-through peak; then elute at a volume flow rate of 5 ml / min, 100% buffer B for 8CV, collect the elution peak, and pre-add 5% volume of buffer C to the elution peak collection tube; start collecting samples when AU is not less than 0.015, and stop collecting samples when AU is not greater than 0.01, to obtain affinity chromatography 1 collection liquid, and store it at 4°C;

[0021] The crude pure equilibration buffer was a solution containing 20 mM PB, 150 mM NaCl, pH 7.4;

[0022] Buffer A was a solution containing 20 mM PB, 0.15 M NaCl, pH 7.0;

[0023] Buffer B was 0.1 M glycine solution, pH 3.2;

[0024] Buffer C was 1 M Tris-HCl solution, pH 8.5.

[0025] Further, in step S3, the specific steps of affinity chromatography 2 are: in a protein purifier, first use the equilibrium buffer to balance for 10CV, the flow rate is 5mL / min, and then load the sample after the UV absorption baseline is stable, the loading flow rate is 5mL / min, and after the loading is completed, use buffer A to balance for 10CV, and then use 100% pure eluent to elute the human growth hormone target antibody at a volume flow rate of 5ml / min. Start collecting samples when AU is not less than 0.050, and stop collecting samples when AU is not greater than 0.05; obtain affinity chromatography 2 collection liquid, adjust the sample pH to 7.0 with 1M Tris-HCl and 1M NaOH after elution, and store it at 4°C;

[0026] The equilibration buffer was a solution containing 20 mM PB, 150 mM NaCl, pH 7.4;

[0027] The purified eluent was 0.1 M Gly solution, pH 2.4.

[0028] Furthermore, the bio-labeling in step S4 is specifically as follows: 500 μl of the animal serum affinity chromatography 1 collection liquid to be coupled is added to a 1.5 ml centrifuge tube, 250 μl of biotin coupling liquid and 250 μl of dissolved activated biotin are added, and the reaction is carried out on a rotary suspension instrument at room temperature for 2 hours to obtain a coupling product; the desalting column is centrifuged; the top and bottom covers of the desalting column are removed, and the column is placed in a centrifuge tube, centrifuged, and the resin preservation liquid is removed; 5 times the column volume of biotin coupling liquid is added to balance the desalting column, and the coupling liquid is removed by centrifugation after balance; the desalting column is then transferred to a new centrifuge tube, the coupling product is transferred to the center of the resin, centrifuged, and the liquid in the centrifuge tube is collected, which is the animal serum affinity chromatography 1 labeled antibody;

[0029] The collected solution of animal serum affinity chromatography 2 is operated in the same manner as above to obtain animal serum affinity chromatography 2 labeled antibody.

[0030] Furthermore, the specific method for determining the content of human growth hormone is as follows:

[0031] (1) Dilute the purified affinity chromatography 2 animal collection solution 100 times with the coating solution, add 100 ul / well to a 96-well ELISA plate, seal with tin foil and leave overnight;

[0032] (2) Wash the plate with washing solution, 300 μl / well, pat dry on non-ciliated paper, add 200 μl of blocking solution containing 5% bovine albumin to each well, incubate and wash the plate several times, pat dry on non-ciliated paper for the last time;

[0033] (3) diluting human growth hormone to 50 ng with a diluent containing 1% bovine albumin, taking the sample into an ELISA plate with a pipette, 100 ul / well, sealing with tin foil and incubating;

[0034] (4) Take out the ELISA plate, discard the solution in the wells, wash the ELISA plate several times, and pat dry on non-lint paper for the last time; add 100 μl of diluted animal serum affinity chromatography 2 labeled antibody to each well, seal the ELISA plate with tin foil and incubate;

[0035] (5) Take out the ELISA plate, discard the solution in the wells, and wash the ELISA plate several times; use an eight-channel pipette to draw up the substrate solution and add it to the wells of the ELISA plate, 100 μl / well, seal the ELISA plate with tin foil and place it in a dark place;

[0036] (6) When the color development is appropriate, add stop solution, 50 μl / well, to terminate the enzyme reaction, read the sample at 450 nm on an ELISA reader, measure the light absorbance of each well, and determine the human growth hormone content.

[0037] Furthermore, the coating solution preparation method is as follows: taking 1000 ml as an example, take 1.6 g Na2CO3 and 2.9 g NaHCO3, dissolve them in water, adjust the pH to 9.6 with NaOH solution, and make the volume to 1000 ml;

[0038] The preparation method of washing buffer is as follows: taking 1000ml as an example, weigh 2.42g of Tris, dissolve it in water, add 13ml of 1N HCl, adjust the pH value to 7.6, make the volume to 1000ml, add 0.5ml of Tween 20, mix well, and you have it;

[0039] The preparation method of the diluent containing 1% bovine albumin is as follows: taking 1000ml as an example, first prepare 10×PBS buffer solution containing 80g NaCl, 2g KCl, 34.9g Na2HPO4·12H2O, and 2g KH2PO4, add water to dissolve, and make up to 1000ml; then measure 100ml of 10×PBS buffer solution, add 900ml water, 1ml Tween20, and 10g bovine albumin to fully dissolve and mix, and the dilution solution is obtained;

[0040] The preparation method of the blocking solution containing 5% bovine albumin is as follows: taking 1000ml as an example, first take 80g NaCl, 2g KCl, 34.9g Na2HPO4·12H2O, and 2g KH2PO4 to prepare 10×PBS buffer; accurately weigh the above reagents, add water to dissolve, and make up to 1000ml; then measure 100ml of 10×PBS, add 900ml of water, and fully dissolve 50g bovine albumin and mix well to obtain;

[0041] The preparation method of substrate solution is as follows: taking 100 ml as an example, accurately weigh 1.84 g Na2HPO4·12H2O and 0.51 g citric acid, add 80 ml water to dissolve, dilute to 100 ml after dissolution, store at 2-8 ° C in dark, before color development, add 4 mg o-phenylenediamine and 15 ul 30% hydrogen peroxide per 10 ml to obtain;

[0042] The stop solution is 2 mol / L H2S04 solution.

[0043] The beneficial effects of the present invention are:

[0044] The high-purity growth hormone is immunized with animals to obtain polyclonal antibodies, and then two steps of ammonium sulfate crude purification and two steps of affinity chromatography are used to purify the specific antibodies to improve the purity of the specific antibodies; biotin coupling is used to label the high-purity antibodies, which can reduce coupling with other impurities and improve the purity of the labeling; the antibody concentration is optimized by chessboard titration method, and the optimal capture antibody and enzyme-labeled antibody working concentration are selected to reduce the experimental background value and improve the specificity of the experiment; then the prepared specific antibody and high-specificity enzyme-labeled antibody are used to perform the double antibody sandwich method to detect the content of human growth hormone. This method is simple, short cycle, high sensitivity, good characteristics, economical and affordable, and provides an effective detection method for human growth hormone research. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 is the chromatogram of affinity chromatography 1;

[0046] Figure 2 This is the detection curve of growth hormone content in the double antibody sandwich after optimization. DETAILED DESCRIPTION

[0047] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0048] 1. Animal immunization

[0049] 1.1 Purpose

[0050] Human growth hormone protease polyclonal antibody was prepared by immunizing rabbits and guinea pigs, and the residual amount of growth hormone protease was detected using the two prepared polyclonal antibodies (double antibody sandwich method).

[0051] 1.2 Experimental animals and materials

[0052] 1.2.1 Animals: 5 rabbits (clean grade) and 10 guinea pigs (SPF grade).

[0053] 1.2.2 Materials: 1ml syringe, hemostatic forceps, alcohol cotton balls, etc.

[0054] 1.3 Immunization regimen

[0055] 1.3.1 Immunization schedule and dosage

[0056] After the first immunization, the second immunization was carried out 2 weeks apart, and then the third immunization was carried out 1 week apart, for a total of 3 immunizations; whole blood was collected within 1 to 2 weeks after the last immunization; rabbits were immunized by subcutaneous injection in the armpits and back, and guinea pigs were immunized by subcutaneous injection in the abdomen, and the injection dose for each immunization of rabbits and guinea pigs was 0.2 ml / animal. Before immunization, rabbits and guinea pigs were both 0.5 to 1 ml, and serum was separated as pre-immunization serum control.

[0057] 1.3.2 Specific operation methods

[0058] (1) The rabbit weighed about 2 kg and was in normal condition. Before immunization, 0.5 ml of blood was collected and centrifuged at 1000 rpm for 5 min to separate the serum, which was frozen at -20°C as a negative control before immunization.

[0059] (2) Guinea pigs weighing 250-350 g and in normal condition. Before immunization, 0.5 ml of blood was collected and centrifuged at 1000 rpm for 5 min to separate serum, which was frozen at -20°C as a negative control before immunization.

[0060] (3) The antigen (human growth hormone protein 4.034 mg / ml) was diluted with 0.9% sterile sodium chloride to obtain an antigen dilution with a concentration of 120 ug / ml; the antigen dilution was mixed with Freund's complete adjuvant in a volume ratio of 2:1 and then ground to obtain an antigen solution A with a final injection concentration of 80 ug / ml; the antigen dilution was mixed with Freund's incomplete adjuvant in a volume ratio of 2:1 and then ground to obtain an antigen solution B with a final injection concentration of 80 ug / ml.

[0061] (4) Primary immunization: Use a disposable sterile syringe to draw up the ground antigen solution A and inject the animal. Rabbits are given one injection each in the armpit and back, each injection is 100ul; guinea pigs are given one injection in the abdomen, each injection is 200ul.

[0062] (5) Secondary immunization: 14 days after the initial immunization, 0.5 ml of blood was collected from rabbits and guinea pigs, and the serum was separated by centrifugation at 1000 rpm for 5 min and then frozen at -20°C. Then, for secondary immunization, antigen liquid B was aspirated and injected into the rabbit's armpit and back with one injection each, 100 ul; and guinea pigs were injected into the abdomen with one injection each, 200 ul.

[0063] (6) Three immunizations: 7 days after the second immunization, 0.5 ml of blood was collected from rabbits and guinea pigs, and the serum was separated by centrifugation at 10,000 rpm for 5 min and stored at -20°C. Then, three immunizations were performed, and antigen liquid B was aspirated and injected into the rabbit's armpit and back, with each injection being 100 ul; and into the guinea pig's abdomen, with each injection being 200 ul.

[0064] The antibody titer was monitored during the immunization process. If the antibody titer monitoring reached the use requirement one week after the third immunization, whole blood was immediately collected to obtain guinea pig serum and rabbit serum. The results of the three immunization titer tests are shown in Table 1.

[0065] Table 1 Three times immunization titer

[0066]

[0067] 2. Antibody Purification

[0068] 2.1 Preparation of hGH coupling filler

[0069] Solution preparation:

[0070] a. Coupling buffer A: prepare 1mM HCl buffer, filter through 0.45μm water membrane under vacuum, store at 2-8℃ for future use;

[0071] b. Coupling buffer B: Prepare a buffer with pH = 8.3 and containing 0.1M NaHCO3 and 0.5M NaCl, filter it under vacuum with a 0.45μm water membrane, and store it at 2-8°C for later use;

[0072] c. Blocking solution: Prepare 0.1M Tris-HCl blocking solution with pH = 8.0, filter through a 0.45 μm water membrane under vacuum, and store at 2-8°C for later use;

[0073] d. Cleaning solution 1: Prepare cleaning solution 1 (containing 0.1M Tris-HCl, 0.5M NaCl) with pH = 8.5, filter through a 0.45μm water membrane under vacuum, store at 2-8°C, and set aside;

[0074] e. Cleaning solution 2: Prepare cleaning solution 2 with pH = 8.5 and containing 0.1M NaAc and 500mM NaCl, filter through a 0.45μm water membrane under vacuum, store at 2-8°C, and set aside;

[0075] Preparation of ligand solution:

[0076] a. hGH protein preparation: hGH stock solution comes from the purified batch C01-240108-C02. The stock solution contains 2.98% related protein, 0.1% high molecular weight protein, protein concentration of 22.34 mg / ml, and a volume of 10 ml. The purification process belongs to the existing technology and is omitted here.

[0077] b. Chromatography column: GE HiPrep 26 / 10 Desalting 53ml pre-packed column

[0078] c. Chromatography method: Use SDL 030V2 protein purifier from Saipu, first use coupling buffer B to equilibrate the chromatography column for 3CV, flow rate 5mL / min, load the sample after the UV absorption baseline is stable, the loading flow rate is 5mL / min, and then use coupling buffer B to equilibrate for 3CV after loading. Start collecting samples when AU is not less than 0.1, and stop collecting samples when AU is not greater than 0.2; obtain ligand solution with a volume of 18ml, a concentration of 11.79mg / ml, and a total protein of 212.22mg; store at 4℃.

[0079] hGH coupling filler preparation:

[0080] a. Take 10 ml of medium (i.e. filler Bogelon CNBr-activated Bestarose 4B) stored in acetone and put it in a sand core funnel, and wash it with 600 ml of pre-cooled coupling buffer A for 30 minutes.

[0081] b. Dilute the washed medium with 5 ml of coupling solution A and mix with 5 ml of ligand solution (see step ligand solution preparation for the preparation process) in equal volumes, and mix in a shaker at room temperature for 2 hours. Determine the protein content of the uncrosslinked protein.

[0082] c. Blocking: Remove the coupling supernatant and add blocking solution, and block at room temperature for 3 hours.

[0083] d. Cleaning: Use cleaning solution 1 and cleaning solution 2 to wash alternately 6 times, with the amount of cleaning solution used each time being 5 times the volume of the medium.

[0084] e. After washing with PBS buffer (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM K2HPO4, pH 7.2), the mixture was stored in 20% ethanol. Finally, CNBr-hGH was a filler medium with a density of 5.3 mg / ml.

[0085] 2.2 Ammonium sulfate primary precipitation

[0086] Take hGH serum obtained by animal immunization, measure 20 ml after thawing, add an equal volume of water for injection, add ammonium sulfate (the final concentration of ammonium sulfate in the system is 15%) under stirring, stir at room temperature for 20 minutes, centrifuge at 20°C and 8000 rpm for 15 minutes, collect the supernatant, and obtain ammonium sulfate primary precipitation supernatant.

[0087] 2.3 Secondary purification of ammonium sulfate

[0088] Take 18 ml of the supernatant of the ammonium sulfate primary precipitation, adjust the pH to 7.25±0.1 with 1 M NaOH, add solid ammonium sulfate according to a final concentration of 30%, stir at room temperature for 15 minutes, centrifuge at 20°C and 8000 rpm for 15 minutes, collect the precipitate, and obtain the ammonium sulfate secondary purification precipitate.

[0089] 2.4 Precipitate re-dissolution

[0090] The ammonium sulfate secondary purification precipitate was dissolved with 10 ml of dissolution buffer (20 mM PB + 0.15 M NaCl, pH = 7.0) to obtain a reconstituted collection solution.

[0091] 2.5 Desalination

[0092] Chromatography column: HiPrep 26 / 10 Desalting 53ml prepacked column

[0093] Loading sample: Redissolve the collected solution and store at 2-8℃ for later use

[0094] Desalting buffer: 20 mM Pb + 0.15 M NaCl, pH = 7.0

[0095] Chromatographic method: Using SDL 030V2 protein purifier from Saipu, first use desalting buffer to equilibrate HiPrep 26 / 10Desalting desalting column 5CV, flow rate 10ml / min; after the UV absorption baseline is stable, load the sample, flow rate 10ml / min, sample volume 10ml, use desalting buffer as mobile phase; collect the feed solution with peak value ≥10mAU to obtain the desalted collection solution.

[0096] 2.6 Affinity chromatography 1

[0097] Chromatography column: GE Mabselect SuReTM LX prepacked column

[0098] Chromatography Buffer 1:

[0099] Buffer A: 20 mM PB + 0.15 M NaCl, pH = 7.0

[0100] Buffer B: 0.1 M glycine, pH = 3.2

[0101] Buffer C: 1M Tris-HCl, pH = 8.5

[0102] Loading sample: desalted collection solution, stored in a refrigerator at 2-8℃ for future use;

[0103] Chromatographic method: using SDL 030V2 protein purifier of Saipu, first equilibrate the chromatography column (GE Mabselect SuReTM LX pre-packed column) with crude pure equilibration buffer (20mM PB, 150mM NaCl, pH7.4) for 10CV, flow rate 5mL / min, load the sample after the UV absorption baseline is stable, the sample loading flow rate is 5mL / min, and then equilibrate with buffer A (20mM PB, 150mM NaCl, pH7.0) for 10CV after loading, collect the UV280nm>15mAU flow-through peak; then elute with a volume flow rate of 5ml / min and 100% buffer B for 8CV, collect the elution peak, and add 5% volume of buffer C to the elution peak collection tube in advance. Start collecting samples when AU is not less than 0.015, and stop collecting samples when AU is not greater than 0.01, and obtain affinity chromatography 1 collection liquid, which is stored at 4°C.

[0104] 2.7 Sample dilution

[0105] The affinity chromatography 1 collection solution was diluted with a pure diluent (40 mM PB, pH 7.4) at a ratio of 1:1, and then the pH was adjusted to 7.43 with 5 M NaOH to obtain a diluent, which was stored at 4°C.

[0106] 2.8 Affinity chromatography 2

[0107] Chromatographic column: Bogelon CNBr-hGH-Bestarose 4B coupling column (prepared in step 2.1 and loaded into the chromatography column)

[0108] Load sample: Affinity chromatography 1 collect dilution

[0109] Chromatographic method: using SDL 030V2 protein purifier of Saipu, first use equilibrium buffer (20mM PB, 150mM NaCl, pH7.4) to balance the chromatography column for 10CV, flow rate 5mL / min, wait for the UV absorption baseline to stabilize, then load the sample, the sample flow rate is 5mL / min, after loading, balance with equilibrium buffer (20mM PB, 150mM NaCl, pH 7.4) for 10CV, and then use 100% pure eluent (0.1M Gly, pH2.4) at a volume flow rate of 5ml / min to elute the human growth hormone target antibody. When AU is not less than 0.050, start collecting samples, and when AU is not greater than 0.050, stop collecting samples; obtain affinity chromatography 2 collection liquid, adjust the sample pH to 7.0 with 1MTris-HCl (pH9.5) and 1M NaOH after elution, and store at 4°C.

[0110] The guinea pig serum and rabbit serum obtained by cell immunization were respectively purified according to the above-mentioned "2. Antibody Purification" operation. The titer of the collected solution of affinity chromatography 1 was measured, and the results are shown in Table 2; the titer of the collected solution of affinity chromatography 2 was measured, and the results are shown in Table 3.

[0111] Table 2 Affinity chromatography 1 Antibody titer detection

[0112]

[0113] Table 3 Affinity chromatography 2 antibody titer detection

[0114]

[0115]

[0116] 3. Antibody titer detection

[0117] The methods for detecting the three immune titers in the above-mentioned step "1. Animal Immunization" and the antibody titer detection of the affinity chromatography 1 collection fluid and the affinity chromatography 2 collection fluid in the step "2. Antibody Purification" are as follows:

[0118] 3.1. Coating ELISA plate

[0119] Dilute the coating antigen (human growth hormone) to 50ug / ml with coating solution (same as coating solution in 6.1.1), add 100ul / well to the 96-well ELISA plate, seal with tin foil and place at 2-8℃ overnight (16 hours).

[0120] 3.2 Closure

[0121] Wash the plate once with washing buffer (same washing buffer as in 6.1.2), 300ul / well, pat dry on lint-free paper, add 200ul blocking solution (same blocking solution containing 5% bovine albumin as in 6.1.4) to each well, incubate at 37℃ for 1 hour, wash the plate 3 times, pat dry on lint-free paper for the last time.

[0122] 3.3. Test sample dilution

[0123] Use diluent (the same diluent containing 1% bovine albumin as in 6.1.3) to dilute the antibody (antiserum from animal immunization or affinity chromatography 1 collection fluid or affinity chromatography 2 collection fluid) in multiple ratios, then use an eight-channel pipette to pipette the sample into the ELISA plate, 100 μl / well, make two or more wells in parallel, seal with tin foil, and incubate at 37°C for 1 hour.

[0124] 3.4. Adding enzyme-labeled antibodies

[0125] Take out the ELISA plate, discard the solution in the wells, wash the plate 3 times, and pat dry on non-lint paper for the last time. Add 100 μl of diluted (1:10000) ELISA antibody to each well, seal the ELISA plate with tin foil, and incubate at 37°C for 1 hour.

[0126] 3.5. Adding substrate

[0127] Take out the ELISA plate, discard the solution in the wells, and wash the ELISA plate 6 times. Use an eight-channel pipette to draw the substrate solution (TMB) (same as the substrate solution in 6.1.5) and add it to the wells of the ELISA plate, 100μl / well, seal the ELISA plate with tin foil, and place it at 33℃ away from light for 10 minutes before observing the results.

[0128] 3.6. Termination of the reaction

[0129] When the color development is appropriate, add stop solution (same as stop solution in 6.1.6), 50 μl / well, to stop the enzyme reaction, read the sample at 450 nm on a microplate reader, and measure the light absorption value (OD value) of each well.

[0130] 3.7 Result judgment

[0131] The cutoff value was calculated based on the OD value of the negative control group. More than 80% of the serum anti-IgG antibodies in the test group should be higher than the cutoff value.

[0132] Cutoff value = average OD value of negative control group × 2.1. A value greater than the cutoff value is considered positive.

[0133] 4. Antibody Purification Protein Detection

[0134] The purified affinity chromatography 1 collection solution and affinity chromatography 2 collection solution obtained in the step "2. Antibody Purification" were subjected to SDS-PAGE electrophoresis analysis, and two clear and complete bands appeared, and no other impurities were found. In addition, the protein concentration of affinity chromatography 1 collection solution and affinity chromatography 2 collection solution was measured, and the results are shown in Table 4. It was found that the protein concentration was correspondingly increased after the two-step purification.

[0135] Table 4 Antibody concentration of affinity chromatography collection fluid

[0136]

[0137] 5. Antibody Labeling

[0138] 5.1. Biotin labeling

[0139] Principle: Activated biotin cross-links with primary amines on antibodies or proteins to form stable amide bonds, thus achieving the coupling reaction between biotin and antibodies or proteins. The reagents and test solutions used in this experiment are from the purchased biotin conjugation kit.

[0140] 5.1.1. Take 500 μl of the antibody or protein to be coupled (rabbit antibody in affinity chromatography 1, i.e., rabbit serum affinity chromatography 1 collection fluid) and add them into a 1.5 ml centrifuge tube.

[0141] 5.1.2. Add 250 μl of biotin coupling solution and 250 μl of dissolved activated biotin at the same time, and react slowly at room temperature for 2 hours on a rotating suspension machine.

[0142] 5.1.3. Centrifuge the desalting column at 1000 rpm for 1 minute.

[0143] 5.1.4. Remove the top and bottom caps of the desalting column and place it in a suitable centrifuge tube (15 ml centrifuge tube). Centrifuge at 1000 rpm for 2 minutes to remove the resin storage solution.

[0144] 5.1.5. Add 5 column volumes of biotin coupling solution (25 ml) to equilibrate the desalting column. After equilibration, centrifuge to remove the coupling solution: 1000 rpm, 2 minutes.

[0145] 5.1.6. Transfer the desalting column to a new centrifuge tube, slowly transfer the coupling product of step 2 (step 5.1.2) to the center of the resin, and centrifuge at 1000 rpm for 8 minutes.

[0146] 5.1.7. Collect the liquid in the centrifuge tube, which is the labeled product (recorded as rabbit serum affinity chromatography 1 labeled antibody).

[0147] 5.1.8. Affinity chromatography 1 guinea pig antibody (i.e. guinea pig serum affinity chromatography 1 collected solution), affinity chromatography 2 rabbit antibody (i.e. rabbit serum affinity chromatography 2 collected solution), affinity chromatography 2 guinea pig antibody (i.e. guinea pig serum affinity chromatography 2 collected solution) were operated in the same manner to obtain labeled products (respectively recorded as guinea pig serum affinity chromatography 1 labeled antibody, rabbit serum affinity chromatography 2 labeled antibody, guinea pig serum affinity chromatography 2 labeled antibody).

[0148] 6. Human growth hormone (hGH) determination

[0149] Principle: The purified antibody is coated on the ELISA plate, and the sample or growth hormone standard is combined with the coated antibody. Biotin-labeled antibodies are added as detection antibodies to bind to the above antigen-antibody complex. TMB is used as the color development system, and the values ​​are measured at a wavelength of 450nm to compare the degree of biotin labeling at different purification stages.

[0150] 6.1. Preparation of reagents and test solutions

[0151] 6.1.1. Preparation of coating solution (taking 1000ml as an example)

[0152] Na2CO3: 1.6g, NaHCO3: 2.9g

[0153] Accurately weigh the above reagents, add 800 ml of water for injection to dissolve, adjust the pH value to 9.6 with 5 M NaOH solution, make up to 1000 ml, and store at 2-8°C.

[0154] 6.1.2. Washing buffer (taking 1000ml as an example)

[0155] Tris: 2.42g, 1N HCl: 13ml, Tween20: 0.5ml

[0156] Accurately weigh 2.42 g of Tris, add 600 ml of water for injection to dissolve, then add 13 ml of 1N HCl, adjust the pH to 7.6 with HCl or NaOH solution, make up to 1000 ml, add 0.5 ml of Tween 20, mix well and store at 2-8°C.

[0157] 6.1.3. Dilution (1% bovine albumin) (taking 1000ml as an example)

[0158] Preparation of 10×PBS buffer: NaCl 80g, KCl 2g, Na2HPO4·12H2O 34.9g, KH2PO4 2g

[0159] Accurately weigh the above reagents, add 800 ml of water for injection to fully dissolve, and then make up to 1000 ml.

[0160] Take 100 ml of 10×PBS buffer, add 900 ml of water for injection, 1 ml of Tween20 and 10 g of bovine albumin and fully dissolve and mix.

[0161] 6.1.4. Blocking solution (5% bovine albumin) (taking 1000ml as an example)

[0162] Preparation of 10×PBS buffer: NaCl 80g, KCl 2g, Na2HPO4·12H2O 34.9g, KH2PO4 2g

[0163] Accurately weigh the above reagents, add 800 ml of water for injection to fully dissolve, and then make up to 1000 ml.

[0164] Take 100 ml of 10×PBS, add 900 ml of water for injection and 50 g of bovine albumin and mix thoroughly.

[0165] 6.1.5. Substrate solution (taking 100 ml as an example)

[0166] Na2HPO4·12H2O: 1.84 g, citric acid: 0.51 g

[0167] Accurately weigh Na2HPO4·12H2O and citric acid, add 80ml of water for injection to dissolve, and then dilute to 100ml. Store at 2-8℃ away from light. Before color development, add 4mg of o-phenylenediamine and 15ul of 30% hydrogen peroxide per 10ml.

[0168] 6.1.6. Stop solution (2 mol / L H2S04 solution) (taking 100 ml as an example)

[0169] Take 11 ml of concentrated sulfuric acid and slowly drip it into 70 ml of water to make up to 100 ml. Store at 2-8°C.

[0170] Experimental procedures

[0171] 6.2.1. Dilute the purified antibody 100 times with coating solution and add 100ul / well to a 96-well ELISA plate. Seal with tin foil and place at 2-8°C overnight (16 hours).

[0172] 6.2.2. Wash the plate with 300ul / well of washing solution, pat dry on lint-free paper, add 200ul of blocking solution to each well, incubate at 37℃ for 1 hour, wash the plate 3 times, and pat dry on lint-free paper for the last time.

[0173] 6.2.3. Dilute human growth hormone to 50 ng with diluent, take the sample into the ELISA plate with a pipette, 100 ul / well, seal with tin foil and incubate at 37°C for 1 hour.

[0174] 6.2.4. Take out the ELISA plate, discard the solution in the wells, wash the plate 3 times, and pat dry on non-lint paper for the last time. Add 100 μl of diluted (1:5000) biotin-labeled antibody to each well, seal the ELISA plate with tin foil, and incubate at 37°C for 1 hour.

[0175] 6.2.5. Take out the ELISA plate, discard the solution in the wells, and wash the ELISA plate 6 times. Use an eight-channel pipette to absorb the substrate solution (TMB) and add it to the wells of the ELISA plate, 100μl / well. Seal the ELISA plate with tin foil and place it at 33℃ away from light for 10 minutes before observing the results.

[0176] 6.2.6. When the color development is appropriate, add stop solution, 50 μl / well, to terminate the enzyme reaction, read the sample at 450 nm on a microplate reader, and measure the light absorption value (OD value) of each well.

[0177] The purified antibodies (i.e., rabbit serum affinity chromatography 1 collection fluid, guinea pig serum affinity chromatography 1 collection fluid, rabbit serum affinity chromatography 2 collection fluid, guinea pig serum affinity chromatography 2 collection fluid) were coated on ELISA plates, and human growth hormone (hGH) was determined according to the above-mentioned operation method. The results are shown in Table 5.

[0178] It can be seen that through the observation of the absorbance value of the capture degree of the purified biotin-labeled antibody, it was found that anti-GH-M-02-01 (guinea pig serum affinity chromatography 1 collection fluid) and anti-GH-R-02-01 (rabbit serum affinity chromatography 1 collection fluid) may have more impurities, the biotin labeling effect is not good, and the overall background value is high; anti-GH-M-02-02 (guinea pig serum affinity chromatography 2 collection fluid) and anti-GH-R-02-02 (rabbit serum affinity chromatography 2 collection fluid) have the same attribute animal labeling capture effect is very good, there is no interference from impurities, the negative background value is low, and the guinea pig is slightly better than the rabbit; the background value is still high when the antibodies of animals of different attributes are captured, and the proteins of different attributes may cause cross-talk.

[0179] Table 5 Capture degree of antibodies by different biotinylated antibodies after purification

[0180]

[0181]

[0182] 7. Optimization of capture antibody and antibody working concentration

[0183] The working concentration of the best paired antibody combination screened out by the antibody pair experiment was optimized, with anti-GH-M-02-02 as the capture antibody and anti-GH-M-bio-02 as the detection antibody. Five different concentrations of capture antibody anti-GH-M-02 were used for coating (2ug / ml, 4ug / ml, 6ug / ml, 8ug / ml, 10ug / ml), and the detection antibody was detected by titration with anti-GH-M-bio-02 at four different dilutions (1:1000, 1:3000, 1:6000, 1:9000). When the 0D450 value was greater than 1 and P / N>2.1, it was determined to be the best working concentration, as shown in Table 6. It can be seen that the coating concentration of human growth hormone is 6ug / ml and the dilution is 1:6000, which is the best working concentration.

[0184] Table 6 Optimization of antibody concentration by chessboard titration method

[0185]

[0186] 8. Detection of growth hormone content in double antibody sandwich after optimization

[0187] Experimental procedures

[0188] Dilute anti-GH-M-02-02 to 6ug / ml with coating solution and add to the ELISA plate overnight, wash, block for 1 hour, wash, add 10ng, 5ng, 2.5ng, 1.25ng, 0.625ng, 0.312ng, 0.0156ng, 0ng of growth hormone to 100ul / well with diluent, incubate at 37℃ for 1 hour. Take out the ELISA plate, wash, add 1:6000 dilution of anti-GH-M-bio-02 to each well, 100ul / well, incubate for 1 hour, add TMB for color development, stop, and read.

[0189] Table 7 Detection of growth hormone content in double antibody sandwich after optimization

[0190]

[0191] The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention belong to the protection scope of the present invention. It should be pointed out that for ordinary technicians in this technical field, some improvements and modifications without departing from the principle of the present invention should also be regarded as the protection scope of the present invention.

Claims

1. A non-diagnostic detection method for human growth hormone based on a highly specific double antibody sandwich method, characterized in that: The following steps are involved: S1 Animal immunization: human growth hormone antigen is mixed with Freund's complete adjuvant to prepare antigen liquid A, and human growth hormone antigen is mixed with Freund's incomplete adjuvant to prepare antigen liquid A and antigen liquid B. Antigen liquid A is injected for primary animal immunization, and antigen liquid B is injected for secondary and tertiary animal immunization to obtain animal serum; S2 crude serum purification: crudely purify the animal serum after three immunizations with protein A or G to obtain crude purified animal serum; S3 serum purification: the crude animal serum is subjected to two-step chromatography purification, affinity chromatography 1 and affinity chromatography 2, to obtain affinity chromatography 2 animal collection fluid; S4 biomarker: biotin-label the collected fluid from affinity chromatography 2 animals to obtain animal serum affinity chromatography 2 labeled antibodies; S5 Determination of human growth hormone content: The ELISA plate was coated with the animal collection fluid of affinity chromatography 2, and after blocking and washing, the animal serum affinity chromatography 2 labeled antibody was used as the detection antibody, and TMB was used as the color development system to determine the human growth hormone content; In step S3, the specific steps of affinity chromatography 1 are as follows: in a protein purifier, first equilibrate the chromatography column with a crude pure equilibration buffer for 10CV at a flow rate of 5 mL / min, load the sample after the UV absorption baseline is stable, and load the sample at a flow rate of 5 mL / min. After loading, equilibrate with buffer A for 10CV, collect the UV280nm>15mAU flow-through peak; then elute at a volume flow rate of 5 ml / min and 100% buffer B for 8CV, collect the elution peak, and pre-add 5% volume of buffer C to the elution peak collection tube; start collecting samples when AU is not less than 0.015, and stop collecting samples when AU is not greater than 0.01, to obtain affinity chromatography 1 collection liquid, and store it at 4°C; The crude pure equilibration buffer was a solution containing 20 mM PB, 150 mM NaCl, pH 7.4; Buffer A is a solution containing 20 mM PB, 0.15 M NaCl, pH 7.0; Buffer B was 0.1 M glycine solution, pH 3.2; Buffer C was 1 M Tris-HCl solution, pH 8.5; In step S3, affinity chromatography 2 specifically comprises the following steps: in a protein purifier, first equilibrate with an equilibrium buffer for 10CV at a flow rate of 5 mL / min, load the sample after the UV absorption baseline is stable, and load the sample at a flow rate of 5 mL / min. After loading, equilibrate with buffer A for 10CV, and then elute the human growth hormone target antibody with a volume flow rate of 5 ml / min using 100% pure eluent; start collecting samples when AU is not less than 0.050, and stop collecting samples when AU is not greater than 0.05; obtain affinity chromatography 2 collection liquid, adjust the sample pH to 7.0 with 1 M Tris-HCl and 1 M NaOH after elution, and store at 4 ° C; the equilibrium buffer is a solution containing 20 mM PB and 150 mM NaCl, pH 7.4; the pure eluent is a 0.1 M Gly solution, pH 2.4; the chromatography column coupling filler used is prepared by the following method: (1) Solution preparation: a. Coupling buffer A: prepare 1 mM HCl buffer, filter, and store at 2-8°C; b. Coupling buffer B: Prepare a buffer with pH = 8.3 and containing 0.1M NaHCO3 and 0.5M NaCl, filter, and store at 2-8°C; c. Blocking solution: Prepare 0.1M Tris-HCl blocking solution with pH=8.0, filter, and store at 2-8℃; d. Cleaning solution 1: Prepare cleaning solution 1 with pH = 8.5, containing 0.1M Tris-HCl and 0.5M NaCl, filter, and store at 2-8℃; e. Cleaning solution 2: Prepare cleaning solution 2 with pH = 8.5 and containing 0.1M NaAc and 500mM NaCl, filter, and store at 2-8°C; (2) Preparation of ligand solution: First, hGH protein was prepared; then, in a protein purifier, the chromatography column was equilibrated with coupling buffer B for 3CV at a flow rate of 5 mL / min, and the sample was loaded after the UV absorption baseline was stable at a loading flow rate of 5 mL / min. After loading, the coupling buffer B was used for equilibration for 3CV again, and the sample was collected when the AU was not less than 0.1, and the sample was stopped when the AU was not greater than 0.2; the ligand solution was obtained; the chromatography column was a GE HiPrep 26 / 10 Desalting 53 ml pre-loaded column; (3) Preparation of hGH coupling filler: a. Take 10 ml of medium filler Bogelon CNBr-activated Bestarose 4B stored in acetone and put it in a sand core funnel, and wash it with pre-cooled 600 ml coupling buffer A for 30 min; b. Dilute the washed medium with 5 ml of coupling solution A and mix with 5 ml of ligand solution in equal volumes. Mix well in a shaker at room temperature for 2 h. c. Blocking: remove the coupling supernatant and add blocking solution, block at room temperature for 3 hours; d. Cleaning: Use cleaning solution 1 and cleaning solution 2 to wash alternately for multiple times; e. After washing with PBS buffer, store in 20% ethanol to finally obtain hGH coupling filler.

2. According to claim 1, a non-diagnostic detection method for human growth hormone based on a highly specific double antibody sandwich method, characterized in that: In step S2, crude purification of serum includes the steps of primary precipitation with ammonium sulfate, secondary purification with ammonium sulfate, re-dissolution of precipitation, and chromatographic desalting.

3. According to claim 1, a non-diagnostic detection method for human growth hormone based on a highly specific double antibody sandwich method, characterized in that: Step S4 biomarker is specifically as follows: 500 μl of the animal serum affinity chromatography 1 collection solution to be coupled is added to a 1.5 ml centrifuge tube, 250 μl of biotin coupling solution and 250 μl of dissolved activated biotin are added, and the reaction is carried out at room temperature for 2 hours on a rotary suspension instrument to obtain a coupling product; Centrifuge the desalting column; remove the top and bottom caps of the desalting column, place it in a centrifuge tube, centrifuge, and remove the resin preservation solution; add 5 times the column volume of biotin coupling solution to balance the desalting column, and centrifuge to remove the coupling solution after balancing; Then transfer the desalting column to a new centrifuge tube, transfer the coupling product to the center of the resin, centrifuge, and collect the liquid in the centrifuge tube, which is the animal serum affinity chromatography 1 labeled antibody; The collected solution of animal serum affinity chromatography 2 is operated in the same manner as above to obtain animal serum affinity chromatography 2 labeled antibody.

4. According to claim 1, a non-diagnostic detection method for human growth hormone based on a highly specific double antibody sandwich method, characterized in that: The specific method for determining the content of human growth hormone is: (1) Dilute the purified affinity chromatography 2 animal collection solution 100 times with the coating solution, add 100ul / well to a 96-well ELISA plate, seal with tin foil and leave overnight; (2) Wash the plate with washing solution, 300 μl / well, pat dry on non-ciliated paper, and add 200 μl of blocking solution containing 5% bovine albumin to each well. After incubation, wash the plate several times, and pat dry on non-ciliated paper for the last time. (3) Dilute human growth hormone to 50 ng using a diluent containing 1% bovine albumin, pipette the sample into an ELISA plate, 100 ul / well, seal with tin foil and incubate; (4) Take out the ELISA plate, discard the solution in the wells, wash the plate several times, and pat dry on non-lint paper for the last time; add 100 μl of diluted animal serum affinity chromatography 2 labeled antibody to each well, seal the ELISA plate with tin foil and incubate; (5) Take out the ELISA plate, discard the solution in the wells, and wash the plate several times; use an eight-channel pipette to draw up the substrate solution and add it to the wells of the ELISA plate, 100 μl / well, seal the ELISA plate with tin foil and place it away from light; (6) When the color development is appropriate, add stop solution, 50 μl / well, to terminate the enzyme reaction. Read the sample at 450 nm on an ELISA reader and measure the light absorbance of each well to determine the human growth hormone content.

5. According to claim 4, a non-diagnostic detection method for human growth hormone based on a highly specific double antibody sandwich method, characterized in that: The preparation method of coating solution is as follows: taking 1000ml as an example, take 1.6g Na2CO3 and 2.9g NaHCO3, dissolve them in water, adjust the pH to 9.6 with NaOH solution, and make up to 1000ml. The preparation method of washing buffer is as follows: taking 1000ml as an example, weigh 2.42g of Tris, dissolve it in water, add 13ml of 1N HCl, adjust the pH value to 7.6, make the volume to 1000ml, add 0.5ml of Tween 20, mix well, and the solution is ready; The preparation method of the diluent containing 1% bovine albumin is as follows: taking 1000ml as an example, first prepare 10×PBS buffer solution containing 80g NaCl, 2g KCl, 34.9g Na2HPO4·12H2O, and 2g KH2PO4, add water to dissolve, and make up to 1000ml; then measure 100ml of 10×PBS buffer solution, add 900ml water, 1ml Tween20, and 10g bovine albumin to fully dissolve and mix, and the dilution solution is obtained; The preparation method of the blocking solution containing 5% bovine albumin is as follows: taking 1000ml as an example, first take 80g NaCl, 2g KCl, 34.9g Na2HPO4·12H2O, and 2g KH2PO4 to prepare 10×PBS buffer; accurately weigh the above reagents, add water to dissolve, and make up to 1000ml; then measure 100ml of 10×PBS, add 900ml of water, and fully dissolve 50g bovine albumin and mix well to obtain; The preparation method of substrate solution is as follows: taking 100 ml as an example, accurately weigh 1.84 g Na2HPO4·12H2O and 0.51 g citric acid, add 80 ml water to dissolve, dilute to 100 ml after dissolution, store at 2-8 ° C away from light, before color development, add 4 mg o-phenylenediamine and 15 ul 30% hydrogen peroxide per 10 ml to obtain; The stop solution is 2 mol / L H2S04 solution.

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