CN110894208B - Progesterone hapten, artificial antigen and polyclonal antibody as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a progesterone hapten, an artificial antigen and a polyclonal antibody, and a preparation method and application thereof. The invention takes progesterone as raw material, provides a synthesis and purification method for preparing progesterone hapten, couples the synthesized and purified progesterone hapten with carrier protein to obtain progesterone complete immunogen, immunizes New Zealand white rabbits, prepares polyclonal antibody with high potency and strong specificity, and finally establishes an enzyme-linked immunosorbent assay (BA-ELISA) for detecting progesterone based on biotin/streptavidin amplified signal₅₀The detection linearity range is 1.29ng/mL, the detection linearity range is 0.22-7.50 ng/mL, the detection limit is 0.080ng/mL, and the method has a wide application prospect.

Description

Progesterone hapten, artificial antigen and polyclonal antibody as well as preparation method and application thereof

Technical Field

The invention relates to the technical field of immunology, and particularly relates to a progesterone hapten, an artificial antigen and a polyclonal antibody, and a preparation method and application thereof.

Background

Progesterone, also known as progesterone, is a steroid hormone that plays a key role in the complex regulation of female reproductive function. If the cow is likely to be aborted due to the fact that the content of the progesterone in the serum or the milk is too low, the abortion of the cow in the early pregnancy can be prevented by properly supplementing the progesterone, and the huge loss of the cow breeding industry can be recovered. Meanwhile, the detection of the content of progesterone in the milk product is also very meaningful. If women and children drink the milk product with the high content of the progesterone for a long time, the sexual precocity can be caused, and the problems of various aspects of physiology and psychology can be caused. If the content of milk products of a certain brand is detected to be too high, the product can be prevented from being purchased, and the probability of precocious puberty of female children is reduced. Therefore, the detection of progesterone in the milk product is particularly important.

Patent CN108254365A discloses a magnetic immunochemiluminescence detection kit for progesterone, which comprises an enzyme-labeled antigen, an enzyme-labeled antigen diluent, a magnetic labeled antibody diluent, a progesterone series standard solution, a chemiluminescent substrate A solution, a chemiluminescent substrate B solution, a complex solution and a washing solution; the enzyme-labeled antigen is progesterone hapten labeled by horse radish peroxidase, the magnetic labeled antibody is progesterone monoclonal antibody labeled by immunomagnetic beads, and the progesterone monoclonal antibody is prepared by taking conjugate obtained by coupling the progesterone hapten and bovine serum albumin as immunogen immune animals; the progesterone hapten is obtained by reacting 16, 17-epoxy progesterone with mercaptopropionic acid. Because the artificial antigen has low immunogenicity and cannot fully expose the specific site of the progesterone, the progesterone antibody with high specificity cannot be generated, and the accuracy of the progesterone detection result is poor; although the magnetic immuno-chemiluminescence detection is adopted, the detection sensitivity of the progesterone can only reach 0.1 mu g/L, and the micro detection of the progesterone cannot be met.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide a progesterone hapten.

Another object of the present invention is to provide a progesterone complete antigen.

Another object of the present invention is to provide a polyclonal antibody specific for progesterone.

The invention also aims to provide application of the progesterone complete antigen and progesterone specific polyclonal antibody.

The invention further aims to provide a progesterone BA-ELISA detection kit.

The above object of the present invention is achieved by the following technical solutions:

a progesterone hapten having a structural formula as shown in formula (I):

the invention takes progesterone as raw material, modifies functional group on carbon of 1 site of progesterone, connects carboxyl to obtain progesterone derivative, which is used as the progesterone hapten.

The preparation method of the progesterone hapten comprises the following steps:

s1, mixing carboxymethyl amine half hydrochloride and anhydrous pyridine according to a molar ratio of 3: 1 are respectively dissolved in methanol solution;

s2, adding methanol-dissolved progesterone P into the anhydrous pyridine solution of S1₄Solution, progesterone P₄The molar ratio of the pyridine to the anhydrous pyridine is 2: 1;

s3, uniformly mixing the solution to obtain yellow viscous liquid; dissolving the yellow viscous liquid by using dichloromethane, adding deionized water for washing, adding anhydrous magnesium sulfate for dewatering, performing suction filtration to obtain a light yellow liquid, performing rotary evaporation to remove the solvent, and concentrating;

s4, using dichloromethane/methanol (20:1(v/v) → 16:1(v/v) → 12:1(v/v)) as an eluent, purifying the residue by using a silica gel chromatographic column, and repeating the steps for 1-2 times to obtain a white target compound, namely progesterone hapten.

A progesterone complete immunogen is prepared by coupling the above progesterone hapten with carrier protein.

Preferably, the carrier protein is bovine serum albumin.

The preparation method of the progesterone complete immunogen comprises the steps of coupling the progesterone hapten shown in the formula (I) with carrier protein by an active ester method, and dialyzing to obtain the progesterone complete immunogen.

Specifically, the preparation method of the progesterone complete immunogen comprises the following steps:

s1, dissolving a progesterone hapten in N, N-Dimethylformamide (DMF), and respectively dissolving N-hydroxysuccinimide (NHS) and 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the N, N-Dimethylformamide (DMF) to enable the molar ratio of the three to be 1:2: 2;

s2, stirring for 12-16 hours at room temperature by using a magnetic stirrer at the rotating speed of 250-300 rpm to fully activate carboxyl on the hapten;

s3, slowly stirring in an ice bath, and gradually dropwise adding the activated hapten into 5-7 mg/mL Bovine Serum Albumin (BSA) to enable the final concentration of DMF in the solution to be 20-25%;

s4, slowly stirring and coupling for 12-16 hours at low temperature to couple more haptens to carrier proteins;

s5, filling the conjugate of the hapten and the carrier protein into a dialysis bag, dialyzing for three days at 4 ℃, and changing PBS (0.01M; pH 7.4) for 3 times a day to remove redundant DMF;

s6.10,000-12,000 rpm, centrifuging at 4 ℃ for 10min, discarding the precipitate, taking the supernatant, measuring the concentration by using a BCA protein quantification kit, subpackaging in an EP tube, and storing at-20 ℃ for later use.

A method for preparing progesterone specific polyclonal antibody comprises emulsifying progesterone complete immunogen, and immunizing rabbit to obtain progesterone specific polyclonal antibody.

Specifically, the preparation method of the progesterone-specific polyclonal antibody comprises the following steps:

s1, diluting a progesterone complete immunogen to 1.0-1.2 mg/mL, mixing the progesterone complete immunogen with a Freund complete adjuvant and a Freund incomplete adjuvant (in a ratio of 1:1) respectively, fully emulsifying to prepare a Freund complete adjuvant vaccine and a Freund incomplete adjuvant vaccine, and performing multipoint subcutaneous injection on a New Zealand white rabbit close to the dorsal ridge to co-inject 500-600 mu g of the complete immunogen;

s2, starting from the first round of immunization, carrying out a new round of immunization every 14-15 days for 6 rounds. During immunization, blood was taken from the vein of rabbit ear rim on day 7 after immunization;

s3.4 ℃, standing overnight, centrifuging, taking the supernatant, detecting the titer of the specific polyclonal antibody by an enzyme-linked immunosorbent assay (ELISA), purifying serum with higher titer by an octanoic acid-ammonium sulfate method, and determining the purity of the specific polyclonal antibody by SDS-PAGE electrophoresis.

The invention also provides a progesterone-specific polyclonal antibody prepared by any one of the methods.

The progesterone complete immunogen or the progesterone specific polyclonal antibody is applied to BA-ELISA detection of progesterone or preparation of a progesterone BA-ELISA detection kit.

A progesterone BA-ELISA detection kit comprises a progesterone standard, any progesterone complete immunogen which is coating antigen, any biotinylated progesterone specific polyclonal antibody, coating solution, washing solution, confining liquid, HRP-labeled streptavidin, substrate developing liquid and stop solution.

A method for detecting progesterone by using the kit comprises the following steps:

s1, coating: diluting the coating antigen to 1 mu g/mL by using a coating buffer solution, coating the antigen on an ELISA plate at 100 mu L/hole for 12-16 hours at 4 ℃, washing the plate for 3-5 times by using PBST, and sucking water;

s2, sealing: sealing by 0.01mol/L PBST containing 30% skimmed milk powder at 270 mu L/hole, incubating at 37 ℃ for 1-2 h, and washing in step S1;

s3, adding a biotinylated antibody and a progesterone standard: diluting a biotinylated polyclonal antibody by 8,000 times by 0.01mol/L PBST, adding the diluted antibody into an enzyme label plate at 50 mu L/hole, simultaneously adding a progesterone standard substance dissolved in 10% DMF PBS solution and a sample solution to be detected at 50 mu L/hole respectively, taking 10% DMF PBS solution as a blank control, incubating at 37 ℃ for 1-2 h, and washing in step S1;

s4, adding streptavidin marked by HRP: adding 100 mu L of HRP-labeled streptavidin diluted at the ratio of 1:10,000 into each hole, incubating for 1-2 h at 37 ℃, washing as in step S1, and blotting water of the ELISA plate;

s5, color development and termination reaction: adding a freshly prepared TMB color developing solution, incubating for 10-20 min at 37 ℃ in 100 mu L/hole, and then adding a 2M sulfuric acid stop solution in 50 mu L/hole;

s6, detecting a light absorption value: and immediately detecting the light absorption value of each hole of the ELISA plate at 450nm, obtaining a competition inhibition curve by using software, substituting the light absorption value of the sample liquid to be detected into the competition inhibition curve, and obtaining the content of the progesterone in the sample liquid to be detected.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a progesterone hapten which is obtained by modifying functional groups on carbon at the 1-position of progesterone and connecting carboxyl. The method comprises the steps of coupling synthesized and purified progesterone hapten with carrier protein to obtain complete immunogen of progesterone, immunizing New Zealand white rabbits to prepare polyclonal antibodies with high titer and strong specificity, and finally establishing an enzyme-linked immunosorbent assay (BA-ELISA) for detecting the progesterone based on amplified signals of biotin/streptavidin₅₀The detection linearity range is 1.29ng/mL, the detection linearity range is 0.22-7.50 ng/mL, the detection limit is 0.080ng/mL, and the method has a wide application prospect.

Drawings

FIG. 1 is a nuclear magnetic hydrogen spectrum characterization and identification chart of the progesterone hapten obtained after modification and purification.

FIG. 2 is a first-order mass spectrum characterization and identification chart of the modified and purified progesterone hapten.

FIG. 3 is a SDS-PAGE electrophoresis of purified anti-progesterone polyclonal antibodies of the present invention. Wherein, the band 1 is 1.2mg/mL before serum purification, the band 2 is 1.2mg/mL before serum purification, the purified IgG is composed of two chains, namely a heavy chain at about 50KDa and a light chain at about 25KDa, and the band at about 25KDa is slightly blurred in 2, which is probably caused by lower concentration.

FIG. 4 shows the competitive inhibition curve of progesterone detection using the BA-ELISA method of the present invention, the IC of progesterone detection₅₀The detection linearity range is 1.29ng/mL, the detection linearity range is 0.22-7.50 ng/mL, and the detection limit is 0.080 ng/mL.

Fig. 5 is a cross-reactivity experiment, wherein hydrocortisone, dehydroepiandrosterone, dexamethasone, androstenedione, estradiol benzoate and testosterone are selected for a specificity experiment, the cross-reactivity rate of progesterone reaches 100%, and the cross-reactivity rates of analogues of progesterone such as hydrocortisone, dehydroepiandrosterone, dexamethasone, androstenedione, estradiol benzoate and testosterone are all less than 0.1%, which indicates that the prepared anti-progesterone polyclonal antibody has high specificity.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1 preparation of Progesterone hapten

A modified preparation of progesterone hapten, which is synthesized by the following steps:

the method specifically comprises the following steps:

(1) 164mg of carboxymethoxylamine hemihydrochloride and 160. mu.L of anhydrous hard pyridine were added and dissolved in 5mL of methanol solution.

(2) At 25 deg.C, 5mL of methanol-solubilized progesterone P was added₄And (3) solution.

(3) Stirring the mixture for 5h, dissolving the yellow viscous liquid by using dichloromethane, adding deionized water for washing for 5 times, adding anhydrous magnesium sulfate to remove water, performing suction filtration to obtain light yellow liquid, removing the solvent under a rotary evaporator, and concentrating.

(4) The residue was purified by silica gel column chromatography using dichloromethane/methanol (20:1(v/v) → 16:1(v/v) → 12:1(v/v)) as an eluent, and the process was repeated 2 times to obtain the objective compound in white. Solid (yield: + 60%, + 90% NMR purity, Rf ═ 0.278).

The nuclear magnetic hydrogen spectrum characterization and identification of the purified hapten (shown in figure 1) and the primary mass spectrum characterization and identification (shown in figure 2) show that the progesterone hapten is successfully prepared.

EXAMPLE 2 preparation of Progesterone complete immunogen

(1) 7.8mg of progesterone hapten were dissolved in 23. mu. L N, N-Dimethylformamide (DMF), and 4.6mg of N-hydroxysuccinimide (NHS) and 8.3mg of 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) were dissolved in 45. mu. L N, N-Dimethylformamide (DMF), respectively.

(2) Stirring the mixture for 12 hours at room temperature by a magnetic stirrer at the rotating speed of 250rpm to fully activate the carboxyl on the hapten.

(3) The activated hapten was gradually added dropwise to 5mg/mL Bovine Serum Albumin (BSA) with slow stirring in an ice bath to give a final DMF concentration of 20%.

(4) Coupling was performed with slow stirring at low temperature for 12 hours to allow more hapten to couple to the carrier protein.

(5) The conjugate of hapten and carrier protein was placed in a dialysis bag and dialyzed at 4 ℃ for three days, with 3 changes of PBS (0.01M; pH 7.4) a day to remove excess DMF.

(6) Centrifuging at 10,000rpm and 4 deg.C for 10min, discarding the precipitate, and collecting the supernatant. The concentration was measured using the BCA protein quantification kit, dispensed into EP tubes, and stored at-20 ℃ until use.

EXAMPLE 3 preparation of polyclonal antibodies specific for anti-Progesterone

(1) Diluting the progesterone complete immunogen to 1.0/mL, mixing with Freund's complete adjuvant and Freund's incomplete adjuvant (in a ratio of 1:1), emulsifying completely to obtain Freund's complete adjuvant vaccine and Freund's incomplete adjuvant vaccine, performing multi-point subcutaneous injection at the position close to the dorsal ridge of New Zealand white rabbit, and co-injecting 500 μ g of complete immunogen.

(2) Starting with the first round of immunization, a new round of immunization was performed every 14 days for 6 rounds. During immunization, blood was taken from the vein of the rabbit ear rim at day 7 after immunization, as shown in table 1.

(3) After standing overnight at 4 ℃, the supernatant was centrifuged and the titer of the specific polyclonal antibody was measured by enzyme-linked immunosorbent assay (ELISA) at 1:640,000, the results of which are shown in table 2. The serum having a high titer was purified by the octanoic acid-ammonium sulfate method, and the purity of the specific polyclonal antibody was measured by SDS-PAGE, and the titers of the antibodies are shown in Table 2, and shown in FIG. 3.

TABLE 1 immunization New Zealand white rabbits timetable

TABLE 2 potency of specific anti-progesterone polyclonal antibodies

1:10,000	2.8456	2.8755	2.9030
				1:20,000	2.7316	2.7657	2.7398
1:40,000	2.4326	2.4576	2.4576
				1:80,000	2.0823	2.0676	2.0230
1:160,000	1.5729	1.5282	1.5961
				1:320,000	1.1812	1.1912	1.1762
1:640,000	0.8061	0.7983	0.7851
				0	0.0710	0.0738	0.0692

Example 4 enzyme-linked immunoassay based on Biotin/streptavidin amplified Signal (BA-ELISA)

1. Chessboard titration method for optimizing concentration of coating antigen and dilution times of biotinylated antibody

(1) Coating: the coated antigen was diluted to 0.125-8 μ g/mL with coating buffer (CBS, pH 9.6, 0.05mol/L) each (as in table 2), diluted in duplicate, coated on 96-well elisa plates, 100 μ L/well, coated at 4 ℃ for 16 hours, washed 5 times with PBST and blotted dry with absorbent paper.

(2) And (3) sealing: blocking (270. mu.L/well) was performed with 30% nonfat dry milk in PBST (0.01mol/L) and incubated in a water bath at 37 ℃ for 1h, as in step 1 washing procedure above.

(3) Adding a biotinylated antibody: after the biotinylated polyclonal antibodies were diluted in duplicate (1:2,000-1: 64,000, Table 2) with PBST (0.01mol/L), they were added to a 96-well microplate (100. mu.L/well) using a pipette gun, while adding PBST as a blank, and incubated in a water bath at 37 ℃ for 1h, as in the washing procedure of step 1 above.

(4) HRP-labeled streptavidin: 100 μ L of HRP-labeled streptavidin diluted at 1:10,000 per well was added, incubated in a water bath at 37 ℃ for 1h, washed as above in step 1, and blotted dry using absorbent paper.

(5) Color development and termination reaction: adding 100 μ L/well of freshly prepared TMB color developing solution, incubating at 37 deg.C for 15min in a water bath, and adding 50 μ L/well of 2M sulfuric acid stop solution.

(6) Detecting a light absorption value: the absorbance at 450nm of each well of the microplate was immediately detected. When the absorbance no longer increases with increasing concentration of the coating antigen and reaches a saturation state, this concentration is selected as the optimum concentration of the coating antigen. When the absorbance value is about 1.0 (between 0.8 and 1.2), the ELISA has higher sensitivity, and the dilution factor in the absorbance value range is selected as the optimal dilution factor of the biotinylated antibody. According to Table 3, the optimal coating antigen concentration was selected to be 0.25. mu.g/mL, and the optimal antibody dilution factor was 1:8,000.

TABLE 3 checkerboard titration optimization of dilution factor for coating antigen and anti-progesterone biotinylated polyclonal antibody

2. Progesterone was detected using ELISA of biotin/streptavidin amplified signal.

(1) Coating: the coating antigen was diluted to 0.25. mu.g/mL with coating buffer (CBS, pH 9.6, 0.05mol/L), coated on a 96-well microplate, 100. mu.L/well, coated at 4 ℃ for 16 hours, washed 5 times with PBST and blotted dry with absorbent paper.

(2) And (3) sealing: blocking (270. mu.L/well) was performed with 30% nonfat dry milk in PBST (0.01mol/L) and incubated in a water bath at 37 ℃ for 1h, as in step 1 washing procedure above.

(3) Addition of biotinylated antibody and progesterone standard: the biotinylated polyclonal antibody was diluted 8,000-fold with PBST (0.01mol/L), added to a 96-well microplate (50. mu.L/well) using a pipette gun, and at the same time, a progesterone standard (50. mu.L/well) dissolved in 10% DMF in PBS was added, and 10% DMF in PBS was used as a blank, and incubated in a water bath at 37 ℃ for 1h, as in the washing procedure of step 1 above.

(4) HRP-labeled streptavidin: 100 μ L of HRP-labeled streptavidin diluted at 1:10,000 per well was added, incubated in a water bath at 37 ℃ for 1h, washed as above in step 1, and blotted dry using absorbent paper.

(5) Color development and termination reaction: adding 100 μ L/well of freshly prepared TMB color developing solution, incubating at 37 deg.C for 15min in a water bath, and adding 50 μ L/well of 2M sulfuric acid stop solution.

(6) Detecting a light absorption value: the absorbance at 450nm of each well of the microplate was immediately detected. The competition inhibition curve was obtained using software. The results are shown in FIG. 4, and the IC of progesterone is detected by BA-ELISA established by the above method₅₀The detection sensitivity is 1.29ng/mL, the detection linear range is 0.22-7.50 ng/mL, the detection limit is 0.080ng/mL, and the detection sensitivity is very strong.

(7) Cross-reactivity experiments: meanwhile, hydrocortisone, dehydroepiandrosterone, dexamethasone, androstenedione, estradiol benzoate and testosterone are selected for specific experiments, and the cross-reaction rate of the progesterone analogues is less than 0.1%. The results are shown in FIG. 5, which indicates that none of the above-mentioned target substances can be detected, and thus indicates that the BA-ELISA established by the above-mentioned method of the present invention has high specificity.

Claims (2)

1. A progesterone BA-ELISA detection kit is characterized by comprising a progesterone standard, a progesterone complete immunogen coating antigen, a biotinylated progesterone specific polyclonal antibody, a coating solution, a washing solution, a confining liquid, HRP-labeled streptavidin, a substrate developing liquid and a stop solution; the progesterone complete immunogen is obtained by coupling progesterone hapten with bovine serum albumin through an active ester method; the progesterone specific polyclonal antibody is obtained by emulsifying the complete immunogen of progesterone and then immunizing rabbits; the structural formula of the progesterone hapten is shown as a formula (I):

the preparation method of the progesterone hapten comprises the following steps:

s1, mixing carboxymethyl amine half hydrochloride and anhydrous pyridine according to a molar ratio of 3: 1 are respectively dissolved in methanol solution;

s2, adding methanol-dissolved progesterone P into the anhydrous pyridine solution of S1₄Solution, progesterone P₄The molar ratio of the pyridine to the anhydrous pyridine is 2: 1;

s3, uniformly mixing the solution to obtain yellow viscous liquid; dissolving the yellow viscous liquid by using dichloromethane, washing by using deionized water, removing water by using anhydrous magnesium sulfate, carrying out suction filtration to obtain a light yellow liquid, carrying out rotary evaporation to remove the solvent, and concentrating;

s4, using dichloromethane/methanol 20:1 → 16:1 → 12:1 as an eluent, purifying the residue by using a silica gel chromatographic column, and repeating for 1-2 times to obtain a white target compound, namely progesterone hapten.

2. A method for detecting progesterone using the kit of claim 1, comprising the steps of:

s1, coating: diluting the coating antigen to 1 mu g/mL by using a coating buffer solution, coating the antigen on an ELISA plate at 100 mu L/hole for 12-16 hours at 4 ℃, washing the plate for 3-5 times by using PBST, and sucking water;

s2, sealing: sealing by 0.01mol/L PBST containing 30% skimmed milk powder at 270 mu L/hole, incubating at 37 ℃ for 1-2 h, and washing in step S1;

s3, adding a biotinylated antibody and a progesterone standard: diluting a biotinylated polyclonal antibody by 8,000 times by 0.01mol/L PBST, adding the diluted antibody into an enzyme label plate at 50 mu L/hole, simultaneously adding a progesterone standard substance dissolved in 10% DMF PBS solution and a sample solution to be detected at 50 mu L/hole respectively, taking 10% DMF PBS solution as a blank control, incubating at 37 ℃ for 1-2 h, and washing in step S1;

s4, adding streptavidin marked by HRP: adding 100 mu L of HRP-labeled streptavidin diluted at the ratio of 1:10,000 into each hole, incubating for 1-2 h at 37 ℃, washing as in step S1, and blotting water of the ELISA plate;

s5, color development and termination reaction: adding a freshly prepared TMB color developing solution, incubating for 10-20 min at 37 ℃ in 100 mu L/hole, and then adding a 2M sulfuric acid stop solution in 50 mu L/hole;

s6, detecting a light absorption value: and immediately detecting the light absorption value of each hole of the ELISA plate at 450nm, obtaining a competition inhibition curve by using software, substituting the light absorption value of the sample liquid to be detected into the competition inhibition curve, and obtaining the content of the progesterone in the sample liquid to be detected.

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