CN111551748A

CN111551748A - Enzyme-linked immunosorbent assay kit for trichosanthin and use method thereof

Info

Publication number: CN111551748A
Application number: CN202010482012.8A
Authority: CN
Inventors: 张波; 辛杰; 王振; 陈敏
Original assignee: Linyi University
Current assignee: Linyi University
Priority date: 2020-05-29
Filing date: 2020-05-29
Publication date: 2020-08-18

Abstract

The invention discloses an enzyme-linked immunoassay kit for trichosanthin and a use method thereof. The kit consists of a TCS reference substance, a sample diluent, an ELISA plate, anti-TCS rabbit polyclonal antiserum, an anti-TCS monoclonal antibody, a goat anti-mouse enzyme-labeled secondary IgG-HRP, a coating buffer solution, a washing solution, a developing solution and a termination buffer solution. The kit can be used for quantitative detection of trichosanthin, has the advantages of simple and convenient operation, quick detection, high flux and low cost, and has very important significance for ensuring the quality of the traditional Chinese medicine trichosanthin and related preparations of the trichosanthin.

Description

Enzyme-linked immunosorbent assay kit for trichosanthin and use method thereof

Technical Field

The invention relates to an enzyme-linked immunosorbent assay kit for trichosanthin and a method for quantitatively detecting the trichosanthin in traditional Chinese medicine trichosanthin.

Background

Trichosanthin (TCS) is an alkaline (isoelectric point PI is 9.4) ribosome inactivating protein with molecular weight of 27KD in Trichosanthin, has various pharmacological activities such as termination of pregnancy, anti-tumor and anti-virus, and is regarded as an important active substance of traditional Chinese medicine Trichosanthin and an important index component for authenticity identification and quality evaluation of Trichosanthin.

TCS is essentially a protein, and the conventional detection method for TCS mainly adopts a general protein detection method, such as a Kjeldahl method and an ultraviolet spectrophotometry (Folin-phenol method, Coomassie brilliant blue staining method, biuret method, biquinoline formic acid method and the like), but the methods have low sensitivity, and the TCS extract contains heteroprotein, so that the detection result is high. The TCS injection with higher purity can be detected by an HPLC method, but is not applicable to Chinese herbal pieces. Therefore, a simple, convenient, rapid and low-cost TCS qualitative and quantitative detection method is established, and has important significance for quality evaluation of traditional Chinese medicine radix trichosanthis decoction pieces and Chinese patent medicine containing radix trichosanthis.

Disclosure of Invention

The invention aims to provide an enzyme linked immunosorbent assay kit for TCS detection, which can be used for quantitatively detecting the TCS content in the traditional Chinese medicine radix trichosanthis decoction pieces.

The invention also aims to provide a double-antibody sandwich enzyme-linked immunoassay method for detecting the TCS content in the trichosanthes root by using the TCS enzyme-linked immunoassay kit.

The invention also aims to provide an indirect competitive enzyme-linked immunosorbent assay method for detecting the TCS content in the trichosanthes root by using the TCS enzyme-linked immunosorbent assay kit.

In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:

an enzyme linked immunosorbent assay kit for trichosanthin detection comprises a TCS reference substance, a sample diluent, an ELISA plate, anti-TCS rabbit polyclonal antiserum, an anti-TCS monoclonal antibody, a goat anti-mouse enzyme-labeled secondary antibody IgG-HRP, a coating buffer solution, a washing solution, a developing solution and a termination buffer solution;

the rabbit polyclonal antiserum for resisting TCS is prepared by adopting the following method:

(a1) dissolving TCS control product to 1.0mg/mL with PBS buffer solution with pH value of 7.5 to obtain immunogen;

(a2) immunizing a New Zealand big ear rabbit with the TCS immunogen of step (a 1); the immunization method is subcutaneous multipoint injection on soles and backs, the immunization dose is 1.0 mg/injection, the immunization period is 2 weeks/time, and the immunization times are 4 times;

(a3) after 4 times of immunization, collecting rabbit blood, and centrifuging to obtain anti-TCS rabbit polyclonal antiserum;

the anti-TCS monoclonal antibody is prepared by adopting the following method:

(b1) immunizing Balb/c female mice with the TCS immunogen of step (a 1); the immunization method is subcutaneous multipoint injection of abdominal cavity and back, the immunization dose is 0.2 mg/mouse, the immunization period is 2 weeks/time, and the immunization times are 4 times;

(b2) after 4 times of immunization, collecting mouse serum, and detecting the positive of the mouse by a direct enzyme-linked immunosorbent assay method; selecting the spleen of a mouse with the highest positive as a spleen cell source for cell fusion;

(b3) taking the spleen cells of the mice in the step (b2), and comparing the number ratio of the spleen cells to the SP2/0 myeloma cells by 9: 1, carrying out cell fusion by a PEG method; screening hybridoma cell strains secreting monoclonal antibodies by using a limiting dilution method; measuring the antibody titer by adopting a direct enzyme-linked immunosorbent assay method, selecting a high-titer monoclonal antibody capable of identifying a TCS standard solution, and finally screening to obtain a monoclonal hybridoma cell strain which stably secretes and identifies the TCS antibody and is named as 2B 10;

(b4) the hybridoma cell line 2B10 obtained in step (B3) was expanded and cultured, and then injected into the abdominal cavity of a mouse in an injection amount of 10⁶Preparing ascites, collecting ascites, and purifying by saturated ammonium sulfate method to obtain anti-TCS monoclonal antibody.

Na in the sample dilution₂HPO₄、KH₂PO₄And NaCl concentrations of 0.02M, 0.0015M and 0.14M, respectively; the volume percentage content of the Tween-20 in the sample diluent is 0.1 percent; the mass percentage of the gelatin in the sample diluent is 0.5%.

The bagIs Na in buffer solution₂CO₃And NaHCO₃The concentrations of (A) and (B) were 0.01M and 0.04M, respectively, and the pH of the solution was 9.6.

Na in the washing solution₂HPO₄、KH₂PO₄The concentration of NaCl in the washing liquid is 0.02M, 0.0015M and 0.14M respectively, the volume percentage content of Tween-20 in the washing liquid is 0.1 percent, and the pH value is 7.5.

The solvent of the color developing solution is substrate buffer solution, the solutes are o-phenylenediamine and 30% hydrogen peroxide, and the concentrations of the o-phenylenediamine and the hydrogen peroxide in the color developing solution are 2mg/mL and 1:2500(v: v), respectively; trisodium citrate and Na in the substrate buffer₂HPO₄The concentration of the color developing solution is 0.01M and 0.03M respectively, the pH value is 5.5, and the color developing solution needs to be prepared for use.

The stop buffer is a 2M aqueous solution of sulfuric acid.

A double antibody sandwich enzyme-linked immunoassay method for quantitatively detecting the TCS content in the trichosanthin by adopting the enzyme-linked immunoassay kit for detecting the trichosanthin comprises the following steps:

(c1) crushing a traditional Chinese medicine sample to be detected, sieving the crushed traditional Chinese medicine sample by a No. 4 sieve, weighing 2g of powder, extracting the powder for 60 minutes by using 50mL of PBS (phosphate buffer solution) under the condition of 4 ℃ under magnetic stirring, and centrifuging the extract to obtain a supernatant to obtain a sample extracting solution to be detected;

(c2) diluting anti-TCS rabbit polyclonal antiserum by 500 times by using a coating buffer solution, coating the diluted anti-TCS rabbit polyclonal antiserum on a 96-hole enzyme label plate at a concentration of 100 mu L/hole, coating the anti-TCS rabbit polyclonal antiserum for 3 hours at 37 ℃, and washing the plate for 3 times by using a washing solution;

(c3) adding the traditional Chinese medicine sample extract to be tested and TCS reference substance solutions with different concentrations to a 96-well enzyme label plate, reacting at 100 mu L/well for 30 minutes at 37 ℃, and washing the plate for 3 times by using a washing solution;

(c4) dissolving the anti-TCS monoclonal antibody with a sample diluent, adjusting the mass concentration to be 2.0 mu g/mL, adding the solution to a 96-well enzyme label plate, reacting at 100 mu L/well for 30 minutes at 37 ℃, and washing the plate for 3 times by using a washing solution;

(c5) adding 2 mu g/mL goat anti-mouse enzyme-labeled secondary IgG-HRP to a 96-hole enzyme-labeled plate, reacting at the temperature of 100 mu L/hole for 30 minutes, and washing the plate for 3 times by using a washing solution;

(c6) 20mg of o-phenylenediamine solution was takenTo 10mL of substrate dilution, 8. mu.L of 30% H was added₂O₂Adding the substrate solution into an enzyme label plate, wherein each hole is 100 mu L, and performing light-shielding color development for 10 min;

(c7) adding 50 mu L of stop buffer solution into each well, and measuring the light absorption value A of each well at the position 492nm of an enzyme-labeling instrument;

(c8) and taking the concentration of the TCS reference substance as an abscissa and the light absorption value A as an ordinate, establishing a standard curve, and substituting the light absorption value A corresponding to the sample to be detected into the standard curve to calculate the content of the TCS in the sample to be detected.

Wherein the concentration of the TCS control solution of step (c3) is 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL, 15.63. mu.g/mL, 7.81. mu.g/mL, 3.91. mu.g/mL, 1.95. mu.g/mL, 0.98. mu.g/mL, 0. mu.g/mL.

An indirect competitive enzyme-linked immunoassay method for quantitatively detecting the TCS content in the trichosanthin by adopting the enzyme-linked immunoassay kit for detecting the trichosanthin comprises the following steps:

(d1) crushing a traditional Chinese medicine sample to be detected, sieving the crushed traditional Chinese medicine sample by a No. 4 sieve, weighing 2g of powder, extracting the powder for 60 minutes by using 50mL of PBS (phosphate buffer solution) under the condition of 4 ℃ under magnetic stirring, and centrifuging the extract to obtain a supernatant to obtain a sample extracting solution to be detected;

(d2) dissolving a TCS reference substance by using a coating buffer solution, adjusting the mass concentration to be 0.05mg/mL, coating the TCS reference substance on a 96-hole enzyme label plate at 37 ℃, washing the enzyme label plate for 3 times by using a washing solution after 3 hours, wherein the concentration of the TCS reference substance is 100 mu L/hole;

(d3) adding the traditional Chinese medicine sample extract to be tested and TCS reference substance solutions with different concentrations to a 96-well enzyme label plate, reacting at 50 mu L/well for 30 minutes at 37 ℃, and washing the plate for 3 times by using a washing solution;

(d4) dissolving the anti-TCS monoclonal antibody with a sample diluent, adjusting the mass concentration to be 2 mu g/mL, adding the solution to a 96-hole enzyme label plate, reacting at 50 mu L/hole for 30 minutes at 37 ℃, and washing the plate for 3 times by using a washing solution;

(d5) adding 2 mu g/mL goat anti-mouse enzyme-labeled secondary IgG-HRP to a 96-hole enzyme-labeled plate, reacting at the temperature of 100 mu L/hole for 30 minutes, and washing the plate for 3 times by using a washing solution;

(d6) 20mg of o-phenylenediamine was dissolved in 10mL of substrate diluent and 8. mu.L of 30% H was added₂O₂A bottom ofAdding the solution into an enzyme label plate, wherein each well is 100 mu L, and developing for 10min in a dark place;

(d7) adding 50 mu L of stop buffer solution into each well, and measuring the light absorption value A of each well at the position 492nm of an enzyme-labeling instrument;

(d8) TCS standard solution with different concentrations is used as abscissa, A/A₀Is a vertical coordinate; a represents the absorbance value when TCS is contained, A₀Expressing the light absorption value of the control hole, and drawing a standard curve by using originPro 8 software; and substituting the light absorption value A corresponding to the sample to be detected into the standard curve to calculate the TCS content in the sample.

Wherein the concentration of the TCS control solution in step (d3) is 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL, 15.63. mu.g/mL, 7.81. mu.g/mL, 3.91. mu.g/mL, 0. mu.g/mL.

The invention has the beneficial effects that: the invention provides an enzyme linked immunosorbent assay kit for TCS quantitative detection for the first time, and provides two specific detection methods. The kit can be used for rapidly detecting the content of TCS in the radix trichosanthis, has accurate experimental results, has the advantages of simple and convenient operation, rapid detection, high flux and low cost, and has very important significance for ensuring the quality of the traditional Chinese medicine radix trichosanthis and related preparations thereof.

Drawings

FIG. 1 is a standard curve of TCS detection by a double-anti-sandwich enzyme-linked immunosorbent assay;

FIG. 2 is a standard curve of indirect competitive ELISA for TCS detection.

Detailed Description

The technical solution of the present invention is further described below with reference to specific embodiments, but is not limited thereto.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

TCS control: shanghai Jinshan pharmaceutical factory, national Standard of medicine H10870026.

TCS standard solution: TCS was dissolved in PBS buffer to obtain a solution.

PBS buffer: the solvent is water and the solute is Na₂HPO₄、KH₂PO₄And NaCl, solute Na₂HPO₄、KH₂PO₄And the concentration of NaCl in the PBS buffer solution is 0.02M, 0.0015M and 0.14M respectively; the pH was 7.5.

Coating buffer solution: the solvent is water and the solute is Na₂CO₃And NaHCO₃(ii) a Solute Na₂CO₃And NaHCO₃The concentration in the coating buffer was 0.01M and 0.04M, respectively; the pH was 9.6.

Washing liquid: the solvent is water and the solute is Na₂HPO₄、KH₂PO₄NaCl and Tween-20; solute Na₂HPO₄、KH₂PO₄The concentrations of NaCl and Tween-20 in the washing solution were 0.02M, 0.0015M, 0.14M and 0.1% (volume percentage content), respectively; the pH was 7.5.

Sample diluent: the solvent is water and the solute is Na₂HPO₄、KH₂PO₄And NaCl, tween-20 and gelatin; solute Na₂HPO₄、KH₂PO₄And the concentrations of NaCl in the sample diluent were 0.02M, 0.0015M and 0.14M, respectively; the volume percentage content of the Tween-20 in the sample diluent is 0.1 percent; the mass percentage of the gelatin in the sample diluent is 0.5%.

Color development liquid: the solvent is substrate buffer solution, the solutes are o-phenylenediamine and 30% hydrogen peroxide, and the concentrations of the o-phenylenediamine and the hydrogen peroxide in the color development solution are 2mg/mL and 1:2500(v: v), respectively; trisodium citrate and Na in the substrate buffer₂HPO₄The concentrations of (A) and (B) were 0.01M and 0.03M, respectively, and the pH was 5.5. Stop buffer: aqueous sulfuric acid solution with a concentration of 2M.

New Zealand big ear rabbit mice were purchased from the Experimental animals center of the military medical academy of sciences.

Balb/c mice were purchased from the laboratory animals center of the military medical academy of sciences.

Example 1

Preparation of anti-TCS rabbit polyclonal antiserum

1. New Zealand big ear rabbits of 3 months old were used as experimental animals.

2. Basic immunity: after filtering the TCS control solution of 1mg/mL through a sterile filter, injecting the New Zealand big ear rabbits at multiple subcutaneous points on the soles and the backs by adopting the injection dosage of 1mg of antigen for each New Zealand big ear rabbit.

3. And (3) boosting immunity: after 2 weeks of basic immunization, 1mL of 1mg/mL TCS immunogen was taken and injected subcutaneously into New Zealand big ear rabbits at multiple sites in the sole and back at a dose of 1mg antigen per New Zealand big ear rabbit. Repeat 4 times.

4. Preparation of anti-TCS rabbit polyclonal antiserum: 3 days after the last time of the boosting immunization, blood is taken from the heart, the blood plasma is kept stand for 0.5 hour at room temperature, then is kept stand for 2 hours at 4 ℃, and finally is centrifuged to take the blood serum, thus obtaining the anti-TCS rabbit polyclonal antiserum.

Example 2

Preparation of anti-TCS monoclonal antibody

Mono, immunogen

TCS control was dissolved to 1mg/mL in PBS buffer at pH 7.5 as immunogen.

Second, animal immunization

1. Balb/c female mice 8-10 weeks old were used as experimental animals.

2. The immune process comprises the following steps: after 1mg/mL of TCS control solution was filtered through a sterile filter, Balb/c mice were injected subcutaneously in the abdominal and back at multiple points at an injection dose of 0.2mg of antigen per mouse. The immunization period is 2 weeks, and the number of immunizations is 4.

Cell fusion and cloning

The boosting immunization is performed once every 14 days, from the third boosting immunization, blood is collected from the eye socket of the mouse 7 days after each immunization, the serum titer of the mouse is determined by adopting a direct enzyme-linked immunosorbent assay, and the titer is defined as the dilution multiple of the serum when the light absorption value A is 1. After the titer is greater than 1:8000, selecting the mouse with the best serum titer (titer is 1: 12000), taking splenocytes, and carrying out the steps of 9: 1 (quantitative ratio) and SP2/0 myeloma cells; screening hybridoma cell strains secreting monoclonal antibodies by using a limiting dilution method; the titer of cell fluid is measured by adopting a direct enzyme-linked immunosorbent assay method, a monoclonal cell strain with high titer of a secretion antibody is screened by using a TCS standard solution, a monoclonal antibody capable of identifying the TCS standard solution is selected, and finally, a monoclonal hybridoma cell strain which stably secretes the anti-TCS antibody is obtained by screening and is named as 2B 10.

The method for measuring the antibody titer by the direct enzyme-linked immunoassay method comprises the following steps:

1. coating: to a 96-well plate, 100. mu.L of a 0.5mg/mL TCS solution was added, coated at 37 ℃ for 3 hours, and washed 4 times with a washing solution.

2. Adding an antibody: adding antiserum or cell culture fluid with different dilution times into the ELISA plate, placing in a wet box at 37 deg.C for 30min, and washing for 4 times.

3. Adding an enzyme-labeled secondary antibody: a goat anti-mouse enzyme-labeled secondary IgG-HRP (purchased from Jackson, catalog No. 79556) (0.2mg/mL) was diluted 1000-fold, 100. mu.L was added to each well, and the plate was washed 4 times at 37 ℃ for 30min in a wet box.

4. Color development: 20mg of o-phenylenediamine (OPD) was dissolved in 10mL of substrate diluent and 8. mu.L of 30% H was added₂O₂The substrate solution was added to the microplate in 100. mu.L per well. And (4) shading and developing for 10 min. The light absorption value A of each hole is measured at the position of 492nm by using a microplate reader, the result of the titer of the mouse serum is shown in table 1, and the result of the measurement of the cell sap of a part of positive hybridoma cell strains is shown in table 2.

In table 1, a is the absorbance of 5 mice at different dilution times after 4 immunizations. Blank wells had an absorbance of 0.070.

TABLE 1 results of measurement of serum titer of mouse

As can be seen from Table 1, the dilution factor is 8000-16000 times when the absorbance of the serum of the mouse No. 2 is 1.0, and is the highest among 5 mice, which indicates that the serum titer of the mouse No. 2 is the highest and the immune effect is the best.

In table 2, 2B10, 2E8, 2F8 and 5G9 represent hybridoma cells in different wells of a cell culture plate added during the screening process, respectively, a is the absorbance of hybridoma cell fluid, the absorbance of blank wells is 0.054, and higher titer of cell culture fluid indicates better antigen recognition.

TABLE 2 determination of antibody titer by direct competitive enzyme-linked immunoassay

As can be seen from the data in Table 2, when the light absorption value of the culture solution of the 2B10 hybridoma cell line is 1.0, the dilution factor is 4000 to 8000, and is the highest among 4 hybridoma cells, which indicates that the titer of the antibody secreted by the 2B10 hybridoma cell line is the highest, so that the 2B10 hybridoma cells are selected to continue to be subjected to amplification culture, and the antibody is prepared.

Fourthly, freezing and restoring cells

Preparing hybridoma cell line 2B10 into 1 × 10 with frozen stock solution⁶Cell suspension per mL, preserved for long period in liquid nitrogen. During recovery, the cryopreservation tube is taken out, immediately placed into a water bath at 37 ℃ for fast melting, centrifuged to remove the cryopreservation liquid, and then transferred into a culture bottle for culture.

Fifth, preparation and purification of monoclonal antibody

1. Method of incremental culture

The preparation method of the cell culture medium comprises the following steps: calf serum (purchased from GIBCOBRL, catalog No. 26170-043) and sodium bicarbonate were added to the DMEM medium, with a final concentration of 20% (mass%) of calf serum, a final concentration of 0.2% (mass%) of sodium bicarbonate, and a pH of 7.4.

The hybridoma cell line 2B10 was cultured in the above cell culture medium at 37 ℃ for 2 days to prepare a monoclonal antibody solution.

2. Preparation of ascites

Balb/c mice were injected intraperitoneally with sterile paraffin oil (0.2 mL/mouse). The hybridoma cell line 2B10 (10) was injected intraperitoneally 7 days later⁶One/only). Collecting ascites after 7 days, purifying the ascites by a saturated ammonium sulfate method to obtain a monoclonal antibody against TCS, and storing the purified ascites at-20 ℃.

The method for purifying ascites by saturated ammonium sulfate comprises the following specific steps:

1) chamberAscites fluid was mixed with an equal volume of PBS solution at room temperature. Adding equal volume of saturated ammonium sulfate dropwise under stirring (even if the concentration of ammonium sulfate reaches 50%), standing at 4 deg.C for 2 hr, centrifuging at 4 deg.C for 15min at 3000g, removing supernatant, and collecting precipitate. The solvent of the PBS buffer solution is water, and the solute is Na₂HPO₄、KH₂PO₄And NaCl, solute Na₂HPO₄、KH₂PO₄And NaCl in the PBS buffer at concentrations of 0.02M, 0.0015M and 0.14M, respectively, and a pH of 7.5. The solvent of the saturated ammonium sulfate solution is water, and the solute is (NH)₄)₂SO₄The concentration was 4.10M and the pH was 7.2.

2) Adding PBS solution with 2 times of the original ascites volume into the obtained precipitate, fully dissolving, dropwise adding saturated ammonium sulfate with half of PBS volume (namely the concentration of the saturated ammonium sulfate reaches 33%), standing at 4 ℃ for 2h, centrifuging at 3000g at 4 ℃ for 15min, removing supernatant, and taking precipitate.

3) Dissolving the precipitate with PBS solution half the volume of the original ascites, dialyzing at 4 deg.C for 2 days, and replacing the dialysate every 6 h. After dialysis, the mixture was freeze-dried and stored at-40 ℃. The dialysate is PBS buffer.

Example 3

Preparation of TCS enzyme-linked immunoassay kit

The kit comprises a TCS reference substance, a sample diluent, an ELISA plate, anti-TCS rabbit polyclonal antiserum, an anti-TCS monoclonal antibody, a goat anti-mouse enzyme-labeled secondary IgG-HRP, a coating buffer solution, a washing solution, a developing solution and a termination buffer solution. Wherein the anti-TCS rabbit polyclonal antiserum and the anti-TCS monoclonal antibody were prepared as described in example 1 and example 2.

Na in the coating buffer₂CO₃And NaHCO₃The concentrations of (A) and (B) were 0.01M and 0.04M, respectively, and the pH of the solution was 9.6.

The solvent of the color developing solution is substrate buffer solution, the solutes are o-phenylenediamine and 30% hydrogen peroxide, and the concentrations of the o-phenylenediamine and the hydrogen peroxide in the color developing solution are 2mg/mL and 1:2500(v: v), respectively; trisodium citrate and Na in the substrate buffer₂HPO₄The concentrations of (A) and (B) were 0.01M and 0.03M, respectively, and the pH was 5.5.

The stop buffer is a 2M aqueous solution of sulfuric acid.

Example 4

A double antibody sandwich enzyme-linked immunoassay method for quantitatively detecting the TCS content in the Mongolian snakegourd root by using the kit prepared in the embodiment 3 comprises the following steps:

(1) crushing a traditional Chinese medicine sample to be detected, sieving the crushed traditional Chinese medicine sample by a No. 4 sieve, weighing 2g of powder, extracting the powder for 60 minutes by using 50mL of sample diluent under the condition of 4 ℃ through magnetic stirring, and centrifuging the extract to obtain a supernatant to obtain a sample extracting solution to be detected;

(2) diluting anti-TCS rabbit polyclonal antiserum by 500 times by using a coating buffer solution, coating the diluted anti-TCS rabbit polyclonal antiserum on a 96-hole enzyme label plate at a concentration of 100 mu L/hole, coating the anti-TCS rabbit polyclonal antiserum for 3 hours at 37 ℃, and washing the plate for 3 times by using a washing solution;

(3) adding the extract of the traditional Chinese medicine sample to be tested and TCS reference substance solutions (500 mu g/mL, 250 mu g/mL, 125 mu g/mL, 62.5 mu g/mL, 31.25 mu g/mL, 15.63 mu g/mL, 7.81 mu g/mL, 3.91 mu g/mL, 1.95 mu g/mL, 0.98 mu g/mL and 0 mu g/mL) with different concentrations to a 96-well enzyme label plate, 100 mu L/well, reacting at 37 ℃ for 30 minutes, and washing the plate with a washing solution for 3 times;

(4) dissolving the anti-TCS monoclonal antibody with a sample diluent, adjusting the mass concentration to be 2.0 mu g/mL, adding the solution to a 96-well enzyme label plate, reacting at 100 mu L/well for 30 minutes at 37 ℃, and washing the plate for 3 times by using a washing solution;

(5) adding 2 mu g/mL goat anti-mouse enzyme-labeled secondary IgG-HRP to a 96-hole enzyme-labeled plate, reacting at the temperature of 100 mu L/hole for 30 minutes, and washing the plate for 3 times by using a washing solution;

(6) 20mg of o-phenylenediamine solution was takenTo 10mL of substrate dilution, 8. mu.L of 30% H was added₂O₂Adding the substrate solution into an enzyme label plate, wherein each hole is 100 mu L, and performing light-shielding color development for 10 min;

(7) mu.L of stop buffer was added to each well, and the absorbance A of each well was measured at 492nm using a microplate reader.

(8) And taking the concentration of the TCS reference substance as an abscissa and the light absorption value A as an ordinate, establishing a standard curve, and substituting the light absorption value A corresponding to the sample to be detected into the standard curve to calculate the content of the TCS in the sample.

Example 5

An indirect competitive enzyme-linked immunoassay method for quantitatively detecting the TCS content in the trichosanthes root by using the kit prepared in the embodiment 3 comprises the following steps:

(2) dissolving a TCS reference substance by using a coating buffer solution, adjusting the mass concentration to be 0.05mg/mL, coating a 96-hole enzyme label plate (100 mu L/hole) at 37 ℃ for 3 hours, and washing the enzyme label plate for 3 times by using a washing solution;

(3) adding the traditional Chinese medicine sample extract to be tested and TCS reference substance solutions (500 mu g/mL, 250 mu g/mL, 125 mu g/mL, 62.5 mu g/mL, 31.25 mu g/mL, 15.63 mu g/mL, 7.81 mu g/mL, 3.91 mu g/mL and 0 mu g/mL) with different concentrations into a 96-well enzyme label plate, reacting at 37 ℃ for 30 minutes, and washing the plate for 3 times by using a washing solution;

(4) dissolving the anti-TCS monoclonal antibody with a sample diluent, adjusting the mass concentration to be 2 mu g/mL, adding the solution to a 96-hole enzyme label plate, reacting at 50 mu L/hole for 30 minutes at 37 ℃, and washing the plate for 3 times by using a washing solution;

(6) 20mg of o-phenylenediamine was dissolved in 10mL of substrate diluent and 8. mu.L of 30% H was added₂O₂Adding the substrate solution into an enzyme label plate, wherein each hole is 100 mu L, and performing light-shielding color development for 10 min;

(8) TCS standard solution with different concentrations is used as abscissa, A/A₀(A represents an absorbance value when TCS is contained, A₀Representing the absorbance of the control wells) was plotted as the ordinate against OriginPro 8 software. And substituting the light absorption value A corresponding to the sample to be detected into the standard curve to calculate the TCS content in the sample.

Example 6

The kit prepared in example 3 and the detection methods described in examples 4 and 5 were used to detect 4 radix trichosanthis and 17 non-radix trichosanthis (radix trichosanthis, yam, radix stephaniae tetrandrae, pachyrhizus angulatus, poria cocos, platycodon grandiflorum, pokeberry root, polygonum multiflorum, radix paeoniae alba, rhizoma bletillae, rhizoma atractylodis macrocephalae, radix ampelopsis, curcuma zedoary, radix isatidis, rhizoma atractylodis, rhizoma alismatis and radix codonopsitis), respectively. The results are shown in Table 3.

TABLE 3 results of the test kit for TCS content in the samples

^a: not measured out

The results are shown in table 3: the detection results of the double-resistance sandwich enzyme immunoassay method and the indirect competitive enzyme immunoassay method for TCS in the radix trichosanthis medicinal material are basically the same, but TCS is not detected in the radix trichosanthis medicinal material. The results show that the kit and the detection method thereof provided by the invention can be used for measuring the content of TCS in the radix trichosanthis, and have strong specificity and accurate detection result.

It should be noted that the above-mentioned embodiments are only some of the preferred modes for implementing the invention, and not all of them. Obviously, all other embodiments obtained by persons of ordinary skill in the art based on the above-mentioned embodiments of the present invention without any creative effort shall fall within the protection scope of the present invention.

Claims

1. An enzyme linked immunosorbent assay kit for trichosanthin detection is characterized by comprising a TCS reference substance, a sample diluent, an ELISA plate, anti-TCS rabbit polyclonal antiserum, an anti-TCS monoclonal antibody, a goat anti-mouse enzyme labeled secondary antibody IgG-HRP, a coating buffer solution, a washing solution, a color development solution and a termination buffer solution;

the anti-TCS monoclonal antibody is prepared by adopting the following method:

(b4) the hybridoma cell line 2B10 obtained in step (B3) was expanded and culturedInjected into the abdominal cavity of a mouse with the injection amount of 10⁶Preparing ascites, collecting ascites, and purifying by saturated ammonium sulfate method to obtain anti-TCS monoclonal antibody.

2. The ELISA kit for trichosanthin detection of claim 1, wherein Na in the sample diluent is₂HPO₄、KH₂PO₄And NaCl concentrations of 0.02M, 0.0015M and 0.14M, respectively; the volume percentage content of the Tween-20 in the sample diluent is 0.1 percent; the mass percentage of the gelatin in the sample diluent is 0.5%.

3. The ELISA kit for trichosanthin detection of claim 1, characterized in that Na in the coating buffer solution₂CO₃And NaHCO₃The concentrations of (A) and (B) were 0.01M and 0.04M, respectively, and the pH of the solution was 9.6.

4. The ELISA kit for trichosanthin detection of claim 1, wherein Na in the washing solution₂HPO₄、KH₂PO₄The concentration of NaCl in the washing liquid is 0.02M, 0.0015M and 0.14M respectively, the volume percentage content of Tween-20 in the washing liquid is 0.1 percent, and the pH value is 7.5.

5. The ELISA kit for trichosanthin detection of claim 1, characterized in that the solvent of the color development solution is substrate buffer solution, the solutes are o-phenylenediamine and 30% hydrogen peroxide, and the concentrations in the color development solution are 2mg/mL and 1:2500(v: v), respectively; trisodium citrate and Na in the substrate buffer₂HPO₄The concentration of the color developing solution is 0.01M and 0.03M respectively, the pH value is 5.5, and the color developing solution needs to be prepared for use.

6. The ELISA kit for trichosanthin detection of claim 1, wherein the stop buffer is 2M sulfuric acid solution.

7. A double-antibody sandwich enzyme-linked immunoassay method for quantitatively detecting the TCS content in trichosanthes root by using the enzyme-linked immunoassay kit for trichosanthin detection according to any one of claims 1 to 6, which is characterized by comprising the following steps:

(c6) 20mg of o-phenylenediamine was dissolved in 10mL of substrate diluent and 8. mu.L of 30% H was added₂O₂Adding the substrate solution into an enzyme label plate, wherein each hole is 100 mu L, and performing light-shielding color development for 10 min;

8. The double-antibody sandwich enzyme-linked immunoassay method for quantitatively detecting the TCS content in Trichosanthis radix using the ELISA kit for trichosanthin detection of claim 7, wherein the concentration of the TCS control solution of step (c3) is 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL, 15.63. mu.g/mL, 7.81. mu.g/mL, 3.91. mu.g/mL, 1.95. mu.g/mL, 0.98. mu.g/mL, 0. mu.g/mL.

9. An indirect competitive enzyme-linked immunoassay method for quantitatively detecting the TCS content in Trichosanthis radix using the enzyme-linked immunoassay kit for trichosanthin detection according to any one of claims 1 to 6, comprising the steps of:

(d6) 20mg of o-phenylenediamine was dissolved in 10mL of substrate diluent and 8. mu.L of 30% H was added₂O₂Adding the substrate solution into an enzyme label plate, wherein each hole is 100 mu L, and performing light-shielding color development for 10 min;

(d8) TCS standard solution with different concentrations is used as abscissa, A/A₀Is a vertical coordinate; a represents the absorbance value when TCS is contained, A₀The absorbance in the control well was expressed, and a standard curve was drawnA wire; and substituting the light absorption value A corresponding to the sample to be detected into the standard curve to calculate the TCS content in the sample.

10. The indirect competitive enzyme-linked immunoassay method for the quantitative determination of the TCS content in Trichosanthis radix using the ELISA kit for trichosanthin detection of claim, wherein the concentration of the TCS control solution in step (d3) is 500. mu.g/mL, 250. mu.g/mL, 125. mu.g/mL, 62.5. mu.g/mL, 31.25. mu.g/mL, 15.63. mu.g/mL, 7.81. mu.g/mL, 3.91. mu.g/mL, 0. mu.g/mL.