Antibody for testing free gossypol, ELISA (enzyme-linked immune sorbent assay) method and kit

Technical Field

The invention relates to cotton dregs and feed in the field of analytical technique of toxic substances, in particular relates to a free gossypol for detecting antibody, enzyme-linked immunosorbent assay (ELISA) method and kit.

Background Art

Carp often used for feed or feed raw material, is characterized in that the animal, fish important protein raw materials, however, in products containing free gossypol, gossypol has very strong toxicity, the gossypol absorbed into the animal in vivo, refused to the main distribution, liver, spleen, lung, blood in the kidney and. Gossypol can not only in livestock and poultry accumulated in edible organization, can also be metasticized, for example, milk cow in vivo of gossypol can be transferred to the milk, egg-laying hens of gossypol in the body in the eggs can be transferred to, therefore, the human of gossypol pose a serious threat to food safety. The United States, the European union and the China and other countries and regions develop the feed, feed raw material and human food content of gossypol limited standards.

Existing detection reduces free gossypol in and feed the method mainly comprises spectrophotometry (aniline process), high performance liquid chromatography, high efficiency of capillary electrophoresis, high performance liquid chromatography mass spectrometry, such as immune chemical analysis method. Wherein spectrophotometry detection of free gossypol is commonly used, but the method has the problems that the low sensitivity, high toxicity, time-consuming operation and the like. High performance liquid chromatography, high efficiency of capillary electrophoresis, high performance liquid chromatography mass spectrometry method for use of the instrument, although the sensitivity is high, the operation is simple, however, high requirements to the instrument and equipment, high cost, it is very difficult to popularize the use of. Immunochemical analysis method, especially enzyme-linked immunosorbent assay (ELISA) for detection of relatively high sensitivity, the operation is simple, free detection surface gossipol the focus of the study. Scholars have already through the free aldehyde gossypol directly with the carrier protein combined to obtain a free amino group on the immunogen, to the immunogens to prepare polyclonal antibodies and monoclonal antibodies, these antibodies for respectively establishing and detecting free gossypol method and indirect competition ELISA competition ELISA method (hereinafter referred to as ELISA method), but still problems: low antibody titer, wherein the polyclonal antibody the use concentration is 1:1000, the use of monoclonal antibody concentration is 1:10; antibodies to gossypol or gossypol derivatives of the high affinity, to the antibody to establish the immune chemical analysis method of the sensitivity is low, the performance of standard curve the linear range is wide, low curve slope.

Content of the invention

For the defects of the prior art, the purpose of this invention is to provide a free gossypol for detecting antibody, method and kit for ELISA, improve the antibody recognition specificity and affinity of gossypol derivatives, ELISA method and kit for improving the detection sensitivity.

In order to achieve the above purpose, the technical scheme of the present invention is:a method for detecting the antibody free gossypol, gossypol arm by cross-linking with bovine serum albumin obtained by coupling immunogen, the antibody is mouse immunogen, said cross-linked arm is O-carbencillin methyl hydroxylamine, the hydrazine benzoic acid, [...] and 6-aminocaproic acid in a.

In the above-mentioned technology on the basis of the programme, gossypol through the cross-linked arm and the ovalbumin obtained by coupling the original packet is.

A free gossypol for detecting ELISA method, comprising the following steps:

Step 1. Arm by cross-linking the gossypol with bovine serum albumin obtained by coupling immunogen, immunogen to obtain antibody of mouse; crossing linking arm the gossypol with ovalbumin obtained by coupling the packet is the original, by the use the packet with the original preparation custodite coatingen, said cross-linked arm is O-carbencillin methyl hydroxylamine, the hydrazine benzoic acid, [...] and 6-aminocaproic acid in a;

Step 2. The sample to be tested is added to volume ratio of 70% aqueous solution of acetone, filtered the filtrate is diluted with dilution of the sample solution to be measured;

Step 3. ELISA detection condition of the method is determined, using the above-mentioned antibody, a method for detecting enzyme ELISA, using the ELISA method for detecting in a sample solution to be measured is detected the content of free gossypol.

In the above-mentioned technical scheme on the basis of, the diluent to include 8.0g   NaCl, 0.2g   KH₂ PO₄, 2.9g   Na₂ HPO₄ · 12H₂ O, 0.2g   KCl, 0.1g sodium ethylmercurithiosalicylate, and double distilled water to 1000 ml solution.

In the above-mentioned technical scheme on the basis of, of the ELISA detection condition determining method comprises the following steps:

Step 301. Configuration ELISA method, the required reagent;

Step 302. The ELISA method, different concentration antibody diluent, and diluent coatingen OD value of the enzyme target role, according to ELISA OD value difference of adjacent apertures is the largest dilution of the concentration of the antibody, the antibody concentration of work; the difference between the OD value of the adjacent holes of the largest concentration of a dilution coatingen, the original density to be determined;

Step 303. The ELISA method, drawing the standard curve line, find the semi-inhibiting concentration;

Step 304. The ELISA method, using the reagent to gossypol standard to be measured.

A free gossypol for detecting ELISA kit, including horseradish peroxidase-labeled goat anti-mouse IgG antibody working fluid, concentrated phosphate buffer solution, the concentrated cleaning solution, substrate solution A, substrate liquid B and a stopping solution, the kit also includes the: peridium coatingen, antibody working fluid and different concentration of gossypol double-adducts of standard solution, wherein the original is coated with ovalbumin coupled by gossypol crossing linking arm conjugate obtained by reaction of; arm by cross-linking antibody as gossypol with bovine serum albumin obtained by coupling immunogen, an antibody obtained by mouse immunogen; gossypol double-adduct to gossypol with cross-linked arm double-addition reaction of the adduct, said cross-linked arm is O-carbencillin methyl hydroxylamine, the hydrazine benzoic acid, [...] and 6-aminocaproic acid in a.

The on the basis of the above-mentioned technical scheme, the packet through to the gossypol 6-aminocaproate of the ovalbumin coupling reaction coupling gossipol -6-aminocaproic acid-ovalbumin; immunogens to gossypol through the 6-aminocaproic acid with bovine serum albumin- coupling gossipol obtained by reaction of 6-aminocaproic acid-bovine serum albumin; gossypol double-adducts with gossypol to 6-aminocaproic acid-addition of addition reaction thing gossipol -6-aminocaproic acid double-adducts.

In the above-mentioned technical scheme on the basis of, the original packet is coated with the enzyme for the specific process of making: the peridium fluid custodite for the original diluted to 0.5 mg/L of the solution, each hole of the enzyme added to the 100   L the above-mentioned solution, the 4 [...] sleepovers, dumping of removing diaper fluid ; in each hole to the 210   L washing liquid, washing 3 times, shooting stem; the adding of each hole 200 the confining liquid   L, 37 the incubating [...] 1h, the liquid in the leans the hole ; detergent washing 3 times, shoot dry, vacuum sealing preservation of a waveform, the original packet is coated with a prepared ELISA plate.

In the above-mentioned technical scheme on the basis of, the antibody used for the working as the phosphate buffer solution in accordance with the above-mentioned antibody 1:16000 dilution of the solution.

In the above-mentioned technical scheme on the basis of, the standard of gossypol double-adducts the concentration of the solution are respectively 0 subsidence g/l, 5 subsidence g/l, 15 subsidence g/l, 45 subsidence g/l, 135 subsidence g/l, 405 subsidence g/l.

The beneficial effects of this invention are:

1, because the gossypol molecule chemical structure is symmetrical, the two contains in the molecule with the same chemical reaction of aldehyde active, the antigen of the invention in the molecule two of gossypol are coupled with aldehyde cross-linked arm double-adducts obtained, and then coupled to a carrier protein, to obtain the gossypol antibodies recognize double crossing linking arm addition derivatives, increased crossing linking arm gossypol derivatives of the introduction of chemical structure and space the complexity of the structure, thereby greatly improving micromolecule gossypol artificial antigen immunogenicity, improve the antibody specificity, affinity, so as to improve the method and kit for ELISA detection sensitivity.

2, ELISA method of the present invention to the instrument equipment requirement is low, the operation is simple, the kit including the ELISA method for all of the reagent and the enzyme used, which is convenient to carry and use.

Description of drawings

Figure 1 is the embodiment of the invention preparation of the antigenic reaction principle equation;

Figure 2 is the embodiment of the invention the antibody with the gossypol-6-aminocaproic acid double-adducts indirect competitive enzyme-linked immune reaction of the standard curve;

Figure 3 is a schematic diagram of the interrelationship of the present invention with high performance liquid chromatography method for ELISA to different measured value of the content of free gossypol in the cotton dregs.

Mode of execution

The following combination of the embodiment of Figure and further detailed description of this invention.

A free gossypol for detecting antibody, gossypol arm by cross-linking with bovine serum albumin obtained by coupling immunogen, the antibody is mouse immunogen, gossypol through the cross-linked arm and the ovalbumin obtained by coupling the original packet is, the cross-linked arm is O-carbencillin methyl hydroxylamine, the hydrazine benzoic acid, [...] and 6-aminocaproic acid in a.

A free gossypol for detecting ELISA method, comprising the following steps:

Step 1. Through the gossypol crossing linking arm O-carbencillin methyl hydroxylamine, the hydrazine benzoic acid, [...] or 6-aminocaproic acid with bovine serum albumin obtained by coupling immunogen, immunogen to mouse to obtain recognition gossypol derivative of the antibody.

Through the gossypol crossing linking arm O-carbencillin methyl hydroxylamine, the hydrazine benzoic acid, [...] or 6-aminocaproic acid with ovalbumin coupled to obtain packet is the original, the original packet is the gossypol-O-carbencillin methyl hydroxylamine-ovalbumin conjugate, gossypol-the hydrazine benzoic acid-ovalbumin conjugate, gossypol-6-aminocaproic acid-ovalbumin conjugate or gossypol-hexanediol [...] -ovalbumin conjugate; use the packet with the original preparation custodite coatingen.

Step 2. The sample to be tested is added to the volume ratio of 70% aqueous solution of acetone, filtered the filtrate is diluted with dilution of the sample solution to be measured, comprising a diluent 8.0g   NaCl, 0.2g   KH₂ PO₄, 2.9g   Na₂ HPO₄ · 12H₂ O, 0.2g   KCl, 0.1g sodium ethylmercurithiosalicylate, and double distilled water to 1000 ml solution.

Step 3. ELISA detection condition of the method is determined, using the above-mentioned antibody, a method for detecting enzyme ELISA, using the ELISA method for detecting in a sample solution to be measured is detected the content of free gossypol.

The stated step 1 in, gossypol antigen (including immunogen and packet is the original) and antibody preparation, can be prepared by the following embodiment.

Embodiment 1:

Step 1.1 gossypol-O-carbencillin methyl azanol (AOAA-FG-AOAA) preparation of double-adducts. Weighing O-carbencillin methyl hydroxylamine semi-hydrochloride (AOAA) 255.0 mg (2mmol) is added to the 2 ml distilled water, to be dissolved, get A liquid; weighing of gossypol acetic acid (FG) 289.0 mg (0.5mmol) is added to 20 ml of anhydrous ethanol, 40 the heat dissolving [...] , forming B liquid; A liquid and the liquid after mixing B, lucifugous magnetic stirring the reaction overnight at room temperature, precipitation filtering, using anhydrous ethanol 50 ml wash the precipitate, collect precipitation, lucifugous dried in air at room temperature, to obtain AOAA-FG-AOAA gossypol double-adducts.

Step 1.2 gossypol-O-carbencillin methyl hydroxylamine-bovine serum albumin/ovalbumin conjugate (FG-AOAA-BSA/OVA) preparation. Weighing AOAA-FG-AOAA   33.2 mg (0.05mmol) is added to the 2 ml methanol, then adding three n-butylamine 35.5 the  L, the 4 [...] stirring 10 min, then adding 4 the isobutyl chloroformate [...] pre-cooling of the 18  L, room temperature reaction 1h, for A liquid; said takes the cow blood serum albumin (BSA) 75 mg dissolved in 25 ml   0.01M carbonate buffer solution, to B liquid; the liquid dropwise A B liquid in the, side-to-side mixing, magnetic force in the ice water bath stirring reaction is 10h. Taking after centrifugation, 4 the dialysis in physiological saline [...] 3d, changed every day dialysate 2 times, get the gossypol-O-carbencillin methyl hydroxylamine-bovine serum albumin conjugate (FG-AOAA-BSA); centrifugal the supernatant fluid freeze drying, position -20 the preservation [...] , is used as the immunogen. Furthermore, the bovine serum albumin (BSA) with equimolar amount of ovalbumin (OVA), the specific mode is not variable, the gossypol-O-carbencillin methyl hydroxylamine-ovalbumin conjugate (FG-AOAA-OVA), is used as the packet is the original.

Step 1.3 anti-gossypol-O-carbencillin methyl hydroxylamine-BSA conjugate antibody preparation. Use small mouse immune FG-AOAA-BSA Balb/C (bought from hubei experimental animal center of medical Academy of Sciences). Immunization procedures are: heating containing FG-AOAA-BSA50-100 the protein   g with the volume of the solution's complete adjuvant (purchased from sigma Company) after emulsification, the back of the mouse subcutaneous multi-point injection; changing's incomplete adjuvant (purchased from the Company sigma) emulsified, every 2 weeks to strengthen a time; after 3-5 time after, orbitalis blood sampling, separation of serum, is anti-gossipol -O-carbencillin methyl hydroxylamine antibody; adding equivalent glycerin, split after mixing, device -20 the preservation [...].

Embodiment 2:

Step 2.1-gossypol diazanyl (HBA-FG-HBA) the preparation of benzoic acid double-adducts. The hydrazine benzoic acid (HBA) 302.0 mg weighed (2mmol) added to 2.5 ml   N, in N-dimethyl formamide, to be dissolved, get A liquid; weighing of gossypol acetic acid (FG) 289.0 mg (0.5mmol) is added to 20 ml of anhydrous ethanol, 40 the water bath [...] magnetic stirring and heating to be completely dissolved, forming B liquid; A liquid and the liquid after mixing B, the 40 [...] lucifugous magnetic stirring reaction sleepovers, precipitation filtering, using anhydrous ethanol 50 ml wash the precipitate, collect precipitation, lucifugous dried in air at room temperature, to obtain (HBA-FG-HBA) gossypol double-adducts.

Step 2.2 gossypol-the hydrazine benzoic acid-bovine serum albumin/ovalbumin conjugate preparation of (FG-HBA-BSA/OVA). Weighing HBA-FG-HBA   39.2 mg (0.05mmol) is added to the 2 ml   N, in N-dimethyl formamide, then adding three n-butylamine 35.5 the  L, the 4 [...] stirring 10 min, then adding 4 the isobutyl chloroformate [...] pre-cooling of the 18  L, room temperature reaction 1h, for A liquid; said takes the cow blood serum albumin (BSA) 75 mg dissolved in 25mL0.01M carbonate buffer solution, to B liquid. The A liquid dropwise B liquid in the, side-to-side mixing, stirring reaction is magnetic force in the ice water bath 10h; taking after centrifugation, 4 the dialysis in physiological saline [...] 3d, changed every day dialysate 2 times, get the gossypol-the hydrazine benzoic acid-bovine serum albumin conjugate (FG-HBA-BSA); centrifugal the supernatant fluid freeze drying, position -20 the preservation [...] , is used as the immunogen. Furthermore, the BSA replaced with equimolar amount of ovalbumin (OVA), the specific mode is not variable, the gossypol-the hydrazine benzoic acid-ovalbumin conjugate (FG-HBA-OVA), is used as the packet is the original.

Step 2.3 anti-gossipol -the hydrazine benzoic acid-BSA conjugate antibody preparation. The mouse Balb/C FG-HBA-BSA immunization, immunization procedure is with 1.3, get anti-gossipol -the hydrazine benzoic acid antibody.

Embodiment 3:

Step 3.1 gossypol-6-aminocaproic acid-synthesis of adduct (EACA-FG-EACA). Weighing 6-aminocaproic acid (EACA) 262.0 mg (2mmol) is added to the 2 ml distilled water, to be dissolved, get A liquid; weighing of gossypol acetic acid (FG) 289.0 mg (0.5mmol) is added to the 15 ml anhydrous ethanol, 40 the water bath [...] magnetic stirring and heating to be completely dissolved, forming B liquid; the liquid dropwise A B liquid in the, lucifugous magnetic stirring reaction 3h, precipitation filtering, using anhydrous ethanol 50 ml wash the precipitate, collect precipitation, lucifugous dried in air at room temperature, to obtain (EACA-FG-EACA) gossypol double-adducts.

Step 3.2 gossypol-6-aminocaproic acid-bovine serum albumin/ovalbumin conjugate (FG-EACA-BSA/OVA) preparation. In the preparation process, step 2.2 in EACA-FG-EACA HBA-FG-HBA replaced with equimolar (in other words, 37.2 mg), other step and the step 2.2 the consistent, can obtain immunogen gossypol-6-aminocaproic acid-bovine serum albumin conjugate and custodite (FG-EACA-BSA) original gossipol -6-aminocaproic acid-ovalbumin conjugate (FG-EACA-OVA), reaction principle equation as shown in Figure 1.

Step 3.3 anti-gossypol-6-aminocaproic acid-BSA conjugate antibody preparation. The mouse Balb/C FG-EACA-BSA immunization, immunization procedures is the same step 1.3, get anti-gossipol -6-aminocaproic acid antibody.

Embodiment 4:

Step 4.1 gossypol-hexanediol [...] -bovine serum albumin/ovalbumin conjugate preparation of (FG-ADH-BSA/OVA). Said (ADH) 334 mg [...] , bovine serum albumin (BSA) 80 mg and 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) 80 mg dissolved in 16mLMES buffer solution [0.1mol/L of the 2-(N-morpholine) ethyl sulfonic acid] in, room temperature magnetic stirring reaction 1.5h. 2000rpm taking after centrifugation, 4 the dialysis in physiological saline [...] 3d, changed every day dialysate 2 times. After dialysis 2000rpm centrifugal, taking, in other words A liquid; weighing ADH-FG-ADH   8.3 mg added to the DMF   2 ml, to be dissolved, forming B liquid; adding fluid by drop B A the liquid in the, stirring while adding, stirring reaction is magnetic force in the ice water bath 12h. After the reaction, for the 4 dialysis physiological saline [...] 3d, changed every day dialysate 2 times, get the gossypol-hexanediol [...] -bovine serum albumin conjugate (FG-ADH-BSA); cleaning the centrifugal after freeze-drying, position -20 the preservation [...] , is used as the immunogen. Furthermore, to the molar amount of such as the BSA OVA, the specific mode is not variable, the gossypol-caprolactam (FG-ADH-OVA) [...] -ovalbumin conjugate, as packet is the original.

Step 4.2 anti-gossypol-hexanediol [...] -BSA conjugate antibody preparation. The mouse Balb/C FG-ADH-BSA immunization, immunization procedure is with 1.3, antibody [...]anti-gossipol -caprolactam obtained.

The step 3 in, the detection of the ELISA method for determining conditions for the process of:

Step 301. Configuration ELISA method, the required reagent.

The phosphate buffer solution: weighing NaCl   8.0g, KH₂ PO₄ 0.2g, Na₂ HPO₄ · 12H₂ O2.9g, KCl   0.2g, and double distilled water to 1000 ml, adjusted to pH 7.4, forming a solution;

diaper fluid : weighing 1.5g   Na₂ CO₃, 2.9g   NaHCO₃, and three distilled water to 1000 ml, adjusting the pH value to 9.6 the rear, forming a solution;

Washing liquid: weighing 8.0g   NaCl, 0.2g   KH₂ PO₄, 2.9g   Na₂ HPO₄ · 12H₂ O, 0.2g   KCl, 0.1g sodium ethylmercurithiosalicylate, measured 0.5 ml tween -20, and double distilled water to 1000 ml, adjusting the pH to 7.4 the rear, forming a solution;

A confining liquid: weighing 0.1g ovalbumin dissolved in 100 ml phosphate buffer solution, to form a solution;

Substrate solution A: weighing 200mg3, 3', the 5 [...] , 5-tetramethyl-benzidine (TMB), the amount of 100 ml anhydrous ethanol, and double distilled water to 1000 ml the rear, forming a solution.

Substrate solution B: weighing 14.6g   Na₂ HPO₄, 9.3g citric acid, measuring 6.4 ml   0.75% urea hydrogen peroxide, add double-distilled water to 1000 ml the rear, forming a solution;

Stop solution: 2mol/L sulfuric acid solution.

Step 302. Determining packet is the original concentration and antibody working concentration: the ELISA method, different concentration antibody diluent, and diluent coatingen OD value of the enzyme target role, according to ELISA OD value difference of adjacent apertures is the largest dilution of the concentration of the antibody, the antibody concentration of work; the difference between the OD value of the adjacent holes of the largest concentration of a dilution coatingen, the original density for the packet is determined. The embodiment of the invention, the choice of the above-mentioned gossypol-6-aminocaproic acid-ovalbumin (FG-EACA-OVA) as a packet is the original, fluid dilution for a coated 8 mg/L, 4 mg/L, 2 mg/L, 1 mg/L, 0.5 mg/L, 0 . 25 mg/L   6 plurality of concentration, in 96-hole ELISA plate, from 1st to 6th row are sequentially added, the 4 [...] sleepovers; washing 3 times, shoot dry, confining liquid added to the 200  L, 37 the closed [...] 1h; washing 3 times, shoot dry, in the plate 1st to 8th row of sequentially adding the 100   L phosphate buffer dilution dilution multiple is 2000, 4000, 8000, 16000, 32000, 64000, 128000, 256000 monoclonal antibody, 37 the incubating [...] 1h, washing 3 times, shooting stem; to the hole 1:5000 times the phosphate buffer solution diluted horseradish peroxidase-labeled goat anti-mouse IgG antibody (abbreviated as two anti-, bought from Wuhan mitaka biotechnology limited) the 100  L, 37 the incubating [...] 1h, washing 5 times, shooting stem; to each hole 100 the mixed substrate   L, lucifugous color 15 min, to the 50   L stop solution, the enzyme for 450 nm wavelength value measuring optical density (OD value), shown in table 1.

Table 1 packet is the original concentration and antibody working concentration

As can be seen from the above table, to OD value is 1.0 left and right, and the adjacent hole the OD value of the difference of the concentration of antibody diluted solution, as the antibody concentration of work; the difference of adjacent holes of the OD value of the diluted concentration coatingen, as a packet is the original concentration. Therefore, the original packet is determined the coatingen FG-EACA-OVA a concentration of 0.5 mg/L, antibody working fluid concentration is diluted multiple is 1:16000 the concentration of the diluted solution.

Step 303. The ELISA method, drawing the standard curve line, find the semi-inhibiting concentration IC₅₀.

Weighing the gossypol-6-aminocaproic acid double-adducts (EACA-FG-EACA)10 mg, using acetone to constant volume to 100 ml, configuration concentration is 100 mg/L of the standard solution. The phosphate buffer solution for diluting to standard solution AOAA-FG-AOAA 0 subsidence g/l, 5 subsidence g/l, 15 subsidence g/l, 45 subsidence g/l, 135 subsidence g/l, the 405 g/l the   6 series concentration, each concentration repeated 3 hole, ELISA method is determined in accordance with, determination is repeated 5 times. AOAA-FG-AOAA concentration of solution in the value for the abscissa, B/B0 drawing standard curve for the longitudinal, semi-inhibiting concentration IC50 is determined, the reagent kit IC50 value is 52.0 subsidence g/l.

Step 304. The ELISA method, using the reagent to gossypol standard to be measured.

Weighing gossypol standard 10 mg, using acetone to constant volume to 100 ml, compounding concentration is 100 mg/L of gossypol standard acetone solution. Phosphate buffer solution for dilution to the gossypol standard solution 10 subsidence g/l, 50 subsidence g/l, 250 subsidence g/l   3 plurality of concentration. Distilled water for 1mmol/L the O-carbencillin methyl hydroxylamine solution. To apply the above-mentioned 3 plurality of concentration of gossypol standard solution each 1 ml, respectively adding 1mmol/L6-aminocaproic acid solution the 7.7  L, 38.6 the  L, the 192.8  L, 40 the oscillating reactions water bath [...] 2h. After the reaction, according to ELISA method measuring, each concentration repeated 3 times. Calculated according to the standard curve gossypol-6-aminocaproic acid double-cross the concentration of (AOAA-FG-AOAA) (CEACA-FG-EACA, μg/kg), and in accordance with formula 1 is converted into concentration of gossypol (CFG). The detection results are shown in table 2.

Formula 1: CFG=CEACA-FG-EACA×518744

Table 2ELISA to the gossypol on the measurement result of the method

Furthermore, through the experiment to confirm the ELISA method

1, ELISA method measuring in the cotton dregs gossypol standard adding:

Accurate weighing 9 have been known for the content of free gossypol 8.53g/kg (high performance liquid chromatography to detect) the thing cotton dregs of 0.5g in 100 ml centrifuge tube, the average is divided into 3 groups, each group of gossypol acetic acid separately adding 2.8 mg, 5.6 mg, 11.1 mg (converted into gossypol are respectively: 2.5 mg, 5 mg, 10 mg), after mixing, add 70% acetone aqueous solution of 50 ml (V:V), vortex mix, ultrasonic 30 min, filtering. Taking the filtrate 1 ml, by adding 0.1mol/L   6-aminocaproic acid aqueous solution, the 10  L, 40 the oscillating reactions water bath [...] 2h. After the reaction, the reaction liquid of the sample reduces the phosphate buffer solution is diluted 100 times, heating dilution ELISA method is determined in accordance with a reaction solution, each sample repeated 3 times. Calculated according to the standard curve gossypol-6-aminocaproic acid double-cross the concentration of (EACA-FG-EACA), in accordance with formula 1 is converted into gossypol concentration, and calculate the recovery rate of added, the results in table 3.

Table 3 adding gossypol in the cotton dregs recovery rate and coefficient of variation

2, ELISA method to determine standard added gossypol in the feed:

Accurate weighing 9 have been known for the content of free gossypol 0.57g/kg (high performance liquid chromatography to detect) the thing the feed of 0.5g in 100 ml centrifuge tube, the average is divided into 3 groups, each group of separately adding 100 mg/L of gossypol acetic acid acetone solution 2.8 ml, 5.6 ml, 11.1 ml (converted into gossypol addition are: 0.25 mg, 0.5 mg, 1.0 mg), after mixing, add 70% acetone aqueous solution (V:V) to the total volume is 50 ml, vortex mix, ultrasonic 30 min, filtering. Taking the filtrate 1 ml, by adding 0.01mol/L   6-aminocaproic acid aqueous solution, the 10  L, 40 the oscillating reactions water bath [...] 2h. After the reaction, the feed sample reaction solution uses phosphate buffer dilution 10 times, heating dilution ELISA method is determined in accordance with a reaction solution, each sample repeated 3 times. Calculated according to the standard curve gossypol-6-aminocaproic acid double-cross the concentration of (EACA-FG-EACA), in accordance with formula 1 is converted into gossypol concentration, and calculate the recovery rate of added, the results in table 4.

Table 4 adding gossypol in the feed rate and coefficient of variation

3, ELISA method and HPLC method to free gossypol in the sample reduces the measured value comparison:

Accurate weighing 10 parts of sample products from different sources the thing 0.5g in 100 ml centrifuge tube, add 70% acetone aqueous solution of 50 ml (V:V), vortex mix, ultrasonic 30 min, filtering. Taking the filtrate 1 ml, by adding acetonitrile/aqueous solution (80:20, V:V) 9 ml, after mixing, a 0.2 micron microporous membrane, is detected by high performance liquid chromatography (HPLC). The specific method is:a high performance liquid chromatograph zimbabwean island of Japan, chromatographic condition: Agilent   extend-C18 (250 × 4.6 mm, 5 µm), ultraviolet detection wavelength of 235 nm, column temperature 35 the [...]. Whereas the 20  L, A the mobile phase: water/formic acid (100 : 0.1, v/v); mobile phase B: acetonitrile, A:B=20 : (V:V) 80, such as elution degree, flow rate is 1 ml/min.

The other taking the filtrate 1 ml, by adding 0.1mol/L   6-aminocaproic acid aqueous solution, the 10  L, 40 the oscillating reactions water bath [...] 2h. After the reaction, the reaction solution with phosphate buffer solution is diluted 100 times, heating dilution ELISA method is determined in accordance with a reaction solution. Calculated according to the standard curve gossypol-6-aminocaproic acid double-cross the concentration of (EACA-FG-EACA), and in accordance with formula 1 is converted into concentration of gossypol. Figure 3 ELISA for this invention with high performance liquid chromatography (HPLC) method for the 10 different sources the content of free gossypol in the cotton dregs in the mutual relations between the measured value, the measured value of the HPLC method X axis, Y axis as the ELISA method measured value. HPLC on the measurement result of the ELISA method, and table 5,

Table 5HPLC ELISA method and the method for determining the content of free gossypol in the cotton dregs

One kind is used in the kit of detection of free gossypol, including horseradish peroxidase-labeled goat anti-mouse IgG antibody working fluid, concentrated phosphate buffer solution, the concentrated cleaning solution, substrate solution A, substrate solution B, coated with terminating liquid and coatingen, antibody working fluid, different concentrations of gossypol double-adducts of standard solution. Wherein the original to the gossypol with ovalbumin coupled through crossing linking arm conjugate obtained by reaction of; arm by cross-linking antibody as gossypol with bovine serum albumin obtained by coupling immunogen, an antibody obtained by mouse immunogen; gossypol double-adduct to gossypol with cross-linked arm double-addition reaction of the adduct, said cross-linked arm is O-carbencillin methyl hydroxylamine, the hydrazine benzoic acid, [...] and 6-aminocaproic acid in a, a packet with a kit is the original, gossypol double-adducts of standard solution and antibody working fluid to gossypol derivatives with a cross-linking arm.

The embodiment of the invention, packet is the original by gossypol crossing linking arm 6-aminocaproate of the ovalbumin coupling reaction coupling gossipol -6-aminocaproic acid-ovalbumin; coatingen making the specific steps are as follows: the peridium fluid custodite for the original diluted to 0.5 mg/L of the solution, each hole of the enzyme added to the 100   L the above-mentioned solution, the 4 [...] sleepovers, dumping of removing diaper fluid ; in each hole to the 210   L washing liquid, washing 3 times, shooting stem; the adding of each hole 200 the confining liquid   L, 37 the incubating [...] 1h, the liquid in the leans the hole ; detergent washing 3 times, shoot dry, vacuum sealing preservation of a waveform, the original packet is coated with a prepared ELISA plate.

Antibodies as gossypol through the 6-aminocaproic acid with bovine serum albumin obtained by coupling immunogen gossypol-6-aminocaproic acid-bovine serum albumin, an antibody obtained by mouse immunogen, the antibody is the antibody work with the phosphate buffer salt according to 1:16000 diluting the obtained solution,

Gossypol double-adducts with gossypol to 6-aminocaproic acid-addition of addition reaction thing gossipol -6-aminocaproic acid double-adducts, gossypol double-adducts of the concentration of the solution are standard 0 subsidence g/l, 5 subsidence g/l, 15 subsidence g/l, 45 subsidence g/l, 135 subsidence g/l, 405 subsidence g/l.

Concentrated phosphate buffer solution comprises a concentration is 80.0 g/l of NaCl, the concentration of 2.0 g/l the KH₂ PO₄, concentration is 29.0 g/l the Na₂ HPO₄. 12H₂ O, concentration is 2.0 g/l and KCl concentration of 0.1 g/l aqueous solution of the sodium ethylmercurithiosalicylate.

The concentrated cleaning solution comprising a concentration is 80.0 g/l of NaCl, the concentration of 2.0 g/l the KH₂ PO₄, concentration is 29.0 g/l the Na₂ HPO₄ · 12H₂ O, concentration is 2.0 g/l the KCl, concentration is 0.1 g/l and the concentration of the sodium ethylmercurithiosalicylate 5 ml/L tween -20 solution.

Liquid A comprises a substrate concentration of 200 mg/L of 3, 3 the [...] , the 5 [...] , 5-tetramethyl-benzidine and concentration is 100 ml/L aqueous solution of anhydrous ethanol.

Substrate solution B to include concentration is 14.6 g/l the Na₂ HPO₄, concentration is 9.3 g/l of citric acid and the concentration of 6.4 ml/L of 0.75% hydrogen peroxide aqueous solution of urea.

Stop solution to 2mol/L sulfuric acid solution.

The kit utilizes ELISA procedures of the method for detecting free gossypol, comprising the following steps:

F1. The concentration of the reagent kit phosphate buffer three steam water dilution 10 times dilution is formed after use, the concentrated washing three steam water dilution 10 times for forming after washing, the substrate liquid A and substrate solution B according to the volume 1:1 for mixing liquid forming substrate, according to each time with the needed amount is, and other reagent is not required for processing.

F2. Processing the sample to be measured of the sample solution to be measured, the specific process is as follows: weighing 0.5g attrition in Carp sample or the feed sample 100 ml centrifuge tube, add 50 ml volume percentage of 70% acetone aqueous solution, vortex mix, ultrasonic 30 min, filtering; taking 1 ml sample filtrate, to the 10   L aqueous solution of the selected cross-linking arm, 40 the oscillating reactions water bath [...] 2h; sample reaction filtrate is diluted with dilution of the sample solution to be measured, wherein adding the filtrate sample products 0.1mol/L aqueous solution of cross-linked arm, reduces the reaction filtrate diluted with dilution of the sample 100 times the sample solution to be measured obtained; feed sample filtrate by adding 0.01mol/L cross-linked arm aqueous solution, with a liquid diluent feed sample reaction filtrate 10 times the sample solution to be measured obtained.

F3. In the original packet is coated with the enzyme is added to each hole of the 50   L different concentrations of gossypol double-adducts or standard solution of the sample solution to be measured, and then adding the 50   L antibody working fluid, the wet-box the enzyme, 37 the constant temperature incubation [...] 1h; dumping of removing the liquid in each hole, each hole in the to the 210   L washing liquid, washing 3 times and the racket does.

F4. The adding of each hole the 100   L Cochlearia peroxidase-labeled goat anti-mouse IgG antibody working fluid, the wet-box the enzyme, 37 the constant temperature incubation [...] 1h; dumping of removing the liquid in each hole, each hole in the to the 210   L washing liquid, washing 5 times and the racket does.

F5. The adding of each hole 100 the mixed substrate   L, wet box is the enzyme, the 37 [...] constant temperature incubation 15 min, the adding of each hole 50 the termination   L of liquid.

F6. Enzyme in the enzyme assay of each hole 450 nm value of the optical density (OD value).

F7. Obtained by calculation according to the optical density values of the sample solution to be measured in the concentration of free gossypol.

This embodiment, using 6-aminocaproic acid as gossypol double-adduct preparation crossing linking arm , antibody kit, the method of calculating the: X axis as the gossypol-6-aminocaproic acid double-adducts of the concentration of the standard solution, Y axis as the gossypol-6-aminocaproic acid double-adducts of optical density value standard solution with the blank solution ("zero" hole) optical density value (B/B0), manufacturing a standard curve, as shown in Figure 2, and carry on the linear regression, the regression equation is given; OD value of sample solution to be measured by dividing the OD value "zero" hole, the inhibition rate of the sample is calculated; the inhibition rate of sample solution to enter the above-mentioned standard curve in the regression equation, multiplied by the dilution factor and, in a sample solution to be detected is calculated gossypol double addition thing gossipol -6-aminocaproic acid double-adducts the concentration of (EACA-FG-EACA); according to the formula Calculate the concentration of free gossypol (FG), the unit is μg/kg.

The invention is not limited to the above-mentioned embodiment, in the technical field as the ordinary technical personnel, without breaking away from the premise of the principle of the present invention, can also be made a number of improvements and retouches, these improvements and retouches also considered within the scope of protection of this invention. In this specification a detailed description of the content of as belonging to the field of professional technical personnel of the prior art known.