CN102162813A - Reagent kit and method for detecting T-2 toxin by using genetically engineered single-chain antibody - Google Patents

Abstract

The invention provides a kit and method for detecting T-2 toxin by a genetically engineered single-chain antibody. In the kit of the invention, the reagents include: sample extract, standard reagent, enzyme-labeled antigen reagent, washing solution , BSA reagent, chromogenic substrate, stop solution. Through sample processing, reagent balance, washing, sample addition, washing, color development, and termination, the content of T-2 toxin in the sample is calculated. The T-2 toxin single-chain antibody used in the kit of the present invention can be efficiently expressed in Escherichia coli and can be produced on a large scale. The preparation process is simple and easy, saving time, effort and money. Using the kit of the invention to detect T-2 toxin can determine whether a sample is polluted by T-2 toxin and calculate the amount of T-2 toxin contained within 1.5-2 hours, which is convenient and quick to use and low in cost.

Description

Kit and method for detecting T-2 toxin by genetically engineered single-chain antibody

technical field

The present invention relates to a detection kit for detecting mycotoxin by using genetically engineered single-chain antibody, more specifically relates to a kit for detecting T-2 toxin by using genetically engineered single-chain antibody, and further relates to using the kit to detect T-2 toxin Methods.

Background technique

Currently, the antibodies used for immunodetection of T-2 toxin are all monoclonal antibodies. The production of monoclonal antibodies uses antigens to immunize animals, and then uses the spleen cells of the immunized animals to fuse with myeloma cells to form hybridoma cells, and finally screens out hybridoma cells with high antibody activity and large-scale reproduction. The whole production process is complicated, the time consumption is long, and the cost is high, especially the operation process needs skilled professional technicians to be competent.

Contents of the invention

In order to overcome the time-consuming, labor-intensive and cost-intensive shortcomings of the existing monoclonal antibody production, the present invention provides a kit and method for detecting T-2 toxin with a genetically engineered single-chain antibody, which makes the detection process of T-2 toxin simple and easy. , saving time, effort and money,

Technical scheme of the present invention is as follows:

The kit for detecting T-2 toxin by using genetic engineering single chain antibody of the present invention, its reagent comprises:

(1) Sample extract: physiological saline containing 50% (volume ratio) dimethyl sulfoxide;

(2) T-2 toxin standard reagent: sample extract containing T-2 toxin;

(3) Enzyme-labeled antigen reagent: PBS solution containing T-2-HRP coupled protein with a titer of 5000, stored in 50% (volume ratio) glycerol, stored at -20°C; diluted with BSA reagent when used;

(4) Washing solution: TNT solution; formula components: 0.05% (volume ratio) tween-20, 20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4;

(5) BSA reagent: TNT solution containing 5% (weight ratio) BSA;

(6) Chromogenic substrate: formula components: 250 μL 20 mg/mL DAB, 40 μL 40 mg/mL NiCl, 9.75 ml 1.0 mol/L Tris-HCl (pH 6.8), stored at -20°C; when used Add 7 μL 30% H ₂ O ₂

(7) Stop solution: deionized water containing: 0.1 mol/L sodium sulfite, 2 mol/L sulfuric acid.

The method for using the above-mentioned genetically engineered single-chain antibody detection kit for T-2 toxin comprises the following steps in turn:

(1) Sample processing:

Add 2-6 ml sample extract solution to 0.5-1.5 g sample, liquid nitrogen or mechanical grinding, after ultrasonic extraction and centrifugation, the supernatant is the sample-containing extract solution;

The extract containing the sample and the enzyme-labeled antigen reagent are mixed evenly in equal volumes to form A reagent; the enzyme-labeled antigen reagent is diluted with BSA reagent first, and 1 μl of T-2-HRP coupled protein PBS is added to 5ml of BSA reagent solution;

The T-2 toxin standard reagent is mixed with the same volume of the enzyme-labeled antigen reagent at a series concentration to form the B reagent at a series concentration;

(2) Reagent balance: equilibrate the kit to room temperature;

(3) Numbering of small wells: Take out the enzyme labeling strip and place it on the reaction plate, mark well 1 as a negative well, wells 2-6 as T-2 toxin standard control wells, and the rest of the wells as sample wells; each well of the enzyme labeling strip There are already immobilized anti-T-2 toxin single-chain antibodies in it;

(4) Washing: Add 200-300 μL of washing liquid into each well, and the washing liquid must not overflow the well. After standing for 2 minutes, remove the washing liquid, pat dry on absorbent paper, and repeat washing once;

(5) Adding samples: Add 50 μL of BSA reagent to No. 1 well, add 50 μL of serial concentration of B reagent to No. 2-6 wells respectively, add 50 μL of corresponding A reagent to the sample well, and shake gently to mix wells. ;

(6) Reaction: put the reaction plate into a 37 ℃ incubator and incubate for 30 min;

(7) Washing: Take out the reaction plate, remove the reaction solution, and pat dry; add 200-300 μL of washing solution to each well, and the washing solution must not overflow. After standing for 2 minutes, remove the washing solution, pat dry on absorbent paper, and repeat washing 4 times ;

(8) Color development: Add 100 μL of color development substrate to each well, shake well, and put the reaction plate in a 37°C incubator for 15 minutes;

(9) Termination and instrument measurement: Add 50 μL of stop solution to each well, and then use a microplate reader to measure the absorbance value A of each well at 450 nm, take the serial concentration of reagent B as the abscissa, and take the corresponding absorbance value A draws a standard curve for the ordinate, and obtains the concentration of T-2 toxin in the sample according to the standard curve, according to the calculation formula T-2 toxin content (μg/g)=C*V/m, wherein the concentration of C-T-2 toxin, μg /mL; V-volume of sample extract, mL; m-sample mass, g; thus calculate the content of T-2 toxin in the sample.

The amino acid sequence of the anti-T-2 toxin single-chain antibody is shown in SEQ ID No.1. The preparation method of the T-2 toxin single-chain antibody is as follows: total RNA is extracted from the spleen of immunized mice, reverse-transcribed into cDNA, and the heavy chain variable region V _H gene and light chain of the cDNA can be amplified by PCR. The V _L gene of the variable region, and then connect the V _H and V _L through a linking peptide to form a single-chain antibody, which is then cloned into a vector to construct a phage antibody library, and then biopanned to obtain a high-quality antibody against T-2 toxin. Affinity anti-T-2 toxin single chain antibody. The amino acid sequence of the anti-T-2 toxin single-chain antibody is shown in SEQ ID No.1.

The structure of the anti-T-2 toxin single chain antibody of the present invention is shown in FIG. 1 . The single-chain antibody (scFv) is made by genetically engineering the heavy chain variable region (V _H ) and light chain variable region (V _L ) of the mouse cDNA through a connecting peptide (Linker, Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser) linked recombinant protein. As shown in Figure 2, the size of the heavy chain variable region (V _H ) is 340 bp; as shown in Figure 3, the size of the light chain variable region (V _L ) is 325 bp; as shown in Figure 4, the anti-T -2 toxin single-chain antibody (scFv) is 750 bp in size; this single-chain antibody (scFv) maintains the antigen-affinity activity and specificity of the parent antibody as the smallest functional antibody fragment, which can be economically produced in bacteria on a large scale, It makes the production of antibodies for immunological detection simple and economical, and greatly reduces the cost of diagnostic reagents.

Significant advantage of the present invention:

The T-2 toxin single-chain antibody used in the kit of the present invention can be efficiently expressed in Escherichia coli and can be produced on a large scale. The preparation process is simple and easy, saving time, effort and money. Using the kit of the present invention to detect T-2 toxin can determine whether a sample is polluted by T-2 toxin and calculate the amount of T-2 toxin contained within 1.5-2 hours, which is convenient and quick to use and low in cost.

Description of drawings

Fig. 1 is a schematic structural view of the anti-T-2 toxin single chain antibody of the present invention.

Fig. 2 is the electrophoresis diagram of V _H amplification of the heavy chain gene.

Fig. 3 is the electrophoresis diagram of light chain gene V _L amplification.

Fig. 4 is an electrophoresis diagram of scFv amplification of the anti-T-2 toxin single-chain antibody gene of the present invention.

Fig. 5 is a competition ELISA detection curve of the anti-T-2 toxin single chain antibody of the present invention.

Detailed ways

The following are specific examples of the present invention to further describe the present invention, but the present invention is not limited thereto.

Example 1

A kit for detecting T-2 toxin using a genetically engineered single-chain antibody was produced according to the following formula:

(1) Sample extract: 50% volume ratio of DMSO in normal saline;

(2) T-2 toxin standard reagents: sample extracts containing T-2 toxin at concentrations of 0.04, 0.08, 0.16, 0.32, and 0.64 μg/mL respectively;

(3) Enzyme-labeled antigen reagent: 0.01M PBS solution containing a titer of 5000T-2-HRP coupled protein, stored in 50% (volume ratio) glycerol, stored at -20°C; diluted with BSA reagent when used, Add 1μl T-2-HRP coupling protein PBS solution to 5ml BSA reagent;

(4) Washing solution: TNT solution; formula components: 0.05% (volume ratio) Tween-20, 20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4;

(5) BSA reagent: TNT solution containing 5% BSA;

(6) Chromogenic substrate: 10 ml chromogenic solution; formula components: 250 μL 20 mg/mL DAB, 40 μL 40 mg/mL NiCl, 9.75 ml 1.0 mol/L Tris-HCl (pH 6.8), - Store at 20°C; add 7 μL 30% H ₂ O ₂ when using;

(7) Stop solution: deionized water containing: 0.1 mol/L sodium sulfite, 2 mol/L sulfuric acid.

Utilize above-mentioned kit to detect the method for T-2 toxin, concrete steps are as follows:

(1) Sample treatment: add 4ml of sample extract to 1g of sample, liquid nitrogen or mechanical grinding, ultrasonic extraction for 10 min, centrifugation at 5000 rpm for 10 min, the supernatant is the extract containing the sample;

Take 100 μl of the sample extract containing the sample and mix evenly with 100 μl enzyme-labeled antigen reagent to form reagent A; dilute the enzyme-labeled antigen reagent before use, and add 1 μl of T-2-HRP coupling protein to every 5ml of BSA reagent PBS solution;

Mix 100 μL each of T-2 toxin standard reagents with serial concentrations of 0.04, 0.08, 0.16, 0.32, and 0.64 μg/mL with 100 μL enzyme-labeled antigen reagent respectively to form B reagents with serial concentrations;

(2) Reagent balance: Take the kit out of the -20°C refrigerator, place it for more than 15 minutes, and equilibrate to room temperature;

(3) Well number: Take out the enzyme labeling strip and place it on the reaction plate support as needed. Well 1 is a negative well, wells 2-6 are T-2 toxin standard control wells, and the remaining wells are sample wells; each well of the enzyme-labeled strip has immobilized anti-T-2 toxin single-chain antibody; the anti-T-2 toxin single-chain antibody The preparation steps of T-2 toxin single-chain antibody are as follows: extract total RNA from the spleen of immunized mice, reverse transcribe it into cDNA, and amplify the antibody heavy chain variable region V _H gene and light chain variable region V _L by PCR Gene, V _H and V _L are connected into a single-chain antibody (scFv) through a linker (Linker), then cloned into a vector to construct a phage antibody library, and then bio-panned to obtain a high-affinity antibody for T-2 toxin single chain antibody.

(4) Washing: Add 250 μL of washing solution to each well, and the washing solution must not overflow. After standing for 2 minutes, remove the washing solution, pat dry on absorbent paper, and wash the plate again;

(5) Adding samples: Add 50 μL of BSA reagent to No. 1 well, add 50 μL of serial concentration of B reagent to No. 2-6 wells respectively, add 50 μL of corresponding A reagent to the sample well, and shake gently to mix wells. ;

(6) Reaction: put the reaction plate into a 37 ℃ incubator and incubate for 30 min;

(7) Washing: Take out the reaction plate, remove the reaction solution, and pat dry. Add 250 μL of washing solution to each well, and the washing solution should not overflow. After standing for 2 minutes, remove the washing solution, pat dry on absorbent paper, and repeat washing the plate 4 times;

(8) Color development: Add 100 μL of color development substrate to each well, shake well, and put the reaction plate in a 37°C incubator for 15 minutes;

(9) Termination and instrument measurement: Add 50 μL of stop solution to each well, and then use a microplate reader to measure the absorbance value A of each well at 450 nm, take the serial concentration of reagent B as the abscissa, and take the corresponding absorbance value A draws a standard curve for the ordinate, and obtains the concentration of T-2 toxin in the sample according to the standard curve, according to the calculation formula T-2 toxin content (μg/g)=C*V/m, wherein the concentration of C-T-2 toxin, μg /mL; V-volume of sample extract, mL; m-sample mass, g; calculate the content of T-2 toxin in the sample.

Combine the T-2 toxin single chain antibody with the anti-E-tag antibody immobilized on the microtiter plate through the E-tag on the T-2 toxin single chain antibody to form a solid-phase antibody; add the sample to be tested and anti-T-2 Toxin enzyme-labeled antigen reagent, the antigen in the sample competes with the enzyme-labeled antigen to bind to the solid-phase antibody. The higher the antigen content in the sample to be tested, the more it binds to the solid-phase antibody, so that the enzyme-labeled antigen binds to the solid-phase antibody. There are fewer opportunities, or even no chance to combine, so that no color or very light color will be developed after adding the substrate, and those with darker color will be negative. As shown in Table 1, as the concentration of the added standard concentration T-2 toxin gradually increased, the corresponding absorbance value OD value gradually decreased. The T-2 toxin standard concentration curve drawn according to this data is shown in Figure 5, and the absorbance value at OD450 nm is inversely proportional to the concentration of the sample to be tested, so the absorbance at OD450 nm of the sample to be tested can be used to determine the Concentration of T-2 toxin.

As shown in Table 1, the analysis was carried out. According to the calculation formula, the T-2 toxin content in the sample (μg/g)=C*V/m, where C-the concentration of T-2 toxin in the sample, μg/mL; V-the volume of the sample extract, mL; m- Sample mass, g; calculate the content of T-2 toxin in the sample. When the volume of the sample extract is 4ml, the sample mass is 1g, and the absorbance value is 0.946, the T-2 toxin content is: 0.04*4 /1=0.16 μg/g.

Table 1 The competition ELISA detection of the T-2 toxin single chain antibody of the present invention

Note: when the concentration is 0 μg/mL, the primary antibody is a mixture of the single-chain antibody of the present invention and an equal volume of PBS, and an equal volume of T-2 toxin standard solution (concentrations of 0.04, 0.08, 0.16, 0.32, 0.64 μg/mL)

<110> Fujian Agriculture and Forestry University

<120> A kit and method for detecting T-2 toxin with a genetically engineered single-chain antibody

<160> 1

<210> 1

<211> 247

<212> PRT

<213> BABA/c mouse ( Mus musculus )

<400>1

Gln Val Gln Leu Gln Gln Ser Gly Thr Val Leu Ala Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Met Ser Cys Lys Thr Ser Ser Phe Tyr Thr Phe Gly Ala

20 25 30

Cys Ser Asn Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Trp Val Asn Asp Ile Ser Thr Thr Ser Trp Gly Asp Pro His Pro

50 55 60

Lys Val Lys Ala Lys Leu Thr Ala Val Thr Ser Thr Asn Thr Ala Tyr

65 70 75 80

Met Glu Val Ser Ser Leu Thr Asn Glu Asp Ser Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Thr Gly Tyr Ser Thr Pro Arg Trp Gln Gly Trp Gly Gln Gly

100 105 110

Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gln Ala Val Val Thr Gln Glu Ser Ala Leu

130 135 140

Thr Thr Ser Pro Gly Glu Thr Val Thr Leu Thr Cys Pro Thr Thr Ala

145 150 155 160

Asp Thr Ser Tyr Thr Asn Ser Asn Arg Trp Trp Val Gln Glu Lys Pro

165 170 175

Asp His Leu Phe Thr Ala Leu Ile Gly Thr Asp Asp Gly Arg Phe Ala

180 185 190

Gly Val Pro Ala Arg Phe Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala

195 200 205

Leu Thr Ile Thr Gly Ala Gln Thr Glu Asp Glu Ala Ile Tyr Phe Phe

210 215 220

Thr Ser Val Ser Cys Trp Tyr Cys Asn Val Phe Gly Gly Gly Thr Lys

225 230 235 240

Leu Thr Val Leu Gly Gln Pro

245

Claims (3)

1. a phage single chain antibody detects the kit of T-2 toxin, and it is characterized in that: its reagent comprises:

(1) sample extracting solution: the physiological saline that contains 50% (volume ratio) dimethyl sulfoxide (DMSO);

(2) T-2 toxin standard reagent: the sample extracting solution that contains the T-2 toxin;

(3) enzyme-labelled antigen reagent: contain the PBS solution of 5000 the T-2-HRP coupling protein of tiring, be stored in the 50%(volume ratio) in the glycerine ,-20 ℃ of preservations; Dilute with BSA reagent during use;

(4) cleansing solution: TNT solution; Formula constituent: the 0.05%(volume ratio) tween-20,20 mmol/L Tris-HCl, 150 mmol/L NaCl, pH 7.4;

(5) BSA reagent: the TNT solution that contains 5% (weight ratio) BSA;

(6) chromogenic substrate: formula constituent: 250 μ L, 20 mg/mL DAB, 40 μ L, 40 mg/mL NiCl, the 1.0 mol/L Tris-HCl (pH 6.8) of 9.75 ml ,-20 ℃ of preservations; Add 7 μ L 30%H during use ₂O ₂

(7) stop buffer: contain in the deionized water: 0.1 mol/L sodium sulphite, 2 mol/L sulfuric acid.

2. method of using the described phage single chain antibody of claim 1 to detect the kit of T-2 toxin is characterized in that: may further comprise the steps successively:

(1) sample preparation:

In 0.5～1.5 g sample, add 2～6 ml sample extracting solutions, after liquid nitrogen or mechanical lapping are pulverized, ultrasonic extraction, centrifugal, supernatant is the extract that contains sample;

It is evenly mixed to contain the extract of sample and enzyme-labelled antigen reagent equal-volume, A reagent; When using, described enzyme-labelled antigen reagent, adds the PBS solution of 1 μ l T-2-HRP coupling protein in the 5ml BSA reagent earlier with the dilution of BSA reagent;

It is mixed evenly with enzyme-labelled antigen reagent equal-volume respectively that T-2 toxin standard reagent is got series concentration, the B reagent of series concentration;

(2) reagent balance: with the kit balance to room temperature;

(3) aperture numbering: take out enzyme mark bar and be placed on the reaction plate, negative hole, No. 1 hole of mark, the 2-6 hole is T-2 toxin standard control hole, all the other holes are sample well; Enzyme mark bar has had the anti-T-2 toxin of immobilization single-chain antibody in every hole;

(4) washing: add 200～300 μ L cleansing solutions every hole in, cleansing solution must not overfolw hole outside, behind the placement 2min, remove cleansing solution, on thieving paper, pat dry, repeated washing is once;

(5) application of sample: No. 1 the hole adds 50 μ L BSA reagent, and the 2-6 hole adds the B reagent 50 μ L of series concentration respectively, and sample well adds corresponding A reagent 50 μ L, and light shaking makes each hole mixing;

(6) reaction: reaction plate is put into 37 ℃ of constant temperature ovens hatch 30 min;

(7) washing: take out reaction plate, remove reactant liquor, pat dry; Every hole adds 200～300 μ L cleansing solutions, and cleansing solution must not overflow, place 2 min after, remove cleansing solution, on thieving paper, pat dry repeated washing 4 times;

(8) colour developing: every hole adds chromogenic substrate 100 μ L, shakes up, and reaction plate is put into 37 ℃ of constant temperature ovens deposit 15 min;

(9) termination and Instrument measuring: every hole adds stop buffer 50 μ L respectively, measure the light absorption value A in each hole then at 450 nm places with microplate reader, series concentration with B reagent is a horizontal ordinate, with its corresponding light absorption value A is ordinate drawing standard curve, obtain the concentration of T-2 toxin in the sample according to typical curve, according to computing formula T-2 content of toxins (μ g/g)=C*V/m, the concentration of C-T-2 toxin wherein, μ g/mL; The volume of V-sample extracting solution, mL; The m-sample quality, g; Thereby the content of T-2 toxin in the calculation sample.

3. phage single chain antibody according to claim 2 detects the using method of the kit of T-2 toxin, and it is characterized in that: the amino acid sequence of described anti-T-2 toxin single-chain antibody is shown in SEQ ID No.1.

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