CN102095849A - Kit and method for detecting mu-conotoxin (CTX) by using single-chain antibody of gene engineering - Google Patents

Abstract

The invention provides a kit and a method for detecting mu-conotoxin (CTX) by using a single-chain antibody of gene engineering. Reagents in the kit comprise sample extracting solution, a standard reagent, an enzyme-labeled antigen reagent, cleaning solution, a bovine serum albumin (BSA) reagent, a color development substrate and stop solution. The method comprises the following steps of: processing samples; balancing the reagents; washing; adding the samples; incubating; washing; developing; stopping; and calculating the content of the mu-CTX in the samples. By the kit and the method, the single-chain antibody of the gene engineering for resisting mu-CTX micromolecules can be effectively expressed in escherichia coli; and the kit can be produced on a large scale, has an extremely simple whole production process, saves time, labor and money, can be conveniently and fast used and has low cost.

Description

Utilize phage single chain antibody to detect the kit and the method for mu-conotoxin

Technical field

The present invention relates to a kind of detection kit of utilizing phage single chain antibody to detect the ocean toxin, related more specifically to utilize phage single chain antibody to detect mu-conotoxin (μ-Conotoxin, the kit of μ-CTX) further relates to the method for using this kit to detect μ-CTX.

Background technology

(Conotoxin is to be come out by carnivorous mollusc cone shell (Conus) secretion of living in the tropic sea midocean CTX) to conotoxin, is used to anaesthetize the small peptide toxin of prey.μ-CTX is similar to tetraodotoxin, is typical sodium-ion channel inhibitor, and symptoms such as extremity numbness, paralysis, serious caused death appear in the poisoner.Detection method commonly used has high performance liquid chromatography/quadrupole rod-flight time mass spectrum (HPLC/Q-TQFMS) coupling technique, but spend big, consuming time, changeability is high, sensitivity is low, be subjected to sample quantitative limitation to be checked, can not provide deterministic evidence to particular toxin, present stage lacks the method for a kind of simple and effective detection μ-CTX.

Summary of the invention

The invention provides a kind of kit that simply, effectively utilizes phage single chain antibody to detect mu-conotoxin.

Technical scheme of the present invention is as follows:

The kit that utilizes phage single chain antibody to detect mu-conotoxin of the present invention, its reagent comprises:

(1) sample extracting solution: deionized water;

(2) standard reagent: TRX-CTX toxin;

(3) enzyme-labelled antigen reagent: the PBS solution for 0.01M, the pH that contains the TRX-CTX-HRP coupling protein is 7.4 is stored in the 50%(volume ratio) glycerine in ,-20 ℃ of preservations, tiring is 5000;

(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: the 10 ml liquid that develops the color; Formula constituent: 250 μ L, 20 mg/mL DAB, 40 μ L, 40 mg/mL NiCl, 1.0 mol/L of 9.75 ml, the Tris-HCl of pH 6.8 ,-20 ℃ of preservations; Add 7 μ L 30%(volume ratios during use) H ₂O ₂

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

Utilize described kit to detect the method for mu-conotoxin, may further comprise the steps successively:

(1) sample preparation: add the 4ml sample extracting solution in the 1g sample, liquid nitrogen or mechanical lapping are pulverized, ultrasonic extraction 10 min, and centrifugal 10 min of 5000rpm, supernatant is the sample extracting solution that contains sample;

The sample extracting solution that contains sample gets 100 μ l and 100 μ l enzyme-labelled antigen reagent are evenly mixed, A reagent;

Each 100 μ L of TRX-CTX toxin standard reagent 0.01,0.1,1.0,10 ng/mL of series concentration are evenly mixed with 100 μ L enzyme-labelled antigen reagent respectively, the B reagent of series concentration;

(2) reagent balance: kit is taken out from-20 ℃ of refrigerators, place more than 15 min, balance is to room temperature;

(3) aperture numbering: take out enzyme mark bar and be placed on the reaction plate support, negative hole, No. 1 hole of mark, the 2-5 hole is TRX-CTX toxin standard control hole, all the other holes are sample well; The anti-μ of immobilization-CTX single-chain antibody in the every hole of enzyme mark bar;

(4) washing: every hole adds 250 μ L cleansing solutions, and cleansing solution must not overflow, and behind the placement 2min, discards cleansing solution, and pats dry on thieving paper, repeats to wash plate 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, adds A reagent 50 μ L in each sample well, and light shaking makes reagent mixing in each hole;

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

(7) washing: take out reaction plate, remove step (5) and add reagent, pat dry, every hole adds 250 μ L cleansing solutions, and cleansing solution must not overflow, place 2 min after, discard cleansing solution, on thieving paper, pat dry, repeat to wash plate 4 times;

(8) colour developing: every hole adds chromogenic substrate 100 μ L, shakes up, and ELISA 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, use the light absorption value A of microplate reader material in 450 nm places measure each hole then, 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 μ-CTX in the sample according to typical curve, according to computing formula μ-CTX content (ng/g)=C*V/m, wherein C is the concentration of μ-CTX, ng/mL; The volume of V-sample extracting solution, mL; The m-sample quality, g; The content of μ-CTX in the calculation sample.

The amino acid sequence of described anti-μ-CTX single-chain antibody is shown in SEQ ID No.1.

Wherein said anti-μ-preparation method is as follows for the CTX single-chain antibody: extract total RNA from the spleen of immune mouse, use the Trizol kit to extract the total RNA of mouse spleen, become cDNA through reverse transcription.Through pcr amplification antibody heavy chain variable region V _HGene and variable region of light chain V _LGene, pcr amplification program are 94 ℃ of 5 min; 94 ℃ of 45s, 55 ℃ of 45s, 72 ℃ of 45s, 33 circulations; 72 ℃ are extended 10 min; 1% agarose gel electrophoresis is verified pcr amplification product then; Because the PCR product all has only a band, reclaim V _HAnd V _LThe PCR product.By one section connection peptides (Linker, Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser) V _HAnd V _LConnect into single-chain antibody (scFv), be cloned into carrier (pCANTAB 5E) then and go up make up phage antibody library, again by biological elutriation with the single-chain antibody of acquisition to μ-CTX tool high-affinity.

In order to overcome the existing monoclonal antibody production expensive deficiency that wastes time and energy, the invention provides a kind of phage single chain antibody, its structure is as shown in Figure 1.This single-chain antibody (scFv) is with antibody heavy chain variable region (V with gene engineering method _H) and variable region of light chain (V _L) recombinant protein that is formed by connecting by one section connection peptides (Linker), as shown in Figure 2, anti-μ-CTX single-chain antibody variable region of heavy chain (V _H) size be 345 bp; As shown in Figure 3, anti-μ-CTX single-chain antibody variable region of light chain (V _L) size be 325 bp; As shown in Figure 4, the size of anti-μ-CTX single-chain antibody (scFv) is 750 bp; This single-chain antibody (scFv) has kept the affine activity of antigen and the functional antibody fragment of specific minimum of parental antibody, can be in bacterium very economical ground large-scale production, make the antibody producing that is used for immune detection become very easy, easy and economical, and then significantly reduce the expense of diagnostic reagent.The minimum detectable activity of this kit is 0.05 μ g/Kg.

Remarkable advantage of the present invention:

Can in Escherichia coli, efficiently express anti-μ of the present invention-CTX micromolecule phage single chain antibody, and large-scale production, whole process of production is simple, time saving and energy saving saves money.Can determine in the 1.5-2 h time whether sample is subjected to μ-CTX endotoxin contamination, and the amount of contained μ-CTX toxin, convenient to use, with low cost.

Description of drawings

Fig. 1 is a single-chain antibody structural representation of the present invention;

Fig. 2 is single-chain antibody heavy chain gene V of the present invention _HThe electrophoretogram of amplification;

Fig. 3 is single-chain antibody light chain gene V of the present invention _LThe electrophoretogram of amplification;

Fig. 4 is the electrophoretogram of single-chain antibody gene scFv amplification of the present invention;

Fig. 5 is single-chain antibody competitive ELISA detection curve figure of the present invention.

Embodiment

Be instantiation of the present invention below, further describe the present invention, but the present invention be not limited only to this.

Embodiment 1

Forming making by following reagent utilizes phage single chain antibody to detect the kit of μ-CTX:

(1) sample extracting solution: deionized water;

(2) standard reagent: be respectively and contain 0.01,0.1, the TRX-CTX toxin sample of 1.0,10 ng/mL; The standard reagent preparation method is as follows:

The structure of A recombinant plasmid pET32a-CTX

Annealing back is formed double-stranded CTXGene is inserted into same warp BamH I and EcoRIn the pET32a carrier of I double digestion, spend the night pET32a-with the connection of T4 dna ligase CTXPlasmid after the reorganization is identified through order-checking.

The abduction delivering of B TRX-CTX fusion

Adopt CaCl ₂Legal system is equipped with competence BL21With pET32a- CTXPlasmid is transformed into Escherichia coli BL21In.Picking transforms successful clone and is inoculated in the LB nutrient culture media that contains the ammonia benzyl, with BL21The empty negative contrast of bacterium and contain pET32a- CTXPlasmid BL2137 ℃ of joltings are spent the night together, and 2% ratio is inoculated in fresh LB nutrient culture media, and being cultured to bacterial density is OD ₆₀₀0.8.Take out 1 mL, all the other add IPTG to final concentration be 0.1 mM, in 37 ℃ of abduction delivering 3 h.

The soluble analysis and the purifying of C TRX-CTX fusion

The centrifugal 5min of reorganization bacterium 5000 r/min with behind the abduction delivering abandons supernatant, and the PBST[that precipitation is resuspended in the ice precooling contains 2 mM EDTA(pH 8.0) and 0.1% beta-mercaptoethanol] in, with the sonioation method cracking, pyrolysis product is placed and is spent the night.Lysate with centrifugal 15 min of 15000 r/min, is taken out supernatant (precipitation account for cumulative volume 2%～4%), and adds and the isopyknic PBS of supernatant in precipitation, resuspended.Respectively get 10 μ L precipitation and supernatant solution, add isopyknic sample-loading buffer respectively, boil 3～5min, carry out SDS-PAGE, to check the solubility of fusion.With supernatant part upper prop purifying, the fusion behind the purifying is identified through SDS-PAGE.Press the content that the Bradford method is measured fusion.

(3) enzyme-labelled antigen reagent: for containing the 0.01M PBS solution of TRX-CTX-HRP coupling protein, be stored in the glycerine of volume ratio 50% ,-20 ℃ of preservations, tiring is 5000; Dilute with PBS reagent during use; And the research that is applied to detect crosslinked of enzyme-labelled antigen preparation method of reagent thereof with reference to yellow rain China ink .HRP and toxin. University Of Agriculture and Forestry In Fujian's B.Sc paper, the preparation method is as follows:

Introduce sulfydryl on the A HRP

A) 1.3 mg HRP are dissolved in 520 μ L Solution 1(pH 7.5), add 7 μ L N-Succinimidyl S-Acetylthioacetate(SATA; 50 mg/mL are dissolved among the DMSO), reaction 30 min under the room temperature;

B) at Solution 1(pH 7.5) in the dialysis, change dislysate twice;

C) reclaim the dialysis product, add 100 μ L Solution 3(pH 7.5), reaction 2 h under the room temperature;

D) dislysate is changed in dialysis in phosphate buffer (contain 1mM EDTA, pH 7.0) twice.

B HRP and TRX-CTX are crosslinked

A) with 54 μ L TRX-CTX(5 mg/ml, be dissolved in DMSO) add in the 1.5 mL centrifuge tubes, add 8 μ L again N-[ p-maleimidophenyl] isocyanate(PMPI; 50 mg/ml are dissolved in DMSO), regulate 8.5,50 ℃ of reactions of pH value with triethylamine and spend the night;

B) with acid for adjusting pH value to 7.0;

C) mix 4 ℃ of reaction 4 h with product in (1);

D) add excessive halfcystine cessation reaction;

E) dialysis in phosphate buffer (PBS) gets cross-linking products ,-20 ℃ of preservations.

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

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

(6) chromogenic substrate: the 10 ml liquid that develops the color; 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.

Utilize the mentioned reagent box to detect μ-CTX toxin according to the following steps:

(1) sample preparation: in 1g sample (pulps such as abalone, hairtail, scallop, little yellow croaker), add the 4ml sample extracting solution, liquid nitrogen or mechanical lapping are pulverized, ultrasonic extraction 10 min, 5000rpm, centrifugal 10 min, supernatant is the sample extracting solution that contains sample;

The sample extracting solution that contains sample gets 100 μ l and 100 μ l enzyme-labelled antigen reagent are evenly mixed, A reagent;

Each 100 μ L of TRX-CTX toxin standard reagent 0.01,0.1,1.0,10 ng/mL of series concentration are evenly mixed with 100 μ L enzyme-labelled antigen reagent respectively, the B reagent of series concentration;

(2) reagent balance: kit is taken out from-20 ℃ of refrigerators, place more than 15 min, balance is to room temperature;

(3) aperture numbering: take out enzyme mark bar as required and be placed on the reaction plate support.Negative hole, No. 1 hole, 2-5 hole are TRX-CTX toxin standard control hole, and all the other holes are sample well; Enzyme mark bar has had the anti-μ of immobilization-CTX single-chain antibody in every hole; Described anti-μ-CTX single-chain antibody preparation process is: extract total RNA from the spleen of immune mouse, become cDNA through reverse transcription, through pcr amplification antibody heavy chain variable region V _HGene and variable region of light chain V _LGene, by one section connection peptides (Linker) V _HAnd V _LConnect into single-chain antibody (scFv), be cloned into then and make up phage antibody library on the carrier, again by biological elutriation to obtain single-chain antibody to μ-CTX tool high-affinity.

(4) washing: every hole adds 250 μ L cleansing solutions, and washing lotion must not be overflowed, and behind the placement 2min, removes cleansing solution, pats dry on thieving paper, repeats to wash plate once;

(5) application of sample: No. 1 the hole adds 50 μ L BSA reagent, and the 2-5 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 250 μ L cleansing solutions, and washing lotion must not be overflowed, place 2 min after, remove cleansing solution, on thieving paper, pat dry, repeat to wash plate 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) stop and Instrument measuring: every hole adds stop buffer 50 μ L respectively, then with the light absorption value A of microplate reader in each hole of mensuration, 450 nm places, is horizontal ordinate with the series concentration of B reagent, is ordinate drawing standard curve with its corresponding light absorption value A.

To resist μ-CTX single-chain antibody and the anti-E-tag antibodies that is fixed on the enzyme mark bar by the E-tag on anti-μ-CTX single-chain antibody, form insolubilized antibody; The enzyme-labelled antigen reagent that adds testing sample and TRX-CTX toxin, antigen in the sample and enzyme-labelled antigen competition combine with insolubilized antibody, antigenic content is high more in the testing sample, what then combine with insolubilized antibody is many more, the chance that makes enzyme-labelled antigen combine with insolubilized antibody is just few more, even the combination of having no chance, adding like this and just do not develop the color or develop the color behind the substrate very shallow, the dark person that develops the color is negative.As shown in table 1, along with the progressively rising of the concentration of the normal concentration TRX-CTX toxin that is added, corresponding light absorption value OD value gradually reduces.The TRX-CTX toxin normal concentration curve of drawing according to this data as shown in Figure 5, therefore the concentration of OD450 nm place's light absorption value and testing sample is inverse relation, but according to the OD450 nm place light absorption value of the testing sample concentration with regard to μ-CTX of containing in the judgement sample.

According to μ in the computing formula sample-CTX content (ng/g)=C*V/m, the concentration of μ-CTX in the C-sample wherein, ng/mL; The volume of V-sample extracting solution, mL; The m-sample quality, g; The content of μ-CTX in the calculation sample.When light absorption value is 0.035, μ-CTX content: 0.005*4/1=0.02 ng/g.

Table 1 The competitive ELISA of single-chain antibody of the present invention detects

Annotate: when concentration is 0ng/mL, be enzyme-labelled antigen reagent and isopyknic PBS, other sample wells also add isopyknic TRX-CTX toxin standard solution (concentration is respectively 0.01,0.1,1.0,10 ng/mL) respectively except enzyme-labelled antigen reagent.

＜110〉University Of Agriculture and Forestry In Fujian

＜120〉utilize phage single chain antibody to detect the kit and the method for mu-conotoxin

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＜213〉the BABA/c mouse ( Mus musculus)

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Met?Ala?Gln?Val?Gln?Leu?Gln?Gln?Ser?Gly?Ala?Glu?Leu?Ala?Arg?Pro

1 5 10 15

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

20 25 30

Asn?Tyr?Thr?Met?His?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu

35 40 45

Trp?Ile?Gly?Tyr?Ile?Asn?Pro?Ser?Ser?Asp?Tyr?Thr?Asn?Tyr?Asn?Gln

50 55 60

Lys?Phe?Lys?Asp?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Glu?Ser?Ser?Ser?Thr

65 70 75 80

Ala?Phe?Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr

85 90 95

Tyr?Cys?Ala?Arg?Phe?Pro?His?Leu?Glu?Asp?Tyr?Trp?Tyr?Phe?Asp?Val

100 105 110

Trp?Gly?Gln?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser

115 120 125

Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Glu?Leu?Thr?Gln

130 135 140

Ser?Pro?Thr?Ile?Met?Ser?Ala?Ser?Leu?Gly?Glu?Lys?Val?Thr?Met?Thr

145 150 155 160

Cys?Arg?Ala?Ser?Ser?Ser?Val?Ser?Ser?Ser?Tyr?Leu?His?Trp?Tyr?Gln

165 170 175

Gln?Lys?Ser?Gly?Ala?Ser?Pro?Lys?Leu?Trp?Ile?Tyr?Ser?Thr?Ser?Asn

180 185 190

Leu?Ala?Ser?Gly?Val?Pro?Ala?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr

195 200 205

Ser?Tyr?Ser?Leu?Thr?Ile?Ser?Ser?Met?Glu?Ala?Glu?Asp?Ala?Ala?Thr

210 215 220

Tyr?Tyr?Cys?His?Gln?Tyr?His?Arg?Ser?Pro?Arg?Thr?Phe?Gly?Gly?Gly

225 230 235 240

Thr?Lys?Leu?Glu?Ile?Lys?Arg

245

Claims (3)

1. kit that utilizes phage single chain antibody to detect mu-conotoxin, it is characterized in that: its reagent comprises:

(1) sample extracting solution: deionized water;

(2) standard reagent: TRX-CTX toxin;

(3) enzyme-labelled antigen reagent: the PBS solution for 0.01M, the pH that contains the TRX-CTX-HRP coupling protein is 7.4 is stored in the 50%(volume ratio) glycerine in ,-20 ℃ of preservations, tiring is 5000;

(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: the 10 ml liquid that develops the color; Formula constituent: 250 μ L, 20 mg/mL DAB, 40 μ L, 40 mg/mL NiCl, 1.0 mol/L of 9.75 ml, the Tris-HCl of pH 6.8 ,-20 ℃ of preservations; Add 7 μ L 30%(volume ratios during use) H ₂O ₂

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

2. method of utilizing the described kit of claim 1 to detect mu-conotoxin is characterized in that: may further comprise the steps successively:

(1) sample preparation: add the 4ml sample extracting solution in the 1g sample, liquid nitrogen or mechanical lapping are pulverized, ultrasonic extraction 10 min, and centrifugal 10 min of 5000rpm, supernatant is the sample extracting solution that contains sample;

The sample extracting solution that contains sample gets 100 μ l and 100 μ l enzyme-labelled antigen reagent are evenly mixed, A reagent;

Each 100 μ L of TRX-CTX toxin standard reagent 0.01,0.1,1.0,10 ng/mL of series concentration are evenly mixed with 100 μ L enzyme-labelled antigen reagent respectively, the B reagent of series concentration;

(2) reagent balance: kit is taken out from-20 ℃ of refrigerators, place more than 15 min, balance is to room temperature;

(3) aperture numbering: take out enzyme mark bar and be placed on the reaction plate support, negative hole, No. 1 hole of mark, the 2-5 hole is TRX-CTX toxin standard control hole, all the other holes are sample well; The anti-μ of immobilization-CTX single-chain antibody in the every hole of enzyme mark bar;

(4) washing: every hole adds 250 μ L cleansing solutions, and cleansing solution must not overflow, and behind the placement 2min, discards cleansing solution, and pats dry on thieving paper, repeats to wash plate 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, adds A reagent 50 μ L in each sample well, and light shaking makes reagent mixing in each hole;

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

(7) washing: take out reaction plate, remove step (5) and add reagent, pat dry, every hole adds 250 μ L cleansing solutions, and cleansing solution must not overflow, place 2 min after, discard cleansing solution, on thieving paper, pat dry, repeat to wash plate 4 times;

(8) colour developing: every hole adds chromogenic substrate 100 μ L, shakes up, and ELISA 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, use the light absorption value A of microplate reader material in 450 nm places measure each hole then, 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 μ-CTX in the sample according to typical curve, according to computing formula μ-CTX content (ng/g)=C*V/m, wherein C is the concentration of μ-CTX, ng/mL; The volume of V-sample extracting solution, mL; The m-sample quality, g; The content of μ-CTX in the calculation sample.

3. method according to claim 2 is characterized in that: the amino acid sequence of described anti-μ-CTX single-chain antibody is shown in SEQ ID No.1.

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