CN111763263A

CN111763263A - Preparation method and detection kit of clostridium difficile antigen and polyclonal antibody

Info

Publication number: CN111763263A
Application number: CN202010655672.1A
Authority: CN
Inventors: 徐广贤; 潘俊斐; 张爱君; 楚元奎; 王红霞; 蒋丹; 屈煜良; 李光琪; 李燕宁
Original assignee: Ningxia Medical University
Current assignee: Ningxia Medical University
Priority date: 2020-07-09
Filing date: 2020-07-09
Publication date: 2020-10-13

Abstract

The invention provides a preparation method and a detection kit of clostridium difficile antigen and polyclonal antibody, relating to the technical field of immunodetection. The invention uses antigenic epitopes TcdB1 and TcdB2 which are exclusively belong to clostridium difficile as antigenic determinants, and immunizes experimental rabbits after single antigenic epitope is connected with keyhole limpet hemocyanin to obtain the polyclonal antibody with high purity, high titer and high specificity. The invention provides a double-antibody sandwich ELISA kit based on the polyclonal antibody, which is used for detecting clostridium difficile, and the established detection method is more scientific and rigorous; and simultaneously, the requirements of the ELISA reagent on important parameters such as sensitivity, specificity, repeatability and the like are met.

Description

Preparation method and detection kit of clostridium difficile antigen and polyclonal antibody

Technical Field

The invention belongs to the technical field of immunodetection, and particularly relates to a preparation method and a detection kit of clostridium difficile antigen and polyclonal antibody.

Background

Clostridium Difficile (CD) is an obligate anaerobe with gram-positive bacillus megaterium, flagella and spores, and is difficult to separate and culture. Difficile infection is mostly asymptomatic carrier, clinical Clostridium Difficile Infection (CDI) can cause mild diarrhea to blood diarrhea, and severely infected patients can cause pseudomembranous colitis, toxic megacolon, intestinal necrosis and even life threatening. Clostridium difficile produces two major toxins: enterotoxin (TcdA) and cytotoxin (TcdB). The enterotoxin can chemotaxis the neutrophil to infiltrate the intestinal wall of the ileum, and release cell factors, so that the intestinal tract is largely dehydrated and hemorrhagic necroses. The cytotoxin can depolymerize actin, damage cytoskeleton, cause local intestinal wall cell necrosis, and directly damage intestinal wall. In recent years, TcdB has been found to have the function of enterotoxin and can cause diseases independently.

The laboratory detection of clostridium difficile is an important basis for clinical diagnosis of CDI, but at present, standard clostridium difficile toxin detection schemes are lacked in China, and related researches are few, so that the clinical data of clostridium difficile are deficient.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing clostridium difficile antigen and polyclonal antibody and a detection kit, so as to establish a complete method for detecting clostridium difficile, which is more scientific and precise and has the advantages of high detection sensitivity, good specificity and repeatability, etc.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a clostridium difficile antigen which comprises an antigen formed by respectively connecting antigen epitopes TcdB1 and TcdB2 with a carrier protein keyhole limpet hemocyanin KLH; the amino acid sequence of the TcdB1 is shown in SEQ ID NO. 1; the amino acid sequence of TcdB2 is shown in SEQ ID NO. 2.

Preferably, the method further comprises adding 1 cysteine modification to the N-terminal of the amino acid sequences of TcdB1 and TcdB2 respectively before the connection.

The invention also provides a preparation method of the polyclonal antibody of clostridium difficile, which comprises the following steps: immunizing a New Zealand rabbit every two weeks, and separating and purifying protein from blood 2 weeks after the 5 th immunization to obtain polyclonal antibody Anti-TcdB1 or Anti-TcdB 2;

during first immunization, the antigen is respectively and independently mixed with Freund's complete adjuvant in equal volume and uniformly mixed, a first immune antigen is obtained after emulsification, and the rabbit is immunized by the first immune antigen;

during the second to fourth immunization, the antigen is respectively and independently mixed with Freund incomplete adjuvant in equal volume and is emulsified to obtain a second immune antigen, and the rabbit is immunized by the second immune antigen;

in the fifth immunization, the rabbits were immunized with the antigen.

Preferably, in the first immunization, 0.1mg of the first immune antigen is injected in 5 parts of armpit, neck and back of the rabbit respectively;

at the time of the second to fourth immunizations, 0.1mg of the second immunizing antigen was injected at the same site as the first immunization, respectively;

at the fifth immunization, 0.1mg of the antigen was injected intravenously via the ear margin.

Preferably, the method also comprises collecting serum 1 week after each immunization, measuring the antibody titer of the serum by an indirect ELISA method and verifying the specificity of the antibody by Western-blot.

Preferably, the separation and purification comprises separating serum from heart blood at 4000r/min, filtering with a 0.45 μm filter, and separating and purifying with a proteinA affinity chromatography column.

The invention also provides a human clostridium difficile TcdB double-antibody sandwich ELISA kit, which comprises the following components: the antibody comprises a primary antibody and a secondary antibody, wherein the primary antibody is Anti-TcdB1 prepared by the preparation method, and the secondary antibody is HRP-labeled Anti-TcdB2 prepared by the preparation method.

Preferably, before the kit is used for detecting human clostridium difficile, the method comprises the following steps of pretreating the 96-well plate, wherein the pretreatment comprises the following steps: diluting Anti-TcdB1 to 0.5 mu g/ml by using the coating solution, adding 100 mu l/hole into a 96-well plate, coating for 2 hours at 37 ℃, and then coating for 8-12 hours at 4 ℃; washing the plate with the PBST buffer solution three times after coating; adding 300 mul of sealing liquid into each hole of the coated plate, sealing for 2 hours at 37 ℃, and then transferring to 4 ℃ for continuous sealing for 8-12 hours; after blocking, plates were washed three times with the PBST buffer and dried.

Preferably, the coating solution comprises carbonate buffer with a molar concentration of 0.05M and a pH value of 9.6.

Preferably, the antibody diluent is a PBST buffer solution containing BSA, and the mass-to-volume ratio of the BSA to the PBST is 0.2 g: 100 mL.

The invention provides a clostridium difficile antigen, which is characterized in that epitope TcdB1 and TcdB2 which are exclusively belong to clostridium difficile are used as antigenic determinants, and a single epitope is used for immunizing an experimental rabbit after being connected with keyhole limpet hemocyanin to obtain a polyclonal antibody with high purity, high cost effectiveness and high specificity. The prepared antibody theoretically has the high specificity of the monoclonal antibody and the high affinity of the polyclonal antibody, can be used for tests such as precipitation, agglutination and the like, and can also be used for detecting denatured proteins. Compared with the monoclonal antibody, the preparation process is more economical and simple, and has the advantages of more stability and convenience in storage.

The invention also provides a double-antibody sandwich ELISA kit based on the polyclonal antibody, which is used for detecting clostridium difficile, and the established detection method is more scientific and rigorous; and simultaneously, the requirements of the ELISA reagent on important parameters such as sensitivity, specificity, repeatability and the like are met.

According to the verification of the epitope and the constructed polyclonal antibody, the result shows that after the fourth immunization, the antibody titers of Anti-TcdB1 and Anti-TcdB2 are respectively greater than 1:128000 and 1:512000, and the serum antibody titers are respectively greater than 1:256000 and 1:1024000 one week after the fifth immunization. The Anti-TcdB1 and the Anti-TcdB2 are respectively used as primary antibodies to carry out Western-blot, a reaction band of an antibody and a supernatant of the cultured toxigenic clostridium difficile is located at 170KDa, and the non-toxigenic clostridium difficile used as a negative control does not have the reaction band, so that the two prepared antibodies have higher specificity. When the clostridium difficile is detected by the double-antibody sandwich ELISA constructed based on the polyclonal antibody, the lowest detection limit is 15.6 ng/ml; detecting clinical common bacteria such as clostridium difficile, PBS, staphylococcus aureus, streptococcus pneumoniae and the like according to an established ELISA double-antibody sandwich method, wherein the A450 value of the clostridium difficile is 0.413, and the clostridium difficile is judged to be positive; the rest is less than the negative critical value 0.1776, the judgment is negative, no cross reaction is found, and the detection method has strong specificity; performing in-batch and inter-batch repeatability verification by using the established double-antibody sandwich ELISA method, wherein the variation coefficient of the in-batch positive detection is 3.62-5.58%, and the variation coefficient of the negative detection is 5.65-8.04%; the variation coefficient of the positive detection among batches is 5.19-7.00%, and the variation coefficient of the negative detection among batches is 6.39-8.21%. The repeatability in batches and among batches is good, and the established double-antibody sandwich ELISA method has certain stability; the detection is carried out once a week according to the established ELISA detection method, and OD is read by a microplate reader₄₅₀The value is that after 6 cycles of detection, although the positive result is reduced, the result is still positive; the negative result is kept stable; the detection kit is proved to have better stability. CDB3 recombinant proteins at final concentrations of 50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml and 250ng/ml were tested in accordance with the established ELIThe SA detection method is used for detection, the recovery rate is 91.17-105.33%, the coefficient of variation of each concentration is 2.35-5.44%, and the established ELISA detection method is high in accuracy.

Drawings

FIG. 1 shows the titers of Anti-TcdB1 and Anti-TcdB 2;

FIG. 2 shows the WB test results for Anti-TcdB1 (left) and Anti-TcdB2 (right), where M: the standard molecular weight of the protein is 1: TcdB1 antibody and toxigenic bacteria reaction band, 2: non-toxigenic bacteria negative control 3: TcdB2 antibody and toxigenic bacteria reaction band, and 4: non-toxigenic bacteria negative control;

FIG. 3 shows the sensitivity and linear regression results;

FIG. 4 shows the results of a specificity test;

FIG. 5 shows the results of a repetitive experiment;

FIG. 6 shows the stability results;

FIG. 7 shows the spatial positions of TcdB1 and TcdB2 in the TcdB protein.

Detailed Description

The screening process of the epitope TcdB1(SEQ ID NO. 1: DGSKYYFDEDTAE) and TcdB2(SEQ ID NO. 2: HQNTLDENFEGESIN) preferably comprises the following steps: the method comprises the steps of obtaining an amino acid sequence CAJ67492.1 of TcdB from NCBI RefSeq dataset, analyzing the sequence by using ABCPred, Bepipred and DNAstar database software, analyzing the hydrophilicity, variability, surface exposure possibility and the like of each peptide segment, selecting the length of 12-20 amino acids as a target antigenic determinant, respectively naming the antigenic determinant as TcdB1 and TcdB2, analyzing the homology of two polypeptides by using a NCBI database protein Blast tool, determining that the polypeptides are only from the TcdB of clostridium difficile, establishing a 3D structure of a toxin B protein by using SWISS-model, and observing the exposure condition of the antigenic epitope. The invention preferably adds 1 cysteine to the N end of the amino acid sequence for modification, and then connects the modified naked peptide with the carrier protein Keyhole Limpet Hemocyanin (KLH) through carboxyl group by glutaraldehyde method to synthesize antigens KLH-TcdB1 and KLH-TcdB 2.

in the fifth immunization, the rabbits were immunized with the antigen.

The invention preferably also comprises blood collection of the ear vein of the New Zealand rabbit as negative serum before the immunization.

In the invention, when the first immunization is carried out, 0.1mg of the first immune antigen is preferably injected into 5 parts of the left oxter, the right oxter, the neck and the back of the rabbit respectively; in the second to fourth immunizations, 0.1mg of the second immunizing antigen is preferably injected separately at the same site as the first immunization; in the fifth immunization, 0.1mg of the antigen of

claim

1 or 2 is preferably administered by intravenous injection into the ear.

The invention preferably also comprises collecting serum 1 week after each immunization, measuring the antibody titer of the serum by an indirect ELISA method, and verifying the specificity of the antibody by Western-blot.

The invention separates and purifies protein from blood after 2 weeks of 5 th immunization, and the blood is preferably heart blood; the separation and purification preferably comprises separating serum from heart blood at 4000r/min, filtering with 0.45 μm filter, and separating and purifying with proteinA affinity chromatography column. After obtaining the polyclonal antibodies Anti-TcdB1 and Anti-TcdB2, the invention preferably further comprises dialysis renaturation desalination, and protein content is quantified by using a BCA kit and stored at-80 ℃.

The invention also provides a human clostridium difficile TcdB double-antibody sandwich ELISA kit, which comprises the following components: the kit comprises a 96-well plate, PBS buffer solution, PBST buffer solution, coating solution, blocking solution, antibody diluent developing solution, stopping solution and antibodies, wherein the antibodies comprise primary antibodies and secondary antibodies, the primary antibodies are Anti-TcdB1, and the secondary antibodies are HRP-labeled Anti-TcdB 2.

In the present invention, before the kit is used for detecting human clostridium difficile, the method preferably comprises the step of pretreating the 96-well plate, wherein the pretreatment comprises the following steps: diluting Anti-TcdB1 to 0.5 mu g/ml by using the coating solution, adding 100 mu l/hole into a 96-well plate, coating for 2 hours at 37 ℃, and then coating for 8-12 hours at 4 ℃; washing the plate with the PBST buffer solution three times after coating; adding 300 mul of sealing liquid into each hole of the coated plate, sealing for 2 hours at 37 ℃, and then transferring to 4 ℃ for continuous sealing for 8-12 hours; after blocking, plates were washed three times with the PBST buffer and dried. The coating solution of the present invention preferably comprises carbonate buffer solution with a molar concentration of 0.05M and a pH value of 9.6. The antibody diluent of the invention is preferably PBST buffer solution containing BSA, and the mass-volume ratio of the BSA to the PBST is preferably 0.2 g: 100 mL.

In the present invention, the sources of the components in the kit are not particularly limited, and the self-configuration preferably includes:

PBS buffer: dissolving PBS phosphate dry powder by using ultrapure water, and fixing the volume to 2L to obtain PBS buffer solution with the pH value of 7.2-7.4;

PBST: adding 500 μ l Tween-20 into 1000ml PBS solution to obtain PBST with final concentration of 0.5%;

coating liquid: weighing Na₂CO₃1.59g and NaHCO₃2.93g, dissolving in ultrapure water, adjusting pH to 9.6 to obtain 0.05M carbonate buffer solution with pH of 9.6, and storing at 4 deg.C under sealed condition;

sealing liquid: adding 5g of skimmed milk into 100ml of PBST, and fully dissolving;

antibody dilution: 0.2g BSA was added to 100ml PBST and dissolved well.

The invention also provides an ELISA detection method of human clostridium difficile TcdB, which specifically comprises the following steps: detecting: taking out the pretreated 96-well plate, adding toxigenic Clostridium difficile culture solution into 100 μ l/well, adding PBS buffer solution into negative control, adding no sample into blank well, and reacting in 37 deg.C thermostat for 60 min;

(II) washing: washing the plate three times with 1 XPBST for standby;

(III) secondary antibody: taking out HRP-labeled Anti-TcdB2(HRP-Anti-TcdB2), diluting with antibody diluent 20000 times, adding 100 μ l/hole, and reacting in 37 deg.C incubator for 30 min;

(IV) washing: the plate was washed five times with 1 XPBST for use.

(V) color development: adding the TMB working solution which is prepared freshly into 50 mul/hole, developing for 5min in a dark place at room temperature, and then adding the stop solution into 50 mul/hole rapidly to stop the reaction. OD 450nm was measured with a microplate reader.

(VI) interpretation of results: OD₄₅₀Values above 0.1872 are positive, values below 0.1776 are negative, and values between 0.1776 and 0.1872 are suspect values.

In the invention, recombinant TcdB (TcdB protein expressed by prokaryotes is utilized by inserting TcdB gene into PET30a plasmid) is diluted by PBS (phosphate buffer solution) in a double-rate mode, and then ELISA (enzyme-linked immunosorbent assay) is carried out by a double-antibody sandwich method, wherein a curve is drawn by taking the logarithm value of the concentration of the recombinant TcdB as the horizontal coordinate and the A450 value as the vertical coordinate, and the curve equation y is 0.0248ln (x) +0.1826, R²When TcdB antigen was diluted to 15.6ng/ml 0.9903, the ELISA result was judged positive. Therefore, the minimum detection limit of the method is 15.6 ng/ml.

The methods for producing clostridium difficile antigens and polyclonal antibodies and the detection kit provided by the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

1. Screening of epitope and preparation of polyclonal antibody

Acquiring an amino acid sequence CAJ67492.1 of TcdB from NCBI RefSeq dataset, analyzing the sequence by using ABCPred, Bepipred and DNAstar database software, analyzing the hydrophilicity, the variability, the surface exposure possibility and the like of each peptide segment, wherein the peptide segment with the ABCPred prediction result score of more than 0.9 is screened out; peptide fragments with an individual amino acid score greater than 0.6 were also selected from the results of the Bepipred prediction, and DNAstar uses predictions of protein secondary structure, which suggest that turns and random coil structures are suitable as epitopes, and hydrophilicity, flexibility and surface accessibility are also indispensable positive influencing factors. Comprehensively analyzing the three groups of data, selecting peptide fragments which appear in the three groups of prediction results as candidate sequences, selecting two peptide fragments as target antigenic determinants which are named as TcdB1 and TcdB2 respectively, analyzing the homology of the two polypeptides by a protein Blast tool of an NCBI database, determining that the peptides are only from the TcdB of clostridium difficile, establishing a 3D structure of toxin B protein by a SWISS-module (figure 7), and observing the exposure condition of the antigenic epitope. Synthesizing naked peptide, adding 1 cysteine to N end of amino acid sequence for modification, connecting with carrier protein Keyhole Limpet Hemocyanin (KLH), and synthesizing the composite proteins of naked peptide TcdB1, TcdB2, KLH-TcdB1 and KLH-TcdB 2.

Before immunization, 2 New Zealand white rabbits were taken, and blood was collected from the marginal vein before immunization as negative serum. Mixing TcdB antigens (KLH-TcdB1 and KLH-TcdB2) with Freund's complete adjuvant 1:1 in equal volume, emulsifying, and injecting 0.1mg antigen into 5 parts of rabbit left oxter, right oxter, neck and back. After two weeks, the antigen was mixed with the same volume of Freund's incomplete adjuvant in equal volume, and the immunization dose and site were the same as above. The 2 nd immunization was repeated 2 times, and 0.1mg of antigen was given by intravenous injection into the ear margin at the 5 th immunization. Serum was collected 1 week after each immunization, and the antibody titer of the serum was determined by indirect ELISA and the specificity of the antibody was verified by Western-blot assay.

Collecting blood from heart 2 weeks after 5 th immunization, separating serum at 4000r/min, filtering with 0.45um filter, separating and purifying with protein A affinity chromatography column, purifying protein, naming Anti-TcdB1 and Anti-TcdB2, dialyzing, desalting, quantifying protein content with BCA kit, and storing at-80 deg.C.

After the fourth immunization, the antibody titers are shown in FIG. 1, wherein A indicates that the titers of Anti-TcdB1 and Anti-TcdB2 are greater than 1:128000 and 1:512000, respectively; b represents the serum antibody titer one week after the fifth immunization, Anti-TcdB1 and Anti-TcdB2 were greater than 1:256000 and 1:1024000, respectively.

Western-blot results using Anti-TcdB1 and Anti-TcdB2 as primary antibodies are shown in FIG. 2, and the reaction band of the antibody and the supernatant of the cultured toxigenic Clostridium difficile is 170KDa, while the reaction band of the non-toxigenic Clostridium difficile as a negative control is absent, so that the 2 prepared antibodies have high specificity.

2. Reagent configuration

PBS: dissolving PBS phosphate dry powder with ultrapure water, and making the volume constant to 2L, namely PBS with pH7.2-7.4.

PBST: add 500. mu.l of Tween-20 to 1000ml of PBS solution, which is the final concentration of 0.5% PBST.

Coating liquid: weighing Na₂CO₃1.59g and NaHCO₃2.93g, dissolved in ultrapure water, and adjusted to pH9.6 to prepare 0.05M carbonate buffer solution of pH9.6, which was stored under sealed conditions at 4 ℃.

Sealing liquid: 5g skim milk was added to 100ml PBST and dissolved well.

Antibody dilution: 0.2g BSA was added to 100ml PBST and dissolved well.

3. Preparation of human clostridium difficile TcdB double-antibody sandwich ELISA kit

Coating: Anti-TcdB1 was diluted to 0.5. mu.g/ml with ELISA coating solution, added to a 96-well plate at 100. mu.l/well, coated for 2 hours at 37 ℃ and then coated overnight at 4 ℃.

Washing: plates were washed three times with 1 × PBST for use.

And (3) sealing: blocking solution was added at 300. mu.l/well, and after blocking for 2 hours at 37 ℃ the cells were transferred to 4 ℃ and blocking was continued overnight.

Washing: the plates were washed three times with 1 XPBST and dried before being placed in sealed bags containing a desiccator.

4. Detection of

And (3) detection: the sealed bag is opened, the 96-well plate is taken out, bacterial culture supernatant is added into 100 mul/well, PBS buffer solution is added into negative control, no sample is added into blank wells, and the reaction is carried out in a constant temperature box at 37 ℃ for 60min.

Washing: plates were washed three times with 1 × PBST for use.

Secondary antibody: the HRP-Anti-TcdB2 was removed, diluted 20000 times with antibody diluent, added at 100. mu.l/well and placed in a 37 ℃ incubator for 30 min.

Washing: the plate was washed five times with 1 XPBST for use.

Color development: adding the TMB working solution which is prepared freshly into 50 mul/hole, developing for 5min in a dark place at room temperature, and then adding the stop solution into 50 mul/hole rapidly to stop the reaction. OD determination with microplate reader₄₅₀The value is obtained.

And (4) interpretation of results: OD₄₅₀Values above 0.1872 are positive, values below 0.1776 are negative, and values between 0.1776 and 0.1872 are suspect values.

Since the culture solution of Clostridium difficile contains a large amount of non-toxin B protein and is difficult to quantify toxin B protein, recombinant toxin B protein is selected to be used for sensitivity experiment, recombinant TcdB is diluted by PBS, two-antibody sandwich ELISA is carried out, the logarithm value of the concentration of the recombinant TcdB is used as the abscissa, the A450 value is used as the ordinate, a curve is drawn, and the curve equation y of 0.0248ln (x) +0.1826, R shown in figure 3 is obtained²When TcdB antigen was diluted to 15.6ng/ml 0.9903, the ELISA result was judged positive. Therefore, the minimum detection limit of the method is 15.6 ng/ml.

Detecting common clinical bacteria such as clostridium difficile, PBS, staphylococcus aureus, streptococcus pneumoniae and the like according to an established ELISA double-antibody sandwich method, wherein the result is shown in figure 4, the A450 value of the clostridium difficile is 0.413, and the clostridium difficile is judged to be positive; and the rest is less than the negative critical value 0.1776, the judgment is negative, and no cross reaction is found, which indicates that the detection method has strong specificity.

The established double-antibody sandwich ELISA method is used for carrying out batch and batch repeatability verification, and the result is shown in figure 5, wherein the variation coefficient of the intra-batch positive detection is 3.62-5.58%, and the variation coefficient of the negative detection is 5.65-8.04%; the variation coefficient of the positive detection among batches is 5.19-7.00%, and the variation coefficient of the negative detection among batches is 6.39-8.21%. The repeatability in batches and among batches is good, and the established double-antibody sandwich ELISA method has certain stability.

Weekly in accordance with established ELISA detection methodPerforming a detection, and reading OD with microplate reader₄₅₀The value is obtained. The results are shown in FIG. 6, and the results are still positive although the positive results are reduced after 6 cycles of detection; the negative results remained stable. The detection kit is proved to have better stability.

CDB3 recombinant proteins were detected at final concentrations of 50ng/ml, 100ng/ml, 150ng/ml, 200ng/ml and 250ng/ml by established ELISA detection methods. The recovery rate is calculated according to a formula (the recovery rate is equal to the detection concentration/the actual concentration multiplied by 100%), and the result is shown in table 1, the recovery rate is 91.17-105.33%, the coefficient of variation of each concentration is 2.35-5.44%, and the ELISA detection method established by the invention is high in accuracy.

TABLE 1 recovery test results

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Ningxia medical university

<120> preparation methods and detection kit of clostridium difficile antigen and polyclonal antibody

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>13

<212>PRT

<213>Clostridium difficile

<400>1

Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu

1 5 10

<210>2

<211>15

<212>PRT

<213>Clostridium difficile

<400>2

His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn

1 5 10 15

Claims

1. A clostridium difficile antigen, which comprises an antigen formed by connecting antigen epitopes TcdB1 and TcdB2 with a carrier protein keyhole limpet hemocyanin KLH respectively; the amino acid sequence of the TcdB1 is shown in SEQ ID NO. 1; the amino acid sequence of TcdB2 is shown in SEQ ID NO. 2.

2. The antigen of claim 1, further comprising, prior to said ligation, the addition of 1 cysteine modification to the N-terminus of the amino acid sequence of TcdB1 and TcdB2, respectively.

3. A method for preparing a polyclonal antibody of Clostridium difficile, comprising the steps of: immunizing a New Zealand rabbit every two weeks, and separating and purifying protein from blood 2 weeks after the 5 th immunization to obtain polyclonal antibody Anti-TcdB1 or Anti-TcdB 2;

in the first immunization, the antigen of claim 1 or 2 is respectively and independently mixed with Freund's complete adjuvant in equal volume and is emulsified to obtain a first immune antigen, and rabbits are immunized by the first immune antigen;

during the second to fourth immunization, the antigen of claim 1 or 2 is respectively and independently mixed with Freund's incomplete adjuvant in equal volume, a second immune antigen is obtained after emulsification, and the rabbit is immunized by the second immune antigen;

in a fifth immunization, the rabbit is immunized with the antigen of claim 1 or 2.

4. The method according to claim 3, wherein the first immunizing antigen is injected at 5 points of the armpit, the neck and the back of the rabbit respectively at the time of the first immunization;

at the time of the fifth immunization, 0.1mg of the antigen of claim 1 or 2 was administered by intravenous injection into the ear margin.

5. The method of claim 3 or 4, further comprising collecting serum 1 week after each immunization, measuring the antibody titer of the serum by indirect ELISA and verifying the specificity of the antibody by Western-blot.

6. The preparation method according to claim 3, wherein the separation and purification comprises separating serum from heart blood at 4000r/min, filtering the serum with a 0.45 μm filter, and separating and purifying the serum with a proteinA affinity chromatography column.

7. A human Clostridium difficile TcdB double-antibody sandwich ELISA kit is characterized by comprising the following components: the antibody comprises a primary antibody and a secondary antibody, wherein the primary antibody is Anti-TcdB1 prepared by the preparation method of any one of claims 3-6, and the secondary antibody is HRP-labeled Anti-TcdB2 prepared by the preparation method of any one of claims 3-6.

8. The kit of claim 7, wherein the kit is used for pre-detection of human clostridium difficile, and comprises a pretreatment of the 96-well plate, wherein the pretreatment comprises: diluting Anti-TcdB1 to 0.5 mu g/ml by using the coating solution, adding 100 mu l/hole into a 96-well plate, coating for 2 hours at 37 ℃, and then coating for 8-12 hours at 4 ℃; washing the plate with the PBST buffer solution three times after coating; adding 300 mul of sealing liquid into each hole of the coated plate, sealing for 2 hours at 37 ℃, and then transferring to 4 ℃ for continuous sealing for 8-12 hours; after blocking, plates were washed three times with the PBST buffer and dried.

9. The kit of claim 8, wherein the coating solution comprises a carbonate buffer at a molarity of 0.05M and a pH of 9.6.

10. The kit of claim 7, wherein the antibody diluent is a PBST buffer containing BSA, and the mass-to-volume ratio of BSA to PBST is 0.2 g: 100 mL.