CN111808824A

CN111808824A - ELISA detection method for alpha toxin of clostridium novyi and application

Info

Publication number: CN111808824A
Application number: CN202010701277.2A
Authority: CN
Inventors: 刘莹; 杜吉革; 陈小云; 印春生; 李倩琳; 王磊; 杨承槐
Original assignee: China Institute of Veterinary Drug Control
Current assignee: China Institute of Veterinary Drug Control
Priority date: 2020-07-20
Filing date: 2020-07-20
Publication date: 2020-10-23
Anticipated expiration: 2040-07-20
Also published as: CN111808824B

Abstract

The invention relates to an ELISA detection reagent for alpha toxin of Novietum, which utilizes inactivated natural toxin of Novietum as immunogen immune mice, and adopts non-toxic 3 non-toxic antigen peptides containing the N end of the alpha toxin of Novietum and recombinant fusion protein (rTnc alpha) at the C end obtained by a prokaryotic expression system to screen an alpha toxin monoclonal antibody hybridoma cell strain, thereby maximally maintaining the natural conformation of the Tnc alpha in the aspect of immunogen and greatly avoiding the complicated purification step and biological potential safety hazard of the natural Tnc alpha; the ELISA detection method of the alpha toxin of the clostridium novyi constructed by the monoclonal antibody obtained by screening and the polyclonal antibody obtained by the recombinant fusion protein (rTnc alpha) provides support for the diagnosis of the clostridium novyi disease and the antigen quantitative method of the vaccine related to the clostridium novyi.

Description

ELISA detection method for alpha toxin of clostridium novyi and application

Technical Field

The invention relates to the field of biological detection, in particular to detection of bacterial toxins.

Background

The clostridium novyi is an anaerobic bacterium which can cause diseases of human beings and various animals, and has great harm to human health and livestock and poultry breeding, and the main pathogenic factor of the clostridium is exotoxin secreted by thalli, mainly comprising alpha, beta, gamma and exotoxin. According to the production of the four toxins, the clostridium novyi can be divided into three types of A, B and C. Among them, types A and B are the main types causing the disease of cattle and sheep, and type C is avirulent (Eeckhaut V, Boyen F, Pasmans F, et al. Clostridium novyitytype B as a practical agent of bovine mean cyclone [ J ]. Anaerobe,2012,18(3): 286) 288.). Because the disease of the clostridium novyi is acute, the patient often dies without treatment in time, and the mortality rate is higher. Thus, early diagnosis of the disease is particularly important, and the C.novyi alpha toxin (Tnc. alpha.) directly related to the disease is an important indicator for diagnosing the disease (Aquino P L, Fonseca F S, Mozzer O D, et al.

Currently, the infection of clostridium novyi can be judged by combining clinical symptoms, pathological anatomy, smear microscopy and neutralization tests, but the steps are complicated and are not easy to operate. In recent years, molecular biology diagnostic techniques have been developed rapidly, and in the field of the detection of molecules of clostridium novyi, there are a PCR technique of the flagellin gene FliA of clostridium novyi (patent "a multiplex PCR detection kit capable of simultaneously detecting clostridium perfringens, clostridium hemolyticum and clostridium novyi type B and application thereof", patent No. CN106148548A) and a loop-mediated isothermal amplification (LAMP) technique of the α toxin gene of clostridium novyi (patent "a method and kit for detecting clostridium novyi by using a loop-mediated isothermal amplification method", patent No. CN 102140505B). The PCR technology is an efficient and rapid diagnosis method, but the result is easy to have the problems of false positive and the like. The loop-mediated isothermal amplification (LAMP) technique is a novel rapid, accurate and economical gene amplification method, but the method has the biggest defect of aerosol pollution. If the test is repeated in the same space for a plurality of times, the accuracy of the subsequent test result can be influenced. For this reason, researchers in the early days established indirect ELISA detection method for alpha toxin of Novo (Natalia L. development of ELISA detection technique for detecting Clostridium novyi alpha toxin [ J ]. Indonesian Journal of Animal and vitamin Sciences, 1997), but indirect ELISA methods were prone to false positives. Therefore, a serological detection method which is simple and convenient to operate, low in cost, high in sensitivity and capable of accurately diagnosing the alpha toxin of the clostridium novyi is urgently needed.

Because the novyi virus disease has the characteristics of acute morbidity, high mortality rate and the like and has no special treatment medicine and the like, the vaccine immunization becomes a main way for preventing the bacterial infection, and the commercialized novyi vaccine is mainly an inactivated vaccine and comprises a sheep black disease, a fast plague bivalent inactivated vaccine and a sheep clostridial disease multi-connected dry powder inactivated vaccine. For veterinary biologies, ensuring the effectiveness of the product is one of its basic requirements, while the antigen content is critical for the effectiveness of veterinary biologies (summer talent, 2018. veterinary biologies (second edition) [ M ]. beijing: chinese agriculture press, page 77). Therefore, the quantification of the antigen content of the vaccine related to the clostridium novyi disease is the key for guaranteeing the effectiveness of the vaccine. The veterinary biological product code of the people's republic of China (two 000 years edition) stipulates that the natural toxin in the clostridium novyi disease-related vaccine should have the toxicity not less than 2000 mice MLD/mL. Although the effective antigen in the natural toxin of the clostridium novyi is alpha toxin, the rapid detection method of the alpha toxin is lacked, so that the mouse tail intravenous injection method with complicated operation and large difficulty coefficient can be selected to carry out the quantification of the natural toxin of the clostridium novyi in the actual production. Therefore, a method is provided for quantifying the toxin content in the component of the clostridium novyi in the related vaccine by establishing an ELISA detection method of the alpha toxin of the clostridium novyi and determining the correlation of the alpha toxin with the natural toxin content (mouse MLD).

Disclosure of Invention

The invention provides a sandwich double-antibody ELISA detection reagent for alpha toxin of clostridium novyi and a preparation method thereof, the detection reagent has the characteristics of simple and convenient sample operation, low cost, quick reaction, high sensitivity, strong specificity and the like, not only can provide reference for the diagnosis of clostridium novyi diseases, but also can provide an effective tool for the quantitative detection of alpha toxin.

According to the invention, inactivated natural toxin of the clostridium novyi is used as immunogen to immunize a mouse, and the nontoxic 3 nontoxic antigen peptides containing the N end of the alpha toxin of the clostridium novyi and the recombinant fusion protein (rTnc alpha) at the C end obtained by a prokaryotic expression system are adopted to screen the hybridoma cell strain of the alpha toxin monoclonal antibody, so that the Tnc alpha natural conformation is maintained to the greatest extent in the aspect of immunogen, and the complicated purification step and biological potential safety hazard of the natural Tnc alpha are greatly avoided; the ELISA detection method of the alpha toxin of the clostridium novyi constructed by the monoclonal antibody obtained by screening and the polyclonal antibody obtained by the recombinant fusion protein (rTnc alpha) provides support for the diagnosis of the clostridium novyi disease and the antigen quantitative method of the vaccine related to the clostridium novyi.

In one embodiment, the invention provides a hybridoma cell line capable of secreting a monoclonal antibody against clostridium novyi alpha toxin. The preservation number is as follows: the hybridoma cell strain is CGMCC No. 19942, and further the hybridoma cell strain is a monoclonal antibody hybridoma cell strain capable of stably secreting anti-alpha toxin of the clostridium novyi, which is named as a murine hybridoma cell of the alpha toxin of the clostridium novyi (CP6h9), obtained by immunizing a mouse by taking inactivated natural toxin of the clostridium novyi as an antigen, fusing spleen cells of the immunized mouse with SP2/0 cells, and then screening positive cloning and subcloning by using non-toxic rTnc alpha.

In another embodiment, the present invention provides a fusion protein, which is obtained by connecting coding genes of non-toxic N-terminal antigenic peptides of Tnc α (amino acids 3 to 17, 965 to 979, and 983 to 997) and the C-terminal (amino acids 1800 to 2178) in series based on a coding gene sequence of clostridium novyi α toxin (Tnc α) using flexible amino acids (GGGGS), and synthesizing the above gene sequence after optimizing the codon according to the e.coli offset, which is called GTnc α 2.

In another embodiment, the invention provides a polyclonal antibody against clostridium novyi alpha toxin, which is obtained by immunization based on the fusion protein GTnc alpha 2, and a preparation method thereof. Further, rTnc alpha is used as an antigen to immunize animals to obtain serum, and then the polyclonal antibody is obtained.

In another embodiment, the invention further provides the use of an ELISA detection reagent for alpha toxin of Clostridium novyi for detecting an infection of Clostridium novyi in a clinical or experimental sample.

The invention has the beneficial effects that:

the invention firstly utilizes the novyi toxoid and the nontoxic recombinant alpha toxin (rTnc alpha) of the novyi to establish the hybridoma cell strain of the alpha toxin monoclonal antibody of the novyi, furthest maintains the natural conformation of the Tnc alpha in the aspect of immunogen, and ensures the sensitivity and the accuracy of the secreted antibody in ELISA detection.

The invention establishes an alpha toxin double-antibody sandwich ELISA quantitative detection method by utilizing the monoclonal antibody of the alpha toxin of the clostridium novyi for the first time, and the method has the characteristics of simple and convenient sample operation, low cost, quick reaction, high sensitivity, strong specificity and the like, thereby not only providing reference for the diagnosis of the disease of the clostridium novyi, but also providing an effective tool for the quantitative detection of the alpha toxin.

The hybridoma cell strain is submitted to China general microbiological culture Collection center for preservation 22 months 05 and 2020, and the detection result shows that the hybridoma cell strain is alive. The classification name of the preserved strain is 'murine hybridoma cell', and the preservation number is CGMCC No. 19942; the name of the depository: institute of microbiology, national academy of sciences; address: xilu No. 1 Hospital No. 3, Beijing, Chaoyang, North.

Drawings

FIG. 1: SDS-PAGE identification of rTnc alpha expression

M₁Protein marker; BSA (1. mu.g); BSA (2. mu.g); 3, empty vector cell lysate; cell lysate induced at 4:15 ℃ for 16 h; 5: cell lysate induced at 37 ℃ for 4 h; 6, supernatant of the cell lysate of the empty vector; 7, inducing cell lysate supernatant for 16h at 15 ℃; cell lysis supernatant induced at 37 ℃ for 4 h; 9, precipitating the cell lysate of the empty carrier; precipitating the cell lysate induced at 10:15 ℃ for 16 h; 11, cell lysis precipitation induced at 37 ℃ for 4 h;

FIG. 2: western blot identification result of rTnc alpha expression

M2 Western blot marker; 1, empty vector cell lysate; cell lysate induced at 2:15 ℃ for 16 h; 3: cell lysate induced at 37 ℃ for 4 h; 4, supernatant of the cell lysate of the empty vector; 5, inducing cell lysate supernatant for 16h at 15 ℃; 6, inducing cell lysis supernatant at 37 ℃ for 4 h; 7, precipitating the cell lysate of the empty carrier; cell lysate precipitation induced at 8:15 ℃ for 16 h; 9, cell lysis precipitation induced at 37 ℃ for 4 h;

FIG. 3: SDS-PAGE identification of rTnc alpha purification

M1:Protein marker；1:BSA；2:rTncα；

FIG. 4: SDS-PAGE identification of monoclonal antibody 6h9 purification

M is Protein marker; 1: purified monoclonal antibody 6h9

FIG. 5: graph of the detection of natural toxin of novyi by double antibody sandwich ELISA.

FIG. 6: linear regression for detecting natural toxin of clostridium novyi by double-antibody sandwich ELISA

Detailed Description

The production of the natural toxin of the clostridium novyi and the determination of the Minimum Lethal Dose (MLD) of the mouse are carried out according to the regulation in the veterinary biological product code of the people's republic of China (two 000 years edition), the strain of the clostridium novyi C61-4 is cultured, and the supernatant of the bacterial liquid culture is taken after centrifugation, namely the natural toxin of the clostridium novyi. The obtained toxin was injected intravenously into mice at different concentrations, and the MLD of the toxin to the mice was determined according to the survival of the mice.

Expression, purification and virulence detection of rTnc alpha:

according to the coding gene sequence of clostridium novyi alpha toxin (Tnc alpha), the coding genes of N-terminal nontoxic antigen peptide (amino acids 3 to 17, 965 to 979 and 983 to 997) and C-terminal (amino acids 1800 to 2178) of the Tnc alpha are connected in series by flexible amino acid (GGGGS), and meanwhile, 6 × His label coding gene is added at the C-terminal of the recombinant fusion protein. The gene sequence is synthesized after being optimized according to the E.coli partial safety codon, and is called as GTnc alpha. The GTnc alpha is constructed to pET30a carrier by the conventional molecular biology method, and the prokaryotic expression carrier pET30a-GTnc alpha is obtained. And transforming the pET30a-GTnc alpha vector into a competent cell, and performing induced expression and purification to obtain the recombinant protein rTnc alpha.

The monoclonal antibody of the clostridium novyi alpha toxin is secreted and generated by a hybridoma cell strain (CP6h 9). The monoclonal antibody is obtained by inoculating a culture solution of the hybridoma cell strain or the hybridoma cell strain to an abdominal cavity of a mouse to generate ascites.

Preparation of polyclonal antibody against clostridium novyi alpha toxin of the present invention: selecting a New Zealand white rabbit as an immune animal, uniformly mixing rTnc alpha as an antigen and an adjuvant (such as 206 adjuvant), and immunizing the rabbit to obtain serum so as to obtain the polyclonal antibody.

The invention provides a preparation method of an ELISA detection reagent for alpha toxin of Novietum, which comprises the following steps:

(1) coating the purified monoclonal antibody 11B3 with an antigen coating solution (coated antibody);

(2) rTnc α was used as a positive control.

Further comprising:

(2) PBST solution of 5% skimmed milk powder is used as a sealing liquid;

(3) after washing with PBST solution for 3 times, the 96-well enzyme-labeled plate is patted dry and stored at-20 ℃ for later use.

(4) Setting negative and positive controls, wherein the positive controls are rTnc alpha and natural toxin of clostridium novyi respectively, the negative controls are escherichia coli culture supernatant and medium of clostridium novyi and PBS buffer solution prepared by a conventional method respectively, and the blank control is not added with any sample;

(5) adding alpha toxin polyclonal antibody diluted by PBS buffer solution in a volume ratio of 1: 1600;

(6) adding HRP-labeled goat anti-rabbit IgG diluted by PBS buffer solution in a volume ratio of 1: 10000;

(7) adding soluble TMB substrate developing solution, and using 2M H₂SO₄The reaction was stopped and absorbance read at 450 nm.

The ELISA detection reagent of the alpha toxin of the novyi can be used for detecting the novyi in clinical suspected sample tissues. And quantification of natural toxin of Clostridium novyi in Clostridium novyi related vaccines.

The present technology is further illustrated by the following examples

Example 1: and (3) preparation and detection of natural toxin of the clostridium novyi.

Inoculating the strain of Clostridium novyi C61-4 preserved by Chinese veterinarian monitoring to anaerobic liver soup, standing at 37 ℃ for anaerobic culture for 72h, inoculating new culture medium for passage once in the same way after recovery, finally inoculating the digestion soup of liver and stomach enzyme (with iron nails) with the inoculation amount of 5%, standing at 37 ℃ for anaerobic culture for 72h, centrifuging at 8000 r/min for 30min, filtering and sterilizing by using a 0.22 mu m filter to obtain the natural toxin of Clostridium novyi, subpackaging and placing at-70 ℃ for low-temperature preservation for later use. After one neotoxin is taken out of the refrigerator each time and ice is melted, the neotoxin is diluted properly by gelatin buffer solution, and the neotoxin is injected into mice by tail vein respectively with different titers, 2 mice are injected at each titer, 0.2 mL/mouse, and the mice are observed for 3 days. The minimum amount of toxin that can cause death of mouse 2/2 is the minimum lethal amount (MLD) of the toxin to the mouse. 1 mouse MLD was determined to be 0.5. mu.L of toxin stock, i.e., the toxin contained 2000 mouse MLDs per mL (2000 MLD/mL).

Example 2: and (3) preparation and detection of rTnc alpha.

1. Gene synthesis

According to the coding gene sequence of clostridium novyi alpha toxin (Tnc alpha), the coding genes of non-toxic antigenic peptides (amino acids 3 to 17, 965 to 979 and 983 to 997) at the N end and the coding genes at the C end (amino acids 1800 to 2178) of Tnc alpha are connected in series by using flexible amino acids (GGGGS), and meanwhile, the coding gene of 6 × His label is added at the C end of the recombinant fusion protein. The gene sequence is synthesized after being optimized according to the E.coli partial safety codon, and is called as GTnc alpha.

2. Construction of fusion protein expression vectors

And (3) carrying out PCR amplification by using the artificially synthesized GTnc alpha as a template.

The PCR system is as follows:

(Mg2+ plus) 10. mu.L, dNTPs 4. mu.L, upstream and downstream primers 1. mu.L each,

polymerase 1. mu.L, DNA template 2. mu.L, supplemented with dd H₂O to 50. mu.L system. The PCR reaction conditions are as follows: pre-denaturation at 98 ℃ for 1 min; denaturation at 98 ℃ for 10s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 2min for 33 cycles; finally, ring extension at 72 ℃ for 10 min.

And recovering the amplified target DNA band, carrying out double digestion by Nde I/Hind III, and connecting the target DNA band with a pET30a vector digested by the same digestion to obtain a positive clone pET30a-GTnc alpha inserted into GTnc alpha.

3. Expression and purification of rTnc alpha

The extracted plasmid was transformed into competent cells of Escherichia coli BL21(DE3), and a single clone was picked up and cultured overnight in LB liquid medium containing kanamycin under shaking at 37 ℃ and, after having been identified by PCR as containing the desired DNA fragment, the strain was named as Escherichia coli BL/Ta strain. The above strains were inoculated in 4mL of LB liquid medium containing kanamycin, cultured with shaking at 37 ℃ and 15 ℃ and then cultured at OD₆₀₀When the concentration is 0.6-0.8, adding IPTG solution with the final concentration of 0.5M for induction culture for 16 h. After the bacterial liquid culture is finished, the thalli are centrifugally collected, and 10mL of lysate [0.02mol/L Tris buffer solution (pH value 7.2) and 0.3mol/L NaCl are added according to the body temperature of each gram of thalli]Resuspending the cells at a ratio of (1) and sonicating the cells in an ice water bathAnd (3) carrying out sound crushing on the thalli for 30min, wherein the crushing conditions are as follows: the operation time is 9s, the pause time is 9s, and the ultrasonic power is 400W. The crushed bacterial liquid is centrifuged at 12000r/min for 10min at 4 ℃, and the supernatant is collected. mu.L of the supernatant was added to 10. mu.L of 4 XSDS-PAGE loading buffer, and subjected to 12% SDS-PAGE electrophoresis at 70 ℃ for 10min, as shown in FIG. 1. As can be seen from FIG. 1, rTnc alpha is mainly expressed in a non-soluble form under the conditions of 37 ℃ and 15 ℃, and the optimum induction expression condition of rTnc alpha is set as 37 ℃ by comprehensively considering the induction time and the expression amount, so that the rTnc alpha is induced and expressed for 4 h. Meanwhile, Western blot identification is carried out on the antibody by adopting an anti-His antibody, and the result (figure 2) is consistent with SDS-PAGE electrophoresis.

4. Purification of rTnc alpha

Escherichia coli BL/Ta strain was inoculated in 1L LB liquid medium containing kanamycin for fermentation culture, followed by shaking culture OD at 37 ℃₆₀₀When the concentration is 0.6-0.8, IPTG solution with the final concentration of 0.5mM is added for induction culture for 4 h. After the bacterial liquid culture is finished, the thalli is collected by centrifugation for 5min at 5000r/min, the thalli is resuspended according to the proportion that 10ml of lysis solution (pH value 7.20.02 mol/LTris buffer solution, 0.3mol/L NaCl) is added to each gram of thalli wet weight, and the thalli is crushed for 3 times by a low-temperature high-pressure homogenizer at the pressure of 800bar under the condition of 4 ℃. The lysate is centrifuged at 10000r/min at 4 ℃ for 30min, and the precipitate (inclusion body) is collected. The obtained inclusion bodies were washed with a washing solution (50mmol/L Tris (pH 8.0), 150mmol/L NaCl, 1% Triton X-100, 2mmol/L EDTA, 2mmol/L DTT) and then dissolved in a dissolving solution (50mmol/L Tris (pH 8.0), 150mmol/L NaCl, 8mol/L urea, 20mmol/L imidazole). After equilibration of the Ni-IDA column, incubation with soluble proteins was performed, the target protein was eluted with equilibration buffer with different concentrations of imidazole, and each eluted fraction was collected for SDS-PAGE analysis. And (3) collecting the washing solution with higher purity, dialyzing and renaturing by using a dialysis bag, filtering and packaging the supernatant by using a 0.22 mu m filter, and finally obtaining the recombinant protein rTnc alpha. As shown in FIG. 3, rTnc. alpha. with a purity of 82% was finally obtained

5. Toxicity test of rTnc alpha to mice

The toxicity of rTnc alpha to mice is measured to verify the actual attenuation effect of the recombinant fusion toxin in vivo. The purified rTnc alpha and the culture supernatant of the clostridium novyi are inoculated to 16-18 g of ICR mice through tail veins at different doses, and each dose is injected with 5 mice, 0.2 mL. Results all mice were healthy and without adverse effects when vaccinated at 0.1mg, whereas vaccination with C.novyi native toxin at 0.5. mu.L resulted in 5/5 death. The results indicate that rTnc α is non-toxic in mice and is identified as a non-toxic recombinant fusion protein.

TABLE 1 virulence of rTnc. alpha. for mice

Example 3: screening of hybridoma cell strain secreting monoclonal antibody against clostridium novyi alpha toxin and preparation of monoclonal antibody ascites

1. Immunization of BALB/c mice and determination of antibody titers

The inactivated natural toxin (toxoid) of the clostridium novyi is used as an immunizing antigen to immunize 3 BALB/c mice with the age of 6-8 weeks for 4 times, and the interval of each time is 2 weeks. The toxin for the first immunization is added with an equal volume of Freund complete adjuvant, and after emulsification, the mice are immunized by a multipoint subcutaneous injection way at the back of the neck, wherein the number of the mice is 500 MLD/mouse; then the adjuvant is replaced by Freund incomplete adjuvant for immunization, and the rest is the same as the prime immune; and (3) collecting a small amount of blood at the tail 7-10 days after the three-immunization, measuring the serum titer of the purified rTnc alpha by using an indirect ELISA method by using a 2 mu g/mL coated ELISA plate, and selecting a reagent with the titer higher than 1:10000 mice were boosted three days prior to fusion and were injected directly intraperitoneally with toxoid at a dose of 500 MLD/mouse. 3-5 days after the last immunization, the splenocytes of the mice with the highest titer are taken for cell fusion.

2. Cell fusion and screening of hybridoma cell lines

And (2) carrying out chemical fusion on myeloma cells SP2/0 with good growth state and splenocytes of the mice with the highest ELISA detection titer by using PEG1500, screening positive hybridoma cells by using an indirect ELISA method taking rTnc alpha as a coating antigen, and carrying out subcloning for 3-5 times by using a limiting dilution method to obtain 1 strain of clostridium novyi alpha toxin-resistant hybridoma cell strain CP6h9, wherein the subtype of a monoclonal antibody secreted by the strain of hybridoma cell strain is G2 a.

3. Preparation of ascites with monoclonal antibody against alpha toxin

Healthy multiparous BALB/c mice of 6-8 weeks old are selected, intraperitoneal injection of Freund's incomplete adjuvant is carried out for sensitization, 0.5mL of mice is used, and hybridoma cells are injected after 7 days. Collecting cells of hybridoma cell strain CP6h9 in logarithmic growth phase, centrifuging at 1000r/min for 1min, washing the cells with DMED culture solution once, re-suspending, and adjusting cell density to 10⁶After one/mL, each mouse was injected intraperitoneally with 0.5 mL. Gently kneading the abdomen of the mouse with alcohol cotton to uniformly disperse cells in the abdominal cavity; after 7-14 days, when the abdomen of the mouse is obviously swollen, aseptically puncturing the lower abdominal swelling part of the mouse with a needle head of a 20ml syringe, and gently kneading and pressing to make ascites flow out or drip out; collecting ascites with 15ml centrifuge tube, centrifuging at 3000r/min for 10min, labeling as 6h9, subpackaging, and storing at-20 deg.C for use.

Example 4: purification and detection of monoclonal ascites

The Protein A (GE Healthcare 17-5079-01) affinity column purification method is adopted for purification, and the purification steps are as follows:

sample pretreatment: ascites fluid was diluted at 1:3 with a coupling buffer (20mM sodium phosphate buffer, pH7.0), centrifuged at 12000rpm4 ℃ for 10min, and then filtered through a 0.22 μm filter to remove fat, cell debris and small particulate matter.

Balancing: the column was equilibrated with 5-10 column volumes of coupling buffer, maintaining a flow rate of 2 s/drop.

Loading: the sample was injected into the upper port of the column using a syringe and the effluent collected in a 50ml centrifuge tube maintaining a flow rate of 4 s/drop.

Impurity washing: the column was run with 5 column volumes of coupling buffer, maintaining a flow rate of 2 s/drop.

And (3) elution: the antibody was eluted with 5 column volumes of elution buffer (0.1M sodium citrate buffer, pH9.0) and collected in the above-mentioned EP tube at a flow rate of 4 s/drop.

And (3) detection of the sample: SDS-PAGE identification is carried out on the monoclonal antibody obtained by purification, and the result is shown in figure 3, and the monoclonal antibody with high purity is obtained by a Protein A affinity column purification method; the BCA method is adopted to measure the concentration of the monoclonal antibody, and the concentration can reach 2.1 mg/mL.

Example 5: establishment of clostridium novyi alpha toxin double-antibody sandwich ELISA detection method based on monoclonal antibody 6h9

1. The monoclonal antibody ascites 6h9 prepared in example 2 was selected as a coating antibody and the rabbit-derived polyclonal antibody against clostridium novyi α toxin was selected as a detection antibody.

2. Preparation of polyclonal antibody against clostridium novyi alpha toxin:

selecting a New Zealand white rabbit as an immune animal, completely mixing rTnc alpha as an antigen and an isometric biphasic oil adjuvant (206 adjuvant) uniformly, injecting 0.2 mg/rabbit subcutaneously at multiple points at the back part, immunizing once at intervals of two weeks, boosting the immunity with toxoid without adjuvant after three-immunization for two weeks, and collecting blood from the heart of the rabbit after 15 days to obtain serum.

3. Determination of novyi alpha toxin double-antibody sandwich ELISA detection method condition

(1) Optimal working concentration of rabbit anti-alpha toxin polyclonal antibody and monoclonal antibody ascites

Diluting the purified monoclonal antibody 6h9 to 10ug/ml, 5ug/ml, 2.5ug/ml, 1.25ug/ml and 0.625ug/ml 100 uL/well with PBS, coating at 4 deg.C for 12h, washing with PBST for three times, adding PBST solution containing 5% skimmed milk powder as blocking solution 200 uL/well, and blocking at 4 deg.C overnight;

after washing with PBST solution for 3 times, the 96-well enzyme-labeled plate is patted dry and stored at-20 ℃ for later use.

Taking out the coated 96-well enzyme label plate from a refrigerator at the temperature of minus 20 ℃, placing the plate at the room temperature for 30min, adding rTnc alpha with the concentration of 0.2ug/mL and 100 mu L/well, and simultaneously carrying out negative control and blank control, wherein the negative control is respectively escherichia coli culture supernatant and PBS prepared according to a conventional method, the blank control is no sample, and the plate is incubated at the temperature of 37 ℃ for 1 h;

after washing with PBST solution for 3 times, adding the obtained polyclonal antibody against the alpha toxin of Clostridium novyi, selecting PBS as the diluent, diluting sequentially at 1:100, 1: 200, 1: 400, 1: 800, 1:1600, 1: 3200 and 1: 6400, 100 muL/well, incubating at 37 ℃ for 1 h.

Washing with PBST solution for 3 times, adding HRP-labeled goat anti-mouse IgG diluted with PBS buffer solution at a volume ratio of 1:10000, incubating for 1h at 37 ℃, and washing with PBST solution for 3 times;

finally adding 50 mu L/hole of soluble TMB substrate color development solution, reacting for 15min at room temperature in a dark place, and reacting with 2M H₂SO₄The reaction was stopped and absorbance read at 450 nm.

The optimal conditions that the P/N value is maximum and the positive value is close to 1.0 are selected, the coating concentration of the monoclonal antibody 6h9 is determined to be 1.25ug/mL, the dilution of the alpha toxin polyclonal antibody is 1: 1600.

(2) optimization of optimal detection conditions

According to the coating concentration of the monoclonal antibody 6h9 and the optimal working concentration of the rabbit anti-alpha toxin polyclonal antibody, under different reaction conditions, the single antibody coating time (12 h at 4 ℃, 2h and 4h at 37 ℃) and the multi-antibody reaction conditions (30 min, 1h and 90min at 37 ℃) are further carried out by a chessboard method; ELISA tests were carried out with the dilution concentrations (1: 2000, 1: 5000, 1: 10000) of the enzyme-labeled secondary antibodies and the reaction conditions (30 min, 1h, 90min at 37 ℃) and the best conditions were selected to have the maximum P/N value and the positive value close to 1.0, the results are shown in Table 2, and finally the multi-antibody action time was determined to be 1h at 37 ℃, the dilution concentration of the enzyme-labeled secondary antibodies was 1: 10000; the action time is 37 ℃ and 1 h.

TABLE 2 determination of optimal reaction conditions

(3) Determination of criteria for decision of results

42 parts of rTnc alpha negative samples are detected by the established double-antibody sandwich ELISA, and OD is determined by setting a positive control (0.2 mu g/mL rTnc alpha)₄₅₀Calculating the average value

And standard deviation of

To obtain

And

the cut-off value was used as the cut-off value for negative and positive.

(4) Specific detection method of clostridium novyi alpha toxin double-antibody sandwich ELISA method

A, B, C and D type natural toxin of clostridium perfringens, tetanus natural toxin, putrefactive clostridium natural toxin, clostridium botulinum natural toxin, emphysema clostridium toxin (all adjusted to 1 mouse MLD/mL), and corresponding recombinant toxin (all adjusted to 0.2 mug/mL) (rCPA, rCPB, rETX, rTTC, rCSA)_m4△11、rMBP-BoH_C、rFLiACctA_N) For antigen ELISA detection, OD₄₅₀The values are all less than 0.24, are negative, show that no cross reaction exists, and are positive when reacting with rTnc alpha and natural toxin of the clostridium novyi, which shows that the specificity of the method is good.

TABLE 3 specific detection of Clostridium novyi alpha toxin diabody sandwich ELISA method

Note: "N" represents a native toxin; "r" represents a recombinant toxin.

(5) And (3) performing sensitivity test on the clostridium novyi alpha toxin double-antibody sandwich ELISA method.

The method takes 0.2 mu g/mL rTnc alpha as stock solution and carries out 1: 2-fold dilution, and the lowest value of the toxin which can be detected by the method is 12.5ng/mL, which indicates that the method has higher sensitivity.

(6) Application of clostridium novyi alpha toxin double-antibody sandwich ELISA method

1) The method comprises the steps of grinding the suspected pathogenic part muscle tissue of the sheep clostridium novyi disease and the attacking control rabbit muscle tissue of the vaccine related to the clostridium novyi disease) and the healthy sheep muscle tissue and the healthy rabbit muscle tissue which are obtained clinically, and then carrying out PCR detection and ELISA detection according to the patent 'a multiple PCR detection kit capable of simultaneously detecting clostridium perfringens, clostridium hemolyticus and B-type clostridium novyi and the application thereof' and the method of the invention. The detection results are shown in table 4, the detection rates of the two methods for the clinical suspected ovine clostridium novyi disease diseased part muscle tissues are the same and are 76.2%, and the detection results for the healthy muscle tissues and the healthy rabbit muscle tissues are negative, which indicates that the method has better accuracy. Compared with a PCR (polymerase chain reaction) method, the alpha toxin double-antibody sandwich ELISA method does not need complex steps such as genome extraction, gene amplification, electrophoresis analysis and the like, and has the advantages of simplicity in operation, short time consumption and the like. In addition, the positive rate of the alpha toxin in the rabbit muscle tissue of the challenge control group of the vaccine related to the clostridium novyi is 100%, and the PCR method cannot detect the sample only containing the clostridium novyi toxin, which indicates that the method has wider diagnosis range of the clostridium novyi disease.

TABLE 4 application of double-antibody sandwich ELISA detection of alpha toxin of Novo

2) 2-fold serial dilution is carried out on natural toxin of the clostridium novyi, the dilution is 5 mouse MLD/0.1mL to 0.195 mouse MLD/0.1mL, the established double-antibody sandwich ELISA method is utilized to detect the samples, the content of the natural toxin is used as an abscissa, OD450nm is used as an ordinate to draw a curve (figure 4), the optimal linear range is selected to establish a standard curve, and the result shows that the linear regression equation y is 0.6728x +0.3994, R2 is 0.9918 (figure 5), the natural toxin of the clostridium novyi of 0.39-12.5 mouse MLD has good linear relation, and the concentration range can be used for quantitative detection of the natural toxin of the clostridium novyi. Therefore, in the process of preparing the vaccine related to the clostridium novyi, the established standard curve can be used for quantifying the natural toxin of the clostridium novyi, thereby replacing a mouse tail vein injection method which is complicated to operate and has a large difficulty coefficient.

Claims

1. A hybridoma cell strain capable of secreting monoclonal antibodies against Clostridium novyi alpha toxin with a deposit number of: CGMCC No. 19942.

2. A fusion protein is based on the coding gene sequence of Clostridium novyi alpha toxin (Tnc alpha), and is synthesized by connecting coding genes of N-terminal nontoxic antigen peptide (3 rd to 17 th, 965 th to 979 th, 983 th to 997 th amino acids) and C-terminal (1800 th to 2178 th amino acids) of Tnc alpha in series through flexible amino acid (GGGGS), and optimizing the gene sequences according to the E.coli partial safety codon.

3. A method for preparing polyclonal antibody against Clostridium novyi alpha toxin, which comprises immunizing animal with the fusion protein obtained in claim 2 as antigen to obtain serum, and obtaining the polyclonal antibody.

4. A preparation method of an ELISA detection reagent for alpha toxin of Novo is provided, which comprises the following steps:

(1) coating the purified monoclonal antibody with an antigen coating solution;

(2) rTnc α was used as a positive control.

5. The method for preparing reagent for ELISA detection of α toxin of Novizium according to claim 4, further comprising the steps of:

(1) coating the purified monoclonal antibody with an antigen coating solution;

(2) PBST solution of 5% skimmed milk powder is used as a sealing liquid;

(4) Setting a negative and positive control and a blank control at the same time, wherein the positive control is rTnc alpha, the negative control is a PBS buffer solution of escherichia coli culture supernatant prepared by a conventional method, and the blank control is that no sample is added;

6. Use of the ELISA detection reagent for alpha toxin of Clostridium novyi prepared according to claim 4 or 5 for detecting Clostridium novyi in clinically suspected sample tissues.

7. Use of the reagent for ELISA detection of alpha toxin of Clostridium novyi prepared according to claim 4 or 5 for quantification of native toxin of Clostridium novyi in a vaccine related to Clostridium novyi.