Colloidal gold detection test strip for detecting clostridium perfringens Beta2 toxin and application of colloidal gold detection test strip

Abstract

The invention relates to the technical field of immunodetection, in particular to a colloidal gold test strip for detecting clostridium perfringens Beta2 toxin and application of the colloidal gold test strip. The colloidal gold test strip provided by the invention has good feasibility and practicability in the aspect of on-site rapid detection, is simple to operate and short in time consumption, and can be used for rapid screening of a large number of samples and rapid diagnosis after Beta2 toxin poisoning. And a simple, convenient, rapid and high-specificity inspection product is provided for detection of the clostridium perfringens Beta2 toxin and infection thereof and epidemic disease investigation.

Description

Technical Field

[0001] The invention relates to the technical field of immune detection, and in particular to a colloidal gold test strip for detecting Clostridium perfringens Beta2 toxin and an application thereof. Background Art

[0002] Clostridium perfringens, also known as Clostridium perfringens, is a Gram-positive, spore-forming anaerobic bacillus. It is widely distributed in nature and can be detected in soil, human and animal intestines. Under a microscope, it can be observed that the bacteria have blunt ends, straight edges and are short rod-shaped, exist alone or in short chains, have capsules but no flagella. The α, Beta1, Beta2, ε, and ι produced by the bacteria are the main lethal toxins. According to the difference in the types of the main exotoxins, Clostridium perfringens can be divided into 7 toxin types from A to G. Among them, type B and C Clostridium perfringens can secrete a variety of lethal exotoxins, which can cause diseases such as sudden death in cattle and sheep, enterotoxemia in calves, lambs, and piglets.

[0003] Beta2 toxin produced by Clostridium perfringens is an important lethal toxin that can cause diseases such as sudden death in cattle and sheep, enterotoxemia in calves, lambs, and piglets. Currently, there are relatively few methods for detecting Beta2 toxin, and most of them are traditional molecular biological diagnosis, including taking samples back to the laboratory to isolate bacteria, identifying them through bacterial metabolites, bacterial morphology, PCR identification, extraction and detection of genes in bacteria, and then comparing them with gene libraries. However, simply detecting the presence of bacteria and toxin genes in the sample cannot determine whether the toxin protein is expressed. In addition, these methods are complicated and time-consuming, and cannot be used for rapid and large-scale detection. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a colloidal gold test strip for detecting Beta2 toxin of Clostridium perfringens and its application. The colloidal gold test strip provided by the present invention has good feasibility and practicality in on-site rapid detection, is simple to operate, and takes a short time, and can be used for rapid screening of a large number of samples and rapid diagnosis after Beta2 toxin poisoning.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The invention provides a colloidal gold test strip for detecting Clostridium perfringens Beta2 toxin, comprising a bottom plate, a nitrocellulose membrane, a conjugation pad, a sample pad and a water absorbent pad, characterized in that the conjugation pad is coated with a gold-labeled IgY antibody and a gold-labeled Beta2 toxin monoclonal antibody; the mass ratio of the gold-labeled IgY antibody to the gold-labeled Beta2 toxin monoclonal antibody is 1:1; a detection line and a quality control line are arranged on the nitrocellulose membrane; the detection line is coated with a Beta2 toxin protein with a concentration of 1.6 μg / μL, and the quality control line is coated with an IgY antibody with a concentration of 1 μg / μL.

[0007] Preferably, the gold-labeled Beta2 toxin monoclonal antibody consists of colloidal gold and the Beta2 toxin monoclonal antibody coupled to the colloidal gold; the Beta2 toxin monoclonal antibody is secreted by E23 hybridoma cells.

[0008] Preferably, the preparation method of the gold-labeled Beta2 toxin monoclonal antibody comprises: mixing a first colloidal gold solution and a Beta2 toxin monoclonal antibody to obtain a gold-labeled Beta2 toxin monoclonal antibody; the volume mass ratio of the first colloidal gold solution to the Beta2 toxin monoclonal antibody is 200 μL:10 μg; the pH value of the first colloidal gold solution is 7.4; the OD value of the first colloidal gold solution is 540 =1.

[0009] Preferably, the Beta2 toxin protein is expressed by the BL21(DE3)pTIG-cpb2 recombinant expression strain.

[0010] Preferably, the preparation method of the gold-labeled IgY antibody comprises: mixing a second colloidal gold solution and an IgY antibody to obtain a gold-labeled IgY antibody; the volume mass ratio of the second colloidal gold solution to the IgY antibody is 200 μL:10 μg; the pH value of the second colloidal gold solution is 8.0; the OD value of the second colloidal gold solution is 540 =1.

[0011] Preferably, the distance between the detection line and the quality control line is 5 to 8 mm.

[0012] Preferably, the structure of the colloidal gold test strip is as follows: a sample pad, a binding pad, a nitrocellulose membrane and a water-absorbing pad are fixed in sequence along the length direction of the bottom plate;

[0013] One end of the conjugate pad in the length direction overlaps the sample pad, and the other end of the conjugate pad in the length direction overlaps the nitrocellulose membrane; the other end of the nitrocellulose membrane in the length direction overlapping the conjugate pad overlaps the water absorbent pad.

[0014] The present invention provides the use of the colloidal gold test strip according to any one of claims 1 to 7 in preparing a product for detecting Beta2 toxin of Clostridium pleuroplastis.

[0015] Preferably, the product comprises a kit for detecting Beta2 toxin of Clostridium perfringens.

[0016] Preferably, the test sample of the product includes one or more of liquid bacterial culture supernatant, feces, and body fluid.

[0017] Beneficial effects:

[0018] The invention provides a colloidal gold test paper strip for detecting Clostridium perfringens Beta2 toxin, comprising a bottom plate, a nitrocellulose membrane, a conjugation pad, a sample pad and a water absorbent pad, wherein the conjugation pad is coated with a gold-labeled IgY antibody and a gold-labeled Beta2 toxin monoclonal antibody; the mass ratio of the gold-labeled IgY antibody to the gold-labeled Beta2 toxin monoclonal antibody is 1:1; a detection line and a quality control line are arranged on the nitrocellulose membrane; the detection line is coated with a Beta2 toxin protein with a concentration of 1.6 μg / μL, and the quality control line is coated with an IgY antibody with a concentration of 1 μg / μL. The colloidal gold test strip provided by the present invention has a sensitivity of 0.6 μg / μL, a complete inhibition concentration of 0.8 μg / μL, a minimum detection limit (LOD value) of 0.6 μg / μL to 0.8 μg / μL, and the test strip does not cross-react with other major lethal toxins of Clostridium perfringens, such as α, β1, ε, etc., can effectively identify and bind Beta2 toxin, is not interfered by other coexisting toxins, has good feasibility and practicality in on-site rapid detection, is simple to operate, and is time-saving, and can be used for rapid screening of a large number of samples and rapid diagnosis after Beta2 toxin poisoning. Provides a simple, fast, and highly specific inspection product for the detection of Clostridium perfringens Beta2 toxin and its infection, and epidemiological investigation. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0020] Figure 1 This is the purification result of Beta2 toxin protein;

[0021] Figure 2 The results of SDS-PAGE electrophoresis of recombinant Beta2 toxin samples; M: protein marker; 1: BL21(DE3)pET32a; 2: flow-through 1; 3: flow-through 2; 4-7: protein peak samples; 8: Beta2 recombinant protein purified mixed sample;

[0022] Figure 3 This is the peak diagram of McAb E23 purification;

[0023] Figure 4 The results of SDS-PAGE of McAb E23 purified samples; M: Marker; 1: 5 μL E23 cell culture supernatant; 2: 5 μL E23 cell culture supernatant flow-through; 3: 5 μL E23 purified peak;

[0024] Figure 5 The experimental results of the optimal pH for colloidal gold labeling; A: the optimal pH result of colloidal gold labeling McAb E23, from left to right are 0μL, 5μL, 10μL, 15μL, 20μL, 25μL, 30μL and 35μL; B: the optimal pH result of colloidal gold labeling chicken IgY;

[0025] Figure 6 The experimental results of the optimal labeling amount of antibodies; A: the experimental results of the optimal labeling amount of McAb E23 labeled with colloidal gold; B: the experimental results of the optimal labeling amount of chicken IgY labeled with colloidal gold;

[0026] Figure 7 This is the experimental result of the optimal spraying amount of gold-labeled IgY;

[0027] Figure 8 The optimal binding amount of the gold-labeled antibody;

[0028] Fig. 9 This is the experimental result of the optimal coating amount of T-line antigen;

[0029] Fig.10 It is the test strip sensitivity test result;

[0030] Fig.11 It is the test result of the test strip specificity. DETAILED DESCRIPTION

[0031] The invention provides a colloidal gold test strip for detecting Clostridium perfringens Beta2 toxin, comprising a bottom plate, a nitrocellulose membrane, a conjugation pad, a sample pad and a water absorbent pad, wherein the conjugation pad is coated with a gold-labeled IgY antibody and a gold-labeled Beta2 toxin monoclonal antibody; the mass ratio of the gold-labeled IgY antibody to the gold-labeled Beta2 toxin monoclonal antibody is 1:1; a detection line and a quality control line are arranged on the nitrocellulose membrane; the detection line is coated with a Beta2 toxin protein with a concentration of 1.6 μg / μL, and the quality control line is coated with an IgY antibody with a concentration of 1 μg / μL.

[0032] The colloidal gold test strip provided by the present invention can prevent Clostridium perfringens Beta2 toxin in a targeted manner and make a timely diagnosis, and the diagnosis result meets the expected effect of the experiment, filling the gap in the diagnosis technology of Clostridium perfringens Beta2 toxin. It can be applied to veterinary clinics and animal husbandry production to achieve rapid diagnosis of pathogens and rapid detection of target substances.

[0033] As an embodiment, the gold-labeled Beta2 toxin monoclonal antibody is composed of colloidal gold and the Beta2 toxin monoclonal antibody coupled to the colloidal gold; the Beta2 toxin monoclonal antibody is obtained by secretion of E23 hybridoma cells. The E23 hybridoma cells described in the present invention are deposited in Ningxia University and disclosed in the literature [Zeng Jin. Study on the cytotoxicity and pathogenic mechanism of Beta2 toxin of Clostridium perfringens [D]. Ningxia University, 2017.].

[0034] As an embodiment, the preparation method of the gold-labeled Beta2 toxin monoclonal antibody comprises: mixing a first colloidal gold solution and a Beta2 toxin monoclonal antibody to obtain a gold-labeled Beta2 toxin monoclonal antibody; the volume mass ratio of the first colloidal gold solution to the Beta2 toxin monoclonal antibody is 200 μL:10 μg; the pH value of the first colloidal gold solution is 7.4; the OD value of the first colloidal gold solution is 540 =1.

[0035] As an embodiment, the Beta2 toxin protein is expressed by the BL21 (DE3) pTIG-cpb2 recombinant expression strain. The BL21 (DE3) pTIG-cpb2 recombinant expression strain of the present invention is deposited in Ningxia University and disclosed in the literature [Zeng Jin. Study on the cytotoxicity and pathogenic mechanism of Beta2 toxin of Clostridium perfringens [D]. Ningxia University, 2017.].

[0036] As an embodiment, the preparation method of the gold-labeled IgY antibody comprises: mixing a second colloidal gold solution and an IgY antibody to obtain a gold-labeled IgY antibody; the volume mass ratio of the second colloidal gold solution to the IgY antibody is 200 μL:10 μg; the pH value of the second colloidal gold solution is 8.0; the OD value of the second colloidal gold solution is 540 =1.

[0037] As an implementation mode, the distance between the detection line and the quality control line is 5 to 8 mm.

[0038] As an embodiment, the structure of the colloidal gold test strip is as follows: a sample pad, a binding pad, a nitrocellulose membrane and a water-absorbing pad are fixed in sequence along the length direction of the bottom plate;

[0039] One end of the conjugate pad in the length direction overlaps the sample pad, and the other end of the conjugate pad in the length direction overlaps the nitrocellulose membrane; the other end of the nitrocellulose membrane in the length direction overlapping the conjugate pad overlaps the water absorbent pad.

[0040] The present invention provides the use of the colloidal gold test strip described in the above technical solution in preparing a product for detecting Beta2 toxin of Clostridium pleurosum.

[0041] As an embodiment, the product includes a kit for detecting Beta2 toxin of Clostridium pleuroplasmosis.

[0042] As an embodiment, the test sample of the product includes one or more of liquid bacterial culture supernatant, feces, and body fluid.

[0043] To further illustrate the present invention, a colloidal gold test strip for detecting Beta2 toxin of Clostridium perfringens provided by the present invention and its application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] 1. Experimental Materials

[0046] Ampicillin was purchased from USBLOANALYZED, USA; IPTG and dialysis bag were purchased from Solebow Biotechnology Co., Ltd.; SDS-PAGE gel kit and Yazyme dual-color pre-stained protein marker (WJ102) were purchased from Shanghai Yazyme Biotechnology Co., Ltd.; BCA protein detection kit was purchased from Nanjing Keygen Biotechnology Co., Ltd.; 6×Protein Loading Buffer was purchased from Quanshijin Biotechnology Co., Ltd.; BL21(DE3)pTIG-cpb2 recombinant expression strain was deposited in Ningxia University and published in the literature [Zeng Jin. Study on the cytotoxicity and pathogenic mechanism of Beta2 toxin of Clostridium perfringens [D]. Ningxia University, 2017.]; ultra-high concentration colloidal gold, chicken IgY, goat anti-chicken IgY, goat anti-mouse IgG, bovine serum albumin, nitrocellulose membrane, binding pad, sample pad, absorbent pad, and PVC bottom plate were purchased from Xibao Biotechnology (Shanghai) Co., Ltd.

[0047] 2. Experimental methods

[0048] 2.1 Preparation of Beta2 toxin antigen and monoclonal antibody for detection

[0049] 2.1.1 Expression and purification of recombinant Beta2 toxin protein for detection

[0050] (1) Recovery culture of strains: Take the correctly identified BL21(DE3)pTIG-cpb2 recombinant expression strain frozen in a -80°C ultra-low temperature refrigerator, inoculate it on an LB solid culture medium plate containing 100 μg / mL ampicillin and place it in a 37°C constant temperature incubator for inversion culture for 16 h.

[0051] (2) Expansion of strains: Pick a single colony of the resuscitated strain and inoculate it into 10 mL of LB liquid culture medium containing ampicillin, and shake and culture it in a shaking incubator at 37°C and 180 rpm for 16 h. After shaking, inoculate it into a 200 mL large bottle of LB culture medium at 1% and shake and culture it in a shaking incubator at 37°C and 180 rpm for 3 h.

[0052] (3) Inducing protein expression: Add the inducer IPTG to the culture flask to a final concentration of 1 mmol / L, and culture in a shaking incubator at 37°C and 180 rpm for 3 h.

[0053] (4) Pour the bacterial solution into a centrifuge bottle and centrifuge at 11000r / min. The collected bacteria are washed once with PBS and stored in a -80℃ refrigerator after centrifugation. During purification, wash the precipitate of every 10mL of bacterial solution with 2mL of PBS, add the prepared balance solution and oscillate to resuspend the cells, freeze and thaw repeatedly in a -80℃ refrigerator twice to form ice crystals in the cells, disrupt them with an ultrasonic cell disruptor for 15 minutes, and then centrifuge at 11000r / min for 15 minutes. Use a 0.22μm pore size filter to remove the coarse suspended matter from the supernatant sample to prepare the sample to be purified.

[0054] (5) The supernatant of bacterial lysis was purified by nickel affinity chromatography using the AKTA purification system. The obtained protein was dialyzed, detected by SDS-PAGE electrophoresis and quantified by BCA, and then packaged and stored at 4°C as an antigen for later use.

[0055] 2.1.2 Preparation of Beta2 toxin monoclonal antibodies for detection

[0056] E23 hybridoma cells (denoted as E23, published in [Zeng Jin. Cytotoxicity and Pathogenic Mechanism of Beta2 Toxin of Clostridium perfringens [D]. Ningxia University, 2017.]) frozen in a liquid nitrogen tank were revived and inoculated into RPMI1640 medium containing 10% fetal bovine serum for resuscitation. Cell subculture was performed. After the cells grew well, the cells were split into 75 cm 2The cells were placed in a culture flask and placed in a CO2 incubator until the cells overgrow to enrich the antibody. After about 5 days, when the culture medium becomes acidic (yellow), the cell culture medium was transferred to a 50 mL centrifuge tube and centrifuged at 10000 r / min for 10 min at room temperature. The supernatant was carefully collected and sterilized by filtration using a 0.22 μm filter. The protein G column was loaded using the AKTA protein purification system and eluted using a 100 mM glycine-HCL solution at pH 2.7 to obtain McAb E23. The purified McAb E23 was analyzed by SDS-PAGE electrophoresis and the protein concentration was determined by the BCA method. Finally, glycerol was added and frozen in a -80°C refrigerator for later use.

[0057] 2.2 Colloidal gold labeled antibody test conditions

[0058] 2.2.1 Selection of the optimal pH for colloidal gold labeling

[0059] Prepare 0.02M K2CO3 solution, 10% NaCl solution, McAb E23 (or chicken IgY) diluted to 1 μg / μL with 1× PBS, and pure water diluted to OD 540 =1 colloidal gold solution. Take 200μL of diluted colloidal gold in 8-well plates, add 0μL, 5μL, 10μL, 15μL, 20μL, 25μL, 30μL and 35μL of 0.02M K2CO3 solution to adjust the pH value, then add 10μgMcAb E23 (or chicken IgY) to the solution, react for 10min, add 40μL of 10% NaCl solution to each well, react for 10min, and observe the color change of the solution. Use precision pH test paper to measure the pH value of the solution and record it.

[0060] 2.2.2 Determination of the optimal binding amount of antibody-conjugated colloidal gold

[0061] Take 12 EP tubes and add 200 μL colloidal gold solution respectively, adjust the colloidal gold to the optimal pH value for labeling McAb E23 (or chicken IgY). Add the gradient of antibodies to be labeled according to the sequence in Table 1, let stand at room temperature for 15 minutes, then add a certain volume of 10% NaCl solution, react for 15 minutes, observe the color change of the labeled colloidal gold and record it.

[0062] Table 1 Optimal binding amount of antibody and colloidal gold conjugate

[0063]

[0064]

[0065] 2.2.3 Colloidal gold labeled antibodies

[0066] 2.2.3.1 Colloidal gold labeling of McAb E23

[0067] Add 300 μL of 1 μg / μL McAb E23 to 5 mL of optimal pH colloidal gold solution to prepare a gold-labeled antibody solution. Add 500 μL of 10% BSA to the gold-labeled antibody solution, stir at room temperature for 30 minutes and then dispense. Centrifuge the dispensed gold-labeled McAb E23 at 11000 rpm / min for 40 minutes, carefully discard the supernatant, and the precipitate is the concentrated gold-labeled antibody. Dissolve the concentrated colloidal gold in 100 μL of the gold-labeled antibody resuspension, and the resulting solution is the colloidal gold-McAb E23 conjugate stock solution; the gold-labeled antibody resuspension is composed of the following components at the following concentrations: 3 g / L bovine serum albumin (BSA), 1 g / L polyethylene glycol 20000, 0.5 mL / LTween-20 and 150 g / L sucrose.

[0068] 2.2.3.2 Colloidal gold labeled chicken IgY

[0069] Determine the optimal pH value and optimal binding amount of colloidal gold labeled chicken IgY according to the above steps. Add 1 / 10 of the amount of 10% BSA solution and stir for 30 minutes, then add 0.01 times the volume of colloidal gold 10% PEG 20000 solution to stabilize the colloidal gold. The remaining steps are the same as 2.2.3.1.

[0070] 2.3 Preparation and assembly of colloidal gold immunochromatographic test strips

[0071] 2.3.1 Treatment of nitrocellulose membrane

[0072] The NC membrane should be cut into a size of 25×3mm. During coating, the distance between the T line and the C line should be controlled at 5-8mm, and finally divided for use.

[0073] 2.3.2 Treatment of the conjugate pad

[0074] The conjugate pad was soaked in the gold-labeled pad treatment solution for 2 h and dried at 37°C and divided for use. The size of each test strip conjugate pad should be maintained at 7×3 mm; the gold-labeled pad treatment solution consisted of the following components: 0.1 g polyvinyl alcohol (PVA), 0.1 g Tween-20, 0.3 g BSA, 4 g sucrose, and Tris-HCl buffer (0.01 M, pH 8.5) was fixed to 100 mL.

[0075] 2.3.3 Sample pad processing

[0076] The sample pad was soaked in the sample pad treatment solution for 2 h and dried at 37 ° C. Finally, it was divided for use. The sample pad of each test strip should be maintained at 17×3 mm; the sample pad treatment solution consisted of the following components: 0.5 g polyvinylpyrrolidone (PVP), 0.1 g Triston-X100, 0.3 g BSA, 4 g sucrose, and Tris-HCl buffer (0.01 M, pH 8.5) was fixed to 100 mL.

[0077] 2.3.4 Assembly of test strips

[0078] Take out the PVC backing as the bottom plate, place the sample pad, conjugate pad (gold label pad), nitrocellulose membrane and absorbent pad in order, overlap by 2mm, adhere to the PVC backing, cut into 3mm strips with a paper cutter, and store at 4-8℃. When testing, just drop the sample on the sample pad.

[0079] 2.4 Optimization of preparation conditions for colloidal gold immunochromatographic test strips

[0080] 2.4.1 Independent C line determines the optimal spraying amount of gold-labeled IgY

[0081] Use 1×PBS to dilute the colloidal gold labeled IgY stock solution to 100% (v / v), 50% (v / v), and 10% (v / v) and spray it on the conjugate pad at a spray volume of 10μL / cm. Coat 2μg / μL of recombinant Beta2 toxin protein (prepared in 2.1.1) and 2μg / μL goat anti-chicken IgY on the NC membrane T line and C line, respectively, with a coating volume of 4μL / cm. Adhere the NC membrane, conjugate pad, sample pad, and absorbent paper to the PVC bottom plate in order. Add 100μL PBS and observe the experimental results.

[0082] 2.4.2 Determination of the optimal spraying amount of Gold Label McAb E23

[0083] Mix the gold-labeled McAb E23 stock solution with a content of 40% (diluted with 1×PBS, the same below), 50% and 60% into 50% gold-labeled IgY and spray it on the binding pad at a spray volume of 10μL / cm and dry it at room temperature. Spray 2μg / μL Beta2 toxin and 2μg / μL sheep anti-chicken IgY at the T line and C line of the NC membrane at a spray volume of 4μL / cm. Adhere the sample pad, binding pad (gold-labeled pad), nitrocellulose membrane and absorbent pad to the PVC bottom plate in order. When adding samples, set 3 groups to add 100μL PBS as a negative control, and 3 groups to add 100μLBeta2 recombinant toxin, and observe the experimental results.

[0084] 2.4.3 Optimal coating amount of T line antigen

[0085] 2μg / μL Beta2 toxin was diluted with 1×PBS to 10%, 20%, 40%, 80%, and 100% and coated on the T line of the NC membrane. The C line pressure concentration was 2μg / μL, and the T line and C line spray volume was 4μL / cm. 50% gold-labeled IgY and 50% gold-labeled E23 binding pads were attached 2mm above the NC membrane. 100μL PBS or Beta2 toxin recombinant protein was added to the sample pad and the experimental results were observed.

[0086] 2.5 Test strip performance test

[0087] 2.5.1 Sensitivity of test strips

[0088] Use 1×PBS to dilute 2μg / μL Beta2 toxin into a series of concentration gradients of 0-2μg / μL (0, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%), add 100μL of Beta2 toxin solution of different concentrations to the sample well of the test strip, observe the color development results after 10 minutes, and evaluate the sensitivity of the test strip to Beta2 toxin.

[0089] 2.5.2 Specificity of test strips

[0090] Different types of toxin standard solutions were used, including α, β1, and ε toxins, and each toxin was repeated three times. This experimental design aims to accurately determine the specificity of the test strip to Beta2 toxin by comparing the differences in the reaction of the test strip to Clostridium perfringens Beta2 toxin and other toxins.

[0091] 3 Experimental results

[0092] 3.1 Preparation of recombinant Beta2 toxin protein antigen for detection

[0093] The culture fluid of the recombinant expression strain that has been revived, expanded and induced to express well was poured into a centrifuge bottle and centrifuged at high speed to harvest the bacteria. After freezing and thawing and breaking the bacteria, the AKTA protein purification system was used for purification. The peak was obtained at 75 mM imidazole. Figure 1 The purified protein was sampled and subjected to SDS-PAGE electrophoresis. The experiment showed that there was a target band at 28 kDa, which was consistent with the size of Clostridium perfringens Beta2 toxin. Figure 2 As shown in the figure, the concentration of Beta2 toxin was 2 μg / μL after quantitative calculation of BCA protein concentration.

[0094] 3.2 Preparation of Beta2 toxin monoclonal antibody for detection

[0095] The E23 hybridoma cells were revived, placed in a CO2 incubator to enrich antibodies, centrifuged at room temperature, and filtered for sterilization. The protein in the E23 hybridoma cell culture supernatant was purified using the AKTA protein purification system. Figure 3 Finally, the purified McAb E23 was subjected to SDS-PAGE gel electrophoresis, and the results showed that corresponding bands appeared at 23kDa and 53kDa, respectively, indicating that the purification result was good. Figure 4 As shown in the figure, the reactivity of E23 and Beta2 toxin recombinant protein was detected by Western blot method. The test results showed that there was a target band at 28kDa, which was consistent with the size of the reported Beta2 toxin protein, indicating that the monoclonal antibody E23 prepared in the experiment can specifically recognize and bind to the Beta2 toxin recombinant protein. The concentration of McAb E23 obtained by BCA protein concentration quantitative calculation was 6.8μg / μL.

[0096] 3.3 Selection of the optimal pH for gold-labeled antibodies

[0097] like Figure 5 As shown, the pH value was adjusted by adding different volumes of 0.02M K2CO3 solution. When labeling McAb E23, 5μL 0.02M K2CO3 was added to reach the optimal labeling pH value (7.4). When labeling chicken IgY, 20μL 0.02M K2CO3 was added to reach the optimal labeling pH value (8.0). At this time, the color of colloidal gold was wine red and stable without changing color. As the amount of K2CO3 added increased, the color of colloidal gold gradually changed.

[0098] 3.4 Selection of the optimal labeling amount of gold-labeled antibodies

[0099] like Figure 6 As shown in the figure, when the antibody dosage is less than 8μg, the color in the EP tube is unstable, and aggregation phenomenon occurs from light to dark, causing the colloidal gold to become dead; when the antibody dosage is higher than 9μg, the color of the colloidal gold gradually tends to be stable; when 10μg of antibody is added, the color of the colloidal gold is bright red and light stable, and it does not fade after being placed for a period of time. Therefore, the optimal concentration of the labeled McAb E23 (or chicken IgY) is 10μg / 200μL.

[0100] 3.5 Optimal spraying amount of gold-labeled IgY on the bonding pad

[0101] Depend on Figure 7 It can be seen that when the spraying concentration of gold-labeled IgY is 50% of the stock solution, the C line is clearly colored, so the optimal spraying concentration of the conjugate pad is 50% of the stock solution.

[0102] 3.6 Optimal spraying amount of Gold Label McAb E23

[0103] By spraying gold-labeled McAb E23 and gold-labeled chicken IgY on the conjugate pad, the T line and C line produce color bands to determine whether there is a positive sample. When testing a positive sample, the Clostridium perfringens Beta2 toxin in the sample binds to the colloidal gold-labeled Clostridium perfringens McAb E23. Through chromatography, the complex formed by the binding moves forward inside the nitrocellulose membrane. When passing through the detection area, the gold-labeled E23 that has bound to the Beta2 toxin antigen cannot bind to the Beta2 toxin antigen coated on the T line again, so no red band appears on the T line. The colloidal gold-labeled chicken IgY cannot be captured by the Beta2 toxin. After chromatography, it is captured by the sheep anti-chicken IgY coated at the C line, causing the C line to produce a red band. When there is no antigen in the sample, the unloaded gold-labeled McAb E23 and gold-labeled chicken IgY are captured by the T line and the C line, respectively, producing two red bands.

[0104] like Figure 8 As shown in the figure, when the added sample is PBS, the empty gold-labeled E23 on the conjugate pad binds to the T line antigen. As the content of gold-labeled E23 increases, the color of the T line gradually darkens, the colloidal gold-labeled chicken IgY in the conjugate pad is captured by goat anti-chicken IgY, and the C line also develops color; when the added sample is Beta2 toxin, the Beta2 toxin in the sample binds to the gold-labeled E23 sprayed on the gold-labeled pad, so the colloidal gold-labeled McAb E23 no longer binds to the Beta2 toxin coated on the detection line, resulting in the T line not showing red, the gold-labeled IgY in the gold-labeled pad is captured by the goat anti-chicken IgY coated on the quality control line, and the C line shows red. According to the experimental results, 50% gold-labeled E23 and 50% gold-labeled IgY are the best combination, and the color development effect is relatively obvious.

[0105] 3.7 Optimal coating amount of T line antigen

[0106] Depend on Fig. 9 As shown in the figure, as the content of Beta2 toxin coated on the nitrocellulose membrane increases, the red color of the T line changes from light to dark. When the coating content is 80%, the color development effect of the T line and C line is obvious. Therefore, the optimal coating amount of the T line antigen is 1.6μg / μL.

[0107] 3.8 Testing of colloidal gold test strip performance

[0108] (1) Sensitivity test results

[0109] When the concentration of Beta2 toxin is less than 0.6μg / μl, the T line develops color, and the color saturation is equivalent to that of the PBS group, which is considered negative; when the concentration of Beta2 toxin is equal to 0.6μg / μl, the T line slightly develops color, and the color saturation is significantly weaker than that of the PBS group, which is considered positive; when the concentration of the standard is greater than 0.6μg / μl, the T line does not develop color, which is considered complete inhibition and a strong positive. It can be judged that the sensitivity of the test strip is 0.6μg / μl, the complete inhibition concentration is 0.8μg / μl, and the minimum detection limit (LOD value) is between 0.6μg / μl-0.8μg / μl. The sensitivity results of the test strip are as follows Fig.10 .

[0110] Table 2 Sensitivity test results

[0111]

[0112] Note: ++ indicates positive, + indicates weakly positive, and -- indicates negative.

[0113] (2) Specificity test results

[0114] The test strips do not cross-react with other major lethal toxins of Clostridium perfringens, α, β1, and ε, indicating that the monoclonal antibody (McAb E23) is highly selective for Beta2 toxin and can effectively recognize and bind to Beta2 toxin without interference from other coexisting toxins. This high specificity ensures the credibility and accuracy of the test results and avoids the occurrence of false positive results.

[0115] In summary, the colloidal gold test strip provided by the present invention has good feasibility and practicality in on-site rapid detection, is simple to operate, and takes a short time, and can be used for rapid screening of a large number of samples and rapid diagnosis after Beta2 toxin poisoning.

[0116] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A colloidal gold test strip for detecting Clostridium perfringens Beta2 toxin, comprising a base plate, a nitrocellulose membrane, a conjugate pad, a sample pad and a water-absorbing pad, characterized in that: The conjugate pad is coated with a gold-labeled IgY antibody and a gold-labeled Beta2 toxin monoclonal antibody; the mass ratio of the gold-labeled IgY antibody and the gold-labeled Beta2 toxin monoclonal antibody is 1:1; a detection line and a quality control line are arranged on the nitrocellulose membrane; the detection line is coated with a Beta2 toxin protein with a concentration of 1.6 μg / μL, and the quality control line is coated with an IgY antibody with a concentration of 1 μg / μL.

2. The colloidal gold test strip according to claim 1, characterized in that: The gold-labeled Beta2 toxin monoclonal antibody consists of colloidal gold and the Beta2 toxin monoclonal antibody coupled to the colloidal gold; the Beta2 toxin monoclonal antibody is obtained by secretion of E23 hybridoma cells.

3. The colloidal gold test strip according to claim 1 or 2, characterized in that: The preparation method of the gold-labeled Beta2 toxin monoclonal antibody comprises: mixing a first colloidal gold solution and a Beta2 toxin monoclonal antibody to obtain the gold-labeled Beta2 toxin monoclonal antibody; the volume mass ratio of the first colloidal gold solution and the Beta2 toxin monoclonal antibody is 200 μL:10 μg; the pH value of the first colloidal gold solution is 7.4; the OD value of the first colloidal gold solution is 540 =1.

4. The colloidal gold test strip according to claim 1, characterized in that: The Beta2 toxin protein is expressed by the BL21 (DE3) pTIG-cpb2 recombinant expression strain.

5. The colloidal gold test strip according to claim 1, characterized in that: The preparation method of the gold-labeled IgY antibody comprises: mixing a second colloidal gold solution and an IgY antibody to obtain the gold-labeled IgY antibody; the volume mass ratio of the second colloidal gold solution to the IgY antibody is 200 μL:10 μg; the pH value of the second colloidal gold solution is 8.0; the OD value of the second colloidal gold solution is 540 =1.

6. The colloidal gold test strip according to claim 1, characterized in that: The distance between the detection line and the quality control line is 5 to 8 mm.

7. The colloidal gold test strip according to claim 1, characterized in that: The structure of the colloidal gold test strip is as follows: a sample pad, a binding pad, a nitrocellulose membrane and a water-absorbing pad are fixed in sequence along the length direction of the bottom plate; One end of the conjugate pad in the length direction overlaps the sample pad, and the other end of the conjugate pad in the length direction overlaps the nitrocellulose membrane; the other end of the nitrocellulose membrane in the length direction overlapping the conjugate pad overlaps the water absorbent pad.

8. Use of the colloidal gold test strip according to any one of claims 1 to 7 in the preparation of a product for detecting Beta2 toxin of Clostridium pleuroplastis.

9. The use according to claim 8, characterized in that: The product comprises a kit for detecting Beta2 toxin of Clostridium pleurops.

10. The use according to claim 8 or 9, characterized in that: The test sample of the product includes one or more of liquid bacterial culture supernatant, feces, and body fluid.

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