A bacterial antigen detection kit and its application

Abstract

The present invention discloses a Staphylococcus aureus β-hemolysin antigen epitope polypeptide and a monoclonal antibody against the epitope, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2. The present invention also discloses a kit for detecting Staphylococcus aureus β-hemolysin, wherein the kit comprises an effective amount of monoclonal antibody, Staphylococcus aureus β-hemolysin mutant protein, Staphylococcus aureus β-hemolysin mutant protein polyclonal antibody and matching detection reagents. The antigen epitope polypeptide prepared by the present invention has good immunogenicity and specificity; the monoclonal antibody prepared by the present invention has good specificity and sensitivity; the diagnostic kit prepared using the monoclonal antibody of the present invention has good specificity, sensitivity and accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of biological diagnosis, and in particular relates to a bacterial antigen detection kit and application thereof. Background Art

[0002] Staphylococcus aureus (SA), also known as "golden Staphylococcus", belongs to the genus Staphylococcus, is a representative of Gram-positive bacteria and a common foodborne pathogen. The most suitable growth temperature for this bacterium is 37°C and pH 7.4. It is tolerant to high salt and can grow in an environment with a salt concentration close to 10%. Staphylococcus aureus often parasitizes on the skin, nasal cavity, throat, stomach, carbuncle, suppurative sores of humans and animals, and is also ubiquitous in the air, sewage and other environments.

[0003] Staphylococcus aureus is spherical in shape. In the culture medium, the colony is round, the colony surface is smooth, the color is colorless or golden yellow, and there is no extended growth. When Staphylococcus aureus is cultured in Columbia blood agar plates, transparent hemolysis circles will be found around the colonies when observed under light. Staphylococcus aureus is arranged in grape clusters under a microscope. Staphylococcus aureus has no spores, flagella, and most of them have no capsules. It is a common pathogen that causes food poisoning. It is commonly found on the skin surface and upper respiratory tract mucosa.

[0004] Staphylococcus aureus is a common foodborne pathogen that is widely present in the natural environment. Under appropriate conditions, Staphylococcus aureus can produce enterotoxins and cause food poisoning. Reports of food poisoning caused by Staphylococcus aureus are endless. Food poisoning caused by Staphylococcus aureus accounts for about 25% of foodborne microbial food poisoning incidents, making Staphylococcus aureus the third largest microbial pathogen after Salmonella and Bacillus parahaemolyticus.

[0005] Staphylococcus aureus can produce a variety of toxins. β-hemolysin (also known as β-toxin) is one of its toxins, which has the characteristics of leukocytotoxicity and hemolytic activity. β-hemolysin is a single-chain polypeptide composed of 330 amino acids, with a molecular weight of 37-39kDa and an isoelectric point (pI) higher than 9. β-hemolysin is a magnesium-dependent neuroesterase with phospholipase C (PLC) activity, which can decompose glycerophosphocholine. β-hemolysin relies on this enzyme activity to hydrolyze the phospholipid bilayer that constitutes the cell membrane, thereby destroying the integrity of the cell membrane, leading to cell lysis and causing hemolysis. Cited from the invention patent (CN 105641689 A); it studied the β-hemolysin of Staphylococcus aureus, and on this basis prepared a subunit vaccine of Staphylococcus aureus. However, there is currently a lack of reagents or raw and auxiliary materials for detecting β-hemolysin. Summary of the invention

[0006] In order to make up for the deficiencies of the prior art, one of the purposes of the present invention is to provide a monoclonal antibody to Staphylococcus aureus β-hemolysin and a preparation method thereof; a second purpose of the present invention is to provide a Staphylococcus aureus β-hemolysin detection kit prepared using the Staphylococcus aureus β-hemolysin monoclonal antibody prepared by the present invention.

[0007] Therefore, the present invention provides a monoclonal antibody against Staphylococcus aureus beta-hemolysin on one hand, wherein the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.2.

[0008] Preferably, the amino acid sequence of the antigenic epitope polypeptide of Staphylococcus aureus β-hemolysin to which the monoclonal antibody of the present invention specifically binds is TQSEDSRCGAGHDRKIRAEQMKEIS.

[0009] In another aspect, the present invention also provides a kit for detecting Staphylococcus aureus β-hemolysin, the kit comprising an effective amount of monoclonal antibody, Staphylococcus aureus β-hemolysin mutant protein, Staphylococcus aureus β-hemolysin mutant protein polyclonal antibody and matching detection reagents.

[0010] Preferably, the monoclonal antibody of the present invention is used as a detection antibody, the detection antibody is labeled with HRP before use, and the HRP-labeled monoclonal antibody is diluted 20,000 times when used.

[0011] Preferably, the Staphylococcus aureus β-hemolysin mutant protein described in the present invention is used as a standard, and the concentrations of the standard are 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.03125 μg / ml, and 0 μg / ml, respectively.

[0012] Preferably, the polyclonal antibody against the mutant protein of Staphylococcus aureus β-hemolysin described in the present invention is used as the coating antibody, and the coating concentration of the coating antibody is 1 μg / ml.

[0013] In another aspect, the present invention also provides a use of the monoclonal antibody in the preparation of a diagnostic reagent for Staphylococcus aureus β-hemolysin.

[0014] The antigen epitope polypeptide prepared by the present invention has good immunogenicity and specificity; the monoclonal antibody prepared by the present invention has good specificity and sensitivity; and the diagnostic kit prepared by using the monoclonal antibody of the present invention has good specificity, sensitivity and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1Monoclonal antibody SDS-PAGE diagram, where M is Marker and 1 is the monoclonal antibody.

[0016] Figure 2 Kit standard curve. DETAILED DESCRIPTION

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0018] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0019] Example 1: Preparation of Staphylococcus aureus β-hemolysin mutant protein

[0020] Referring to the invention patent (CN 105641689 A), Examples 1 to 6 were used to prepare mutant proteins of Staphylococcus aureus β-hemolysin. The prepared proteins were packaged (0.5 ml / tube) and stored below -70°C.

[0021] Example 2: Design and screening of Staphylococcus aureus β-hemolysin antigen epitope polypeptides

[0022] The β-hemolysin protein of GenBank: AGC51114.1 was selected. Through the analysis and study of the protein sequence, the 34 amino acids at the N-terminus were used as signal peptide sequences, and the remaining 296 amino acids were used for epitope peptide design. A total of 12 epitope peptides were designed and synthesized (Table 1). The 12 epitope peptides were used to immunize mice, and the mouse serum was collected for ELISA antibody titer detection to screen the epitope peptide with the highest antibody titer. After testing, the antibody titer of epitope peptide 7 was the highest (Table 1), so epitope peptide 7 was selected as the focus of subsequent research.

[0023] ELISA antibody titer detection (see Example 10 of CN 105641689 A) is briefly described as follows: the β-hemolysin mutant protein prepared in Example 1 was used to coat the ELISA plate, 0.5 μg / ml, 0.1 ml / well, and coated overnight at 4°C; after washing 3 times, it was blocked with 5% skim milk and incubated at 37°C for 2 h; mouse serum was serially diluted 10 times (10-10 7), then add to the coated ELISA plate, 0.1ml / well, incubate at 37℃ for 1h; after washing 3 times, add 1:5000 diluted HRP-labeled goat anti-mouse IgG secondary antibody, 0.1ml / well, incubate at 37℃ for 30min; after washing 3 times, add 0.1ml TMB single-component colorimetric solution, incubate at 37℃ for 10min; add stop solution (2M sulfuric acid), 50μl / well, and measure OD450nm value. The standard for judging antibody positivity: P / N≥2.1 (where P is the sample OD450nm value, N is the blank control OD450nm value), and OD450≥0.1.

[0024] Table 1 Staphylococcus aureus β-hemolysin antigen epitope peptides

[0025]

[0026]

[0027] Example 3: Preparation of monoclonal antibodies against Staphylococcus aureus β-hemolysin antigen epitope polypeptides

[0028] 3.1 Animal immunization: 8-12 week old BalB / C mice were immunized with the synthetic antigen epitope peptide 7:

[0029] Primary immunization: 50-100 μg protein antigen plus an equal volume of Freund's complete adjuvant emulsified until it is insoluble in water can be injected subcutaneously at multiple points on the back of mice. The injection volume is 500 μl / mouse. The second immunization will be performed 2 weeks later.

[0030] Second immunization: 50-100 μg (25-50 μg is half of the initial immunization dose) protein antigen (same amount as the initial immunization), add an equal volume of Freund's incomplete adjuvant and emulsify until it is insoluble in water, then inject it subcutaneously at multiple points on the back of the mouse, with an injection volume of 500 μl / mouse. The third immunization is carried out 2 weeks later.

[0031] The third immunization: 50 μg of protein antigen plus an equal volume of Freund's incomplete adjuvant was emulsified until it could not be dissolved in water and then injected into the mouse peritoneum. The injection volume was 500 μl / mouse. Three days before mouse fusion, 25 μg of antigen was taken for booster immunization. The injection volume was 200 μl / mouse.

[0032] 3.2 Hybridoma cell screening: After three immunizations, mouse spleen cells were separated and fused with myeloma cells Sp2 / 0, and then cloned and screened by limiting dilution method. Cell lines with high antibody titer and good morphology were screened and subcloned by limiting dilution method until monoclonal cells were obtained. Monoclonal cells were expanded and cultured and stored in liquid nitrogen. After multiple rounds of testing and screening, a good positive hybridoma cell was finally obtained, which was named hybridoma cell 1.

[0033] 3.3 Ascites preparation: 6-8 week old BALB / c female mice were intraperitoneally injected with pristane (0.5 ml / mouse). Seven days later, the mice were intraperitoneally injected with 1×10 6 1. After 7 to 10 days, aseptically extract the mouse ascites with a syringe and centrifuge at 12000 rpm at 4°C for 20 min. Collect the supernatant, which is the ascites antibody. Divide the harvested supernatant into 2 ml / tubes and store at -70°C for later use.

[0034] 3.4 Antibody purification: The prepared ascites antibody was purified by conventional caprylic acid-ammonium sulfate precipitation method. 20 μl of the purified ascites antibody was taken and tested for purity using SDS-PAGE. The results showed that ( Figure 1 ), the SDS-PAGE purity of the monoclonal antibody was greater than 90%; the protein concentration of the purified antibody was detected using a BCA kit, and the protein concentration was 1.36 mg / ml.

[0035] 3.5 Determination of monoclonal antibody sequence: The heavy chain variable region and light chain variable region of the prepared monoclonal antibody were analyzed and determined. After detection and analysis, the heavy chain variable region sequence of the monoclonal antibody is shown in SEQ ID NO.1, and the light chain variable region sequence is shown in SEQ ID NO.2.

[0036] SEQ ID NO.1:

[0037] MGVQLQQSGPVTCPGASVKISTKVDKYTFTEYTMHWPCICTVKSLEWIGGIN

[0038] PNNFPPKPKNFKGKATLTVDKSPPKPKDVLTILRSLTSEDSAIYYCSRYDYAMDYWGQGTSVTTTVSS.

[0039] SEQ ID NO.2:

[0040] DIPSSEQPSSASVVSVGEKVTMSCKNFYPKDLLYSSNQIDGSWYQQKGVLNP

[0041] KLLISKDSTRESGVPERHNGSGSTHKTSFTLTISKSFNDLAVYYCQQCYSYPRT FGGG TKLEIKR.

[0042] Example 4: Preparation of anti-Staphylococcus aureus β-hemolysin polyclonal antibodies

[0043] The mutant protein of Staphylococcus aureus β-hemolysin prepared in Example 1 was mixed with an equal volume of complete Freund's adjuvant (first immunization) / incomplete Freund's adjuvant, and the mixture was pipetted back and forth with a syringe for emulsification for 2-3 hours.

[0044] Newly purchased healthy adult rabbits can be immunized after being raised for 4-5 days without abnormalities. Blood samples are collected before immunization for subsequent experiments.

[0045] The vaccine is inoculated by multiple subcutaneous injections on both sides of the spine, and 0.5 ml can be injected at each point. The second immunization is carried out 2 weeks after the first immunization (reference dose: 2 mg), and the second immunization dose is half of the first immunization (1 mg). 7 days after the second immunization, 1 ml can be collected from the ear vein for serum titer detection. The third immunization is carried out after the second immunization, and the dose is the same as the second immunization. Serum titer detection can be carried out 7 days after the third immunization. When the serum titer reaches the requirement, booster immunization can be carried out (the dose is the same as before, and PBS should be used instead of Freund's adjuvant mixed with protein). One week later, the jugular vein is bled to collect serum.

[0046] Immunized animals should be prohibited from eating several hours before blood collection. Collect blood samples in sterile centrifuge tubes (avoid violent shaking), let them coagulate at 37℃ for 1h, and place them at 4℃ overnight. The precipitated antiserum is light yellow. Centrifuge the blood sample in the centrifuge tube at 4000r / min for 10min. Aspirate the supernatant, use ProteinG column for column purification, and store the purified rabbit polyclonal antibody in quantitative aliquots, 0.5ml / tube, at -20℃. At the same time, use SDS-PAGE for purity detection and BCA for concentration detection; after testing, the SDS-PAGE purity of the prepared rabbit polyclonal antibody is 90%, and the concentration is 2.25mg / ml.

[0047] ELISA antibody titer detection (see Example 2) is briefly described as follows: the β-hemolysin mutant protein prepared in Example 1 was used to coat the ELISA plate, 0.5 μg / ml, 0.1 ml / well, and coated overnight at 4°C; after washing 3 times, it was blocked with 5% skim milk and incubated at 37°C for 2 h; the mouse serum was serially diluted 10 times (10-10 7 ), then add to the coated ELISA plate, 0.1ml / well, incubate at 37℃ for 1h; after washing 3 times, add 1:5000 diluted HRP-labeled goat anti-rabbit IgG secondary antibody, 0.1ml / well, incubate at 37℃ for 30min; after washing 3 times, add 0.1ml TMB single-component colorimetric solution, incubate at 37℃ for 10min; add stop solution (2M sulfuric acid), 50μl / well, and measure OD450nm value. The standard for judging antibody positivity: P / N≥2.1 (where P is the sample OD450nm value, N is the blank control OD450nm value), and OD450≥0.1.

[0048] After testing, the titer of the rabbit polyclonal antibody prepared in this study is very high, reaching 10 6 . It can be used for follow-up research.

[0049] Example 5: Detection of Staphylococcus aureus β-hemolysin

[0050] Preparation of standard: The Staphylococcus aureus β-hemolysin mutant protein prepared in Example 1 was diluted in a gradient manner, the diluent was a 2% BSA PBST solution, and the dilution gradient was 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.03125 μg / ml, and 0 μg / ml.

[0051] Preparation of ELISA plate: The rabbit polyclonal antibody prepared in Example 4 was used to coat the ELISA plate. The rabbit polyclonal antibody was coated on the ELISA plate at a dose of 1 μg / ml (0.1 ml / well) and incubated at 4°C overnight. After washing, 0.2 ml / well of blocking solution (PBST solution containing 2% BSA) was added and incubated at 37°C for 2 h. After washing, the plate was patted dry and stored at 4°C for later use.

[0052] Preparation of enzyme-labeled antibody: The monoclonal antibody prepared in Example 3 was HRP-labeled using an HRP labeling kit, and an equal volume of glycerol was added to the HRP-labeled monoclonal antibody and stored at -20°C for later use. Before use, it was diluted 20,000 times with blocking solution and stored at 4°C for later use.

[0053] Kit detection: 1) Dilute the sample to be tested so that its concentration after dilution is below 1μg / ml, add the diluted sample to the ELISA plate, and add the standard at 0.1ml / well, incubate at 37℃ for 30 minutes; 2) After washing, add the enzyme-labeled antibody at 0.1ml / well, incubate at 37℃ for 30 minutes; 3) After washing, add TMB developer at 0.1ml / well, incubate at 37℃ for 10 minutes; 4) Add stop solution (2M H2SO4) at 0.1ml / well, mix well, and immediately place the coated plate in the ELISA reader, and read the OD450nm value at a wavelength of 450nm; 5) Use the standard concentration as the horizontal axis and the corresponding standard OD450nm value as the vertical axis to fit the standard curve, and generate the standard curve function and R 2 , where R 2 Should be ≥0.9 (such as Figure 2 6) Substitute the OD450nm value of the diluted sample into the standard curve function to calculate the concentration of the diluted sample. The concentration of the sample is calculated by multiplying the concentration by the dilution factor of the sample. If there are 2-3 gradients of sample dilution, the average of the concentrations calculated by all gradients is taken as the concentration of the sample.

[0054] Application of the kit: The inventor collected 20 sera of patients infected with Staphylococcus aureus (specific bands were detected by western blot for Staphylococcus aureus β-hemolysin) and 35 normal sera (no specific bands were detected by western blot for Staphylococcus aureus β-hemolysin). The prepared kit was used to test the 55 sera respectively. The results showed that the detection values ​​of the sera of the 20 patients infected with Staphylococcus aureus detected by the kit were all greater than 5ng / ml, and the detection values ​​of the 35 normal sera were all ≤0 (i.e., no Staphylococcus aureus β-hemolysin), indicating that the kit has good accuracy, specificity and sensitivity.

[0055] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A monoclonal antibody against Staphylococcus aureus β-hemolysin, characterized in that: The amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody are shown in SEQ ID NO.1 and SEQ ID NO.

2.

2. The monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the antigen epitope polypeptide of Staphylococcus aureus beta-hemolysin to which the monoclonal antibody specifically binds is TQSEDSRCGAGHDRKIRAEQMKEIS.

3. A kit for detecting Staphylococcus aureus β-hemolysin, characterized in that: The kit comprises an effective amount of the monoclonal antibody according to claim 1, a Staphylococcus aureus β-hemolysin mutant protein, a Staphylococcus aureus β-hemolysin mutant protein polyclonal antibody and a matching detection reagent.

4. The kit according to claim 3, characterized in that The monoclonal antibody is used as the detection antibody. The detection antibody is labeled with HRP before use. The HRP-labeled monoclonal antibody is diluted 20,000 times when used.

5. The kit according to claim 3, characterized in that The Staphylococcus aureus β-hemolysin mutant protein is used as a standard, and the concentrations of the standard are 1 μg / ml, 0.5 μg / ml, 0.25 μg / ml, 0.125 μg / ml, 0.0625 μg / ml, 0.03125 μg / ml, and 0 μg / ml, respectively.

6. The kit according to claim 3, characterized in that The polyclonal antibody against the mutant protein of Staphylococcus aureus beta-hemolysin is used as the coating antibody, and the coating concentration of the coating antibody is 1 μg / ml.

7. Use of the monoclonal antibody according to claim 1 in the preparation of a diagnostic reagent for Staphylococcus aureus β-hemolysin.

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