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Anti-SpA5 protein monoclonal antibody, application thereof, and kit containing monoclonal antibody

A technology of monoclonal antibody and kit, which is applied in the direction of anti-bacterial immunoglobulin, immunoglobulin, biological testing, etc. It can solve the problem that there is no specific anti-SpA5 antibody, and achieve the effect of high sensitivity and wide detection range

Inactive Publication Date: 2020-04-28
CHENGDU OLYMVAX BIOPHARM +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

No specific anti-SpA5 antibody exists

Method used

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  • Anti-SpA5 protein monoclonal antibody, application thereof, and kit containing monoclonal antibody
  • Anti-SpA5 protein monoclonal antibody, application thereof, and kit containing monoclonal antibody
  • Anti-SpA5 protein monoclonal antibody, application thereof, and kit containing monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Example 1: Preparation of mouse monoclonal antibody that specifically recognizes SpA5 protein

[0053] 1. Preparation and screening of monoclonal antibodies

[0054]SpA5 recombinant protein (1.4mg / ml) was mixed with adjuvant CFA and AD11.15 to prepare the immunogen, that is, the recombinant protein was first mixed with CFA at a volume ratio of 5:6 as immunogen A, and then the recombinant protein was mixed with AD11. 15 was mixed at a volume ratio of 1:1 as immunogen B. Immunogen A is the primary immunization and immunogen B is the booster immunization. Three mice were immunized intramuscularly, and the tail blood of the mice was drawn on the 14th day after immunization, and the antibody titer of the tail blood was evaluated by indirect ELISA method.

[0055] Coat the ELISA plate with SPA5 recombinant protein (1 μg / mL), add 100 μL to each well, react overnight at 4°C; wash the plate 3 times with PBS solution, block with 5% milk-PBS at room temperature for 1 hr; then wa...

Embodiment 2

[0078] Example 2: Preparation and use of double antibody sandwich ELISA detection kit for SpA5 protein

[0079] The preparation method of the rabbit polyclonal antibody against SpA5 protein: ① adjust the recombinant protein SpA5 solution to a concentration of 1mg / mL with diluent (10mMHis, 0.9%NaCl, pH=6.0), and mix with Freund’s Adjuvant (complete Freund's adjuvant for the first immunization, incomplete Freund's adjuvant for booster immunization) emulsified. ②The emulsified antigen was injected subcutaneously near the lymph nodes in the limbs of rabbits, 200 μL for each point, 0.4 mg in total. The immunization program was 0, 14, 21 days, a total of 3 times of immunization. ③ On the 28th day after the first immunization, 100 μL of blood was collected from the immunized rabbits through the ear vein, the serum was separated, and the antibody titer corresponding to the recombinant protein SpA5 was detected by indirect ELISA. ④ After the antibody titer corresponding to the recomb...

Embodiment 3

[0115] Embodiment 3: sample detection

[0116] Use the kit prepared in Example 2 to detect the protein content of SpA5 antigen in the 20180903 batches of finished products of Staphylococcus aureus vaccine. The detection steps are as follows:

[0117] 1. Sample pretreatment: Take 20 vaccine finished products, mix each tube, draw 600μL into a 1.5mL EP tube, centrifuge at 5000rpm for 10min, absorb 300μL of the supernatant, add 2mol / L Na 2 CO 3 300 μL of the solution and mix well, after the solution is clear, centrifuge to take an appropriate amount of supernatant for later use;

[0118] 2. according to the operation step of embodiment 2, sample is detected;

[0119] 3. The results of the standard were linearly fitted using the logX-LogY fitting method to obtain a standard curve, and the absorbance value (OD value) of the test sample was substituted into the standard curve to calculate the sample concentration. The test results are as follows:

[0120] Table 6: The results of t...

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Abstract

The invention belongs to the technical field of biology, and provides an anti-SpA5 protein monoclonal antibody, application thereof, and a kit containing the monoclonal antibody. The anti-SpA5 proteinmonoclonal antibody comprises a heavy chain and a light chain, wherein the heavy chain and the light chain respectively comprise three CDR regions, the amino acid sequences of the heavy chain CDR regions are respectively as shown in SEQ ID NO: 1-3, and the amino acid sequences of the light chain CDR regions are respectively as shown in SEQ ID NO: 4-6. The antibody disclosed by the invention can be specifically combined with SpA5 protein and effectively detect the content of the SpA5 protein, and has high sensitivity and wide detection range.

Description

technical field [0001] The invention belongs to the field of biotechnology and relates to a monoclonal antibody. Background technique [0002] Staphylococcus aureus protein A (SpA) is present in most pathogenic Staphylococcus aureus, but not in Staphylococcus epidermidis and Staphylococcus saprophyticus. Blast comparison analysis showed that SpA was a highly conserved antigenic component. Its molecular weight is 55kDa, and it is distributed on the surface of the cell wall. In mouse models of septic arthritis, sepsis, and skin abscesses infected by Staphylococcus aureus, the virulence of Staphylococcus aureus strains with deletion mutations in SpA was reduced, which was thought to be due to the reduction of SpA binding to the IgG-Fc segment, reducing Anti-host phagocytosis of Staphylococcus aureus. [0003] The applicant refers to relevant research literature reports, through molecular design, the five domains of wild-type SpA (wSpA) were screened for multiple mutations at...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/12G01N33/68G01N33/577G01N33/535
CPCC07K16/1271G01N33/68G01N33/577G01N33/535C07K2317/565G01N2333/31
Inventor 邹全明曾浩顾江鲁东水杨峰张卫军徐丽敏程平
Owner CHENGDU OLYMVAX BIOPHARM
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