Competitive enzyme-linked immunosorbent assay method of EV71 neutralizing antibody, kit or reagent and prepration method thereof

A technology of EV71 and reagent kit, applied in the direction of measuring devices, instruments, analytical materials, etc., to achieve wide applicability and ensure specificity

Active Publication Date: 2009-12-23
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The invention discloses an EV71 virus A, B, C three-genotype broad-spectrum neutralizing antibody competitive ELISA detection method, which can quickly and effectively detect EV71 neutralizing antibodies in human and other animal samples, and replace EV71 virus in animals The challenge test a

Method used

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  • Competitive enzyme-linked immunosorbent assay method of EV71 neutralizing antibody, kit or reagent and prepration method thereof
  • Competitive enzyme-linked immunosorbent assay method of EV71 neutralizing antibody, kit or reagent and prepration method thereof
  • Competitive enzyme-linked immunosorbent assay method of EV71 neutralizing antibody, kit or reagent and prepration method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Preparation of EV71 Coating Antigen

[0031] The C4 genotype EV71 virus was inoculated into Vero cells, and after cultivation, a lysate was added to lyse the cells, the culture medium was harvested, the EV71 virus was purified after inactivation, and the purified EV71 solution was obtained after sterilization and filtration, which was the coated antigen. (See: Bishop NE, Hago DL, Borovec SV et al. Rapid and efficient purification of hepatitis Avirus from cell culture (rapid and efficient purification of hepatitis Avirus from cell culture). J Virol Methods, 1994, 47: 203-216). Then the BCA kit (Micro BCA TM Protein Assay Kit PIERCE Company, Cat. No. 23235) was used to detect protein concentration.

[0032]Of course, those skilled in the art understand that the purified solution of EV71 of other genotypes can also be prepared by adopting the above method, and the prepared purified solution can be used in the following examples. Specifically, the coating anti...

Embodiment 2

[0033] Example 2: Preparation and Identification of Anti-EV71 Monoclonal Antibody

[0034] BALB / c mice were immunized three times with the EV71 purified liquid prepared in Example 1, and the splenocytes of the BALB / c mice with the highest titer by indirect ELISA were fused with SP2 / 0 myeloma cells in proportion, and placed on the HAT culture medium. Fusion hybrid tumor cells were cultured. Subcloning was carried out three times. For each subcloning, the purified liquid of A, B3, and C4 genotype EV71 was used to separately coat the microtiter plate, and the indirect ELISA titer of the cell supernatant of each cell well was detected. Wells with OD values ​​of all three genotypes greater than 0.4 were cloned. Thus, a HD6 monoclonal cell line with strong reactions to the three genotypes A, B, and C was obtained. The above-mentioned HD6 monoclonal cell line was expanded and cultured, and the expanded cells were injected into the peritoneal cavity of BALB / c mice. After about 7-14 ...

Embodiment 3

[0051] Embodiment 3: Purification of anti-EV71 monoclonal antibody

[0052] The HD6 monoclonal antibody ascites obtained in Example 2 was subjected to staphylococcal protein A affinity chromatography (see: Valdés Veliz R, García J, Reyes B, et al. A very sensitive enzyme-linked immunoabsorbent assay to staphy-lococcal Protein A in the presence of immunoglobulins (highly sensitive enzyme-linked immunosorbent assay for staphylococcal protein A in the presence of immunoglobulins). Biochem Biophys Res Commun, 2003, 303(3): 863-867.) Purification. The BCA kit was used to detect the protein concentration of the purified antibody to be 1.2 mg / ml; the purified antibody was subjected to SDS-PAGE reducing electrophoresis, and the electrophoresis gel was stained with Coomassie brilliant blue. After decolorization, KODAK Gel200 gel imager was used to scan the electropherogram, and the purity of the purified antibody was analyzed to be 93.2% (higher purity). Thus, high-purity anti-EV71 mo...

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Abstract

The invention discloses a competitive enzyme-linked immunosorbent assay method of an EV71 neutralizing antibody, a kit or a reagent and a preparation method thereof. The kit utilizes the labeled monoclonal antibody which has higher neutralizing potency on three genes of A, B and C of EV71 virus, a reference material of the EV71 neutralizing antibody and a coated plate and can simply, rapidly and accurately carry out qualitative and quantitative assay on the content of the EV71 neutralizing antibody in a human sample or an animal sample. EV71 virus particles are coated on the ELISA plate, the enzyme-labeled EV71 neutralizing antibody is diluted according to a certain proportion, then respectively mixed with the sample to be assayed and the reference material of the EV71 neutralizing antibody and reacted with an EV71 antigen coated on the ELISA plate, a semi-logarithmic standard curve is drawn according to the absorptance percentage of OD value of the reference serum reaction and the logarithm of the potency of the reference material of the EV71 neutralizing antibody after color development, and the absorptance percentage of the OD value of the sample to be assayed is substituted ina standard curve equation to calculate the corresponding neutralizing potency.

Description

technical field [0001] The present invention relates to the detection method and test kit or reagent that are used to carry out qualitative and quantitative competitive ELISA for human and animal EV71 neutralizing antibody (neutralizing antibody against EV71), specifically can relate to serum (serum) A detection method and a kit or reagent for detecting neutralizing antibodies against EV71, particularly involving a competitive enzyme-linked immunosorbent detection method for neutralizing antibodies against EV71. Background technique [0002] The EV71 virus (enterovirus) particle is a spherical structure with icosahedral stereosymmetry, without envelope and protrusion, extremely small, with a diameter of about 24-30 nm, and its genome is a single-stranded positive-strand RNA. The virological classification of EV71 virus belongs to the genus Enterovirus in the Picornaviridae family, and is divided into three genotypes: A, B, and C. The EV71 virus is mainly transmitted through...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/563G01N33/569C12P21/08
Inventor 蔡芳高强张小梅尹卫东
Owner SINOVAC BIOTECH
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