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Anti-venomous-snake PLA2 protein antibodies and application thereof

A PLA2, protein technology, applied in the direction of anti-animal/human immunoglobulin, material testing products, instruments, etc., can solve the problems of failing to identify the snake species that bite, delaying the treatment window, etc., to improve the diagnosis of snakebite The effect of efficiency

Inactive Publication Date: 2019-10-11
KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Numerous cases of fatalities due to failure to identify biting snake species, delaying valuable treatment window

Method used

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  • Anti-venomous-snake PLA2 protein antibodies and application thereof
  • Anti-venomous-snake PLA2 protein antibodies and application thereof
  • Anti-venomous-snake PLA2 protein antibodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] The invention provides a preparation method of an anti-venomous snake PLA2 protein antibody, comprising:

[0067] Step S01, preparing PLA 2 protein antigen.

[0068] Specifically, PLA was synthesized by Jill Biochemical (Shanghai) Co., Ltd. through solid-phase synthesis 2 protein peptide antigen. Among them, the amino acid sequences of the ten small peptide antigens are respectively shown in SEQ ID NO: 6-15, and 6 histidines are added to the C-terminus of each small peptide antigen, for example, added to the C-terminus of SEQ ID NO: 6 6 histidines were obtained; 50 mg of polypeptide antigens were synthesized from each short peptide antigen and the purity was greater than 95%.

[0069] Step S02, use PLA 2 The mice were immunized with the protein antigen, and the splenocytes of the immunized mice were obtained.

[0070] Specifically, using the PLA prepared in step S01 2 Five Balb / c mice were immunized with protein antigens respectively. Among them, the Balb / c mouse...

Embodiment 2

[0080] Phyllostachys anti-PLA 2 Protein Monoclonal Antibody Tritoxomab-PLA 2 -1. Tritoxomab-PLA 2 -2 with Phyllostachys PLA 2 Protein epitope binding test, specific steps:

[0081] 1) Coating: Zhuyeqing PLA 2 Protein (5 μg / ml) was diluted with coating solution and added to 96-well plate in an amount of 100 μl per well, and 100 μl of coating solution was added to blank wells, and coated overnight at 4°C.

[0082] 2) Washing: Pour off the coating solution in the wells of the plate the next day, wash three times with washing solution, 250 μl per well, 3-5 minutes each time, and pat dry as much as possible.

[0083] 3) Blocking: 200 μl of blocking solution per well, 37° C. for 1 hour. Wash three times afterwards and pat dry.

[0084] 4) Add 100 μl antibody Tritoxomab-PLA to the positive well 2 -1 or antibody Tritoxomab-PLA 2 -2 (5 μg / ml), add 100 μl of blocking solution to negative wells and blank wells, and keep at 37° C. for 1 hour. Wash three times afterwards and pat d...

Embodiment 3

[0096] This embodiment provides a double-antibody sandwich ELISA detection kit, including a PLA-coated 2 Solid phase carrier for protein antibody, enzyme-labeled PLA 2 Protein antibody, diluent, chromogenic substrate, stop solution, sealing membrane, and positive standard. Among them, PLA 2 Antibodies to proteins including anti-Pipaphyllum PLA 2 Protein monoclonal antibody panel, anti-tip PLA 2 Protein monoclonal antibody panel, Anti-Viper PLA 2 Protein monoclonal antibody panel, Anti-COBRA PLA 2 Protein monoclonal antibody panel, Anti-Apis viper PLA 2 Protein monoclonal antibody panel.

[0097] Phyllostachys anti-PLA 2 Protein monoclonal antibody group (Tritoxomab-PLA 2 -1 and Tritoxomab-PLA 2 -2), Tritoxomab-PLA 2 -1 and Tritoxomab-PLA 2 -2 as coating antibody and enzyme-labeled antibody respectively, wherein, Tritoxomab-PLA 2 -2 includes a detectable label, the solid phase carrier is a microwell plate, magnetic particles or a plastic tube, and the enzyme label i...

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Abstract

The invention relates to anti-venomous-snake PLA2 protein antibodies and application thereof. The anti-venomous-snake PLA2 protein antibodies comprise monoclonal antibodies of tritoxomab-PLA2, protoxomab-PLA2, viptoxomab-PLA2, najatoxomab-PLA2 or glotoxomab-PLA2, and have high affinity and specificity. The monoclonal antibodies are prepared by adopting PLA2 proteins of the five kinds of venomous snakes as antigens for immunizing mice through a hybridoma fusion technology, antibody screening is conducted by taking the amino acid sequences of of two hypervariable regions in the PLA2 proteins asantigens, and finally, ten monoclonal antibodies of tritoxomab-PLA2, protoxomab-PLA2, viptoxomab-PLA2, najatoxomab-PLA2 and glotoxomab-PLA2 are obtained in total. One or a combination of more of the ten monoclonal antibodies can be used as a diagnostic test reagent for snake wounds. The anti-venomous-snake PLA2 protein antibodies can be prepared into the diagnostic reagent or kit for the snake wounds, and the diagnostic efficiency of the snake wounds is improved.

Description

【Technical field】 [0001] The present invention relates to the field of antibody and immune detection, in particular to anti-venomous snake PLA 2 Protein antibodies and their applications. 【Background technique】 [0002] According to the statistics of the World Health Organization, there are 5,000,000 cases of venomous snake bites in the world every year, of which the mortality rate is 2%, and those who leave serious sequelae account for 8%. Its harm is far greater than the sum of other tropical diseases. The above data are only data with medical records, and the actual number of poisonous snake bites is much larger. In my country, 200,000 people are bitten by venomous snakes every year, and the death rate from snakebite is 5%-10%. The death rate and disability rate are far higher than the global average. [0003] Snake venom contains a large number of toxin molecules, which can act on key proteins in various systems of the human body, such as coagulation factors in the bl...

Claims

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Application Information

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IPC IPC(8): C07K16/18G01N33/68G01N33/577
CPCC07K16/18G01N33/577G01N33/6854G01N2333/4609G01N2800/42
Inventor 赖仞龙承波余永志吕秋敏李家耀
Owner KUNMING INST OF ZOOLOGY CHINESE ACAD OF SCI
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