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Foot-and-mouth disease vaccine host cell protein double-antibody sandwiched enzyme-linked immunosorbent assay kit as well as using method thereof

An enzyme-linked immunosorbent reagent and host cell protein technology, which is applied in the direction of serum immunoglobulin, anti-animal/human immunoglobulin, biological testing, etc., can solve the problems of vaccine detection supervision loopholes and lacks, and achieve the goal of coating antibodies Stable and uniform amount, easy and sufficient emulsification, avoiding cross-effects

Active Publication Date: 2014-05-14
内蒙古必威安泰生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Paradoxical to this need is the absence of products related to the detection of host cell proteins for FMD vaccines, which creates gaps and loopholes in the oversight of vaccine testing

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1B

[0046] Preparation of Example 1 BHK21 cell protein

[0047] Material: 10×Phosphate-NaCl Buffer: 100 mmol / LPBS, pH 7.2. Said NaCl80g, Na 2 HPO 4 12H 2 O29g, KCl2g, KH 2 PO 4 2g, add distilled water to dissolve, add 100mmol / LEDTA20m1, dilute to 1000mL with deionized water;

[0048] BHK21 cells (baby hamster kidney cells) were purchased from China Institute of Inspection, DMEM medium, horse serum, and dialysis bags (8000nm-14000nm).

[0049] Extraction steps:

[0050] BHK21 cells (baby hamster kidney cells) were purchased from the China National Institute of Inspection and Quarantine. They were cultured and passaged in DMEM medium plus horse serum. When the cells were full of the bottom of the culture bottle, they were washed 5 times with 10×PBS with a pH of 7.2, and the culture medium was washed clean. Digest the cells with enzymes and wait for the cells to fall off, then wash 5 times with 10×PBS with a pH of 7.2, put them into a dialysis bag with a pore size of 8000-1400...

Embodiment 2

[0059] Embodiment 2 antigen emulsification

[0060] Material: Freund's adjuvant, the antigen protein solution obtained in Example 1

[0061] Steps:

[0062] (1) Dilute the protein solution according to the required amount (BHK21 cell protein solution 500ug / ml, add 10ml Freund's adjuvant to 10ml);

[0063] (2) Mix Freund's adjuvant with equal volume of protein solution;

[0064] (3) Both ends of the thin silicone tube are connected to a No. 5 needle and tied with a cable tie;

[0065] (4) Add a small peristaltic pump in the middle, and its speed is 2ml / min;

[0066] (5) Place the centrifuge tube containing the antigen-adjuvant mixture in an ice pack;

[0067] (6) Put both ends of the tube into the centrifuge tube at the same time, and one end extends into the bottom of the tube;

[0068] (7) Turn on the peristaltic pump and circulate for 40 minutes until the ice water titration test does not diffuse and the emulsification is complete.

Embodiment 3

[0069] The preparation of embodiment 3 coating antibody serum:

[0070] Materials: emulsified antigen obtained in Example 2, 2ml syringe, 10ml syringe, blood collection tube, tweezers, scissors, alcohol cotton.

[0071] Steps:

[0072] (1) Baoding rabbit (New Zealand big-eared rabbit);

[0073] (2) Remove the rabbit hair on the skin of the rabbit's ear vein;

[0074] (3) Blood collection with a 2ml syringe, 1ml / piece;

[0075] (4) The emulsified antigen obtained in Example 2 was used for immunization. For the first immunization, the emulsified antigen with Freund's complete adjuvant was used, and the rabbit was injected at four points on both sides of the back spine, with a total injection volume of 1000ug / rat. The second immunization was carried out 14 days after the first immunization, the steps and doses were the same as the first immunization, and the antigen was emulsified with Freund's incomplete adjuvant for the second immunization. Three immunizations were carried ...

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Abstract

The invention relates to a double-antibody sandwiched enzyme-linked immunosorbent assay kit for detecting a foot-and-mouth disease vaccine host cell protein. The double-antibody sandwiched enzyme-linked immunosorbent assay kit comprises a standard substance, a 96-pore elisa plate coated with a rabbit anti-BHK21 cell protein polyclonal antibody, horse radish Peroxidase labeled goat-anti-rabbit IgG, an anti-BHK21 cell protein antibody fluid, a diluent, a confining liquid, a washing liquid, a stop liquid 2M H2SO4 and a developing liquid, wherein the standard substance is a baby hamster kidney cell protein; the coated antibody and the detection antibody are both rabbit anti-BHK21 cell protein polyclonal antibodies. The coated antibody quantity in the detection kit provided by the invention is more stable and homogeneous; trace antigens are emulsified more simply, conveniently and fully; a binding antibody and a detection antibody are the same antibody and are more convenient, rapid and efficient for manufacturing the kit; the cross influence among different antibodies is prevented.

Description

technical field [0001] The invention relates to a double-antibody sandwich enzyme-linked immunoassay kit for foot-and-mouth disease vaccine host cell protein and its use method, in particular to a double-antibody sandwich enzyme-linked immunosorbent assay kit for detecting residual protein in host cells (ie young hamster kidney cells) in foot-and-mouth disease vaccines The immunological reagent kit and its application method belong to the field of biological detection. Background technique [0002] Foot-and-mouth disease is an acute, febrile, highly contagious infectious disease common to pigs, cattle, sheep and other domestic and wild cloven-hoofed animals, with more than 70 susceptible animals. The clinical feature is a vesicular eruption on the oral mucosa, hooves, and breast skin. The disease has many transmission routes and fast speed, and has repeatedly broken out in the world, causing huge political and economic losses. In view of this, the World Organization for An...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N33/535C07K16/18C07K16/06
CPCC07K16/06C07K16/18G01N33/54373G01N33/54393G01N33/6803
Inventor 赵炳武赵宏凯任美荣刘金萍王永伟吕宏亮张澍
Owner 内蒙古必威安泰生物科技有限公司
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