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Coating antigen, enzyme-labeled antibody and blocking method kit for detecting porcine pseudorabies virus ge antibody

A technique for porcine pseudorabies and a kit, which is applied in the field of biomedicine, can solve the problems of poor specificity of results, false positive results, false negatives, etc., and achieves the effects of favorable purification, good specificity and high sensitivity

Active Publication Date: 2022-04-08
LUOYANG PULIKE WANTAI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the detection method of PRV gE antibody is mainly blocking ELISA, while the existing commercial blocking ELISA kits are originally coated with protein or whole virus. The PRV gE recombinant protein or its antigenic epitope protein constructed and expressed by bacillus and baculovirus is detected by the kit prepared by it, and the specificity of the result is poor, and false negatives are prone to occur, thereby missing detection; when the original whole virus is encapsulated, the whole virus It is an antigen obtained by a series of inactivation and purification of the classic strain (due to the variant strain becoming popular since October 2011). The sensitivity of the prepared kit to detect the serum results of the variant strain after infection is not good, and it is easy to cause false positive results, and the gray area There are many samples, and it is impossible to accurately judge whether the samples are negative or positive, which is not conducive to the purification of pseudorabies

Method used

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  • Coating antigen, enzyme-labeled antibody and blocking method kit for detecting porcine pseudorabies virus ge antibody
  • Coating antigen, enzyme-labeled antibody and blocking method kit for detecting porcine pseudorabies virus ge antibody
  • Coating antigen, enzyme-labeled antibody and blocking method kit for detecting porcine pseudorabies virus ge antibody

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] The preparation of embodiment 1 porcine pseudorabies virus antigen

[0053] 1.1 Selection of strains for antigen

[0054] Select representative strains among the two types of porcine pseudorabies virus strains to culture and proliferate to obtain their virus liquid, for example: the classic strain is the Fa strain, purchased from the China Veterinary Drug Control Institute; the variant strain is the HN1201 strain.

[0055] 1.2 Porcine pseudorabies virus inactivation, concentration and purification methods

[0056] 1.2.1 Inactivation method

[0057] Inactivation methods include: formaldehyde inactivation method (MH1 for short), formaldehyde concentration range 0.05%-0.5% (v / v), standing at 25-37°C or shaking at 50-200r / min, inactivation time 24-48 Hours; Diethyleneimine BEI inactivation method (referred to as MH2), BEA first 2 hours of cyclization treatment, virus inactivation is preferably in the concentration range of 14mM ~ 70mM, 25 ~ 37 ° C shaker 50 ~ 200r / min, in...

Embodiment 2

[0068] Embodiment 2 Preparation and identification of monoclonal antibody and enzyme-labeled antibody

[0069] 2.1 Preparation and identification of monoclonal antibodies

[0070] 2.1.1 Preparation of monoclonal antibody 11H1

[0071] Preparation of immunogen: The Fa strain with the best evaluation effect in Example 1 was purified to obtain 2.5mg / ml, 91% antigen after purification, HN1201 strain obtained through purification 6 and 8 respectively, and purification 4 and purification 6 A total of 4 immunogens were prepared by combining the mixed antigens of the classic strain and the variant strain.

[0072] Hybridoma preparation: see Ed Harlow et al., "Antibodies A Laboratory Manual", Cold Spring Harbor Laboratory 1988. First, the four immunogens were immunized with multi-point intraperitoneal and back subcutaneous injections at intervals of 2 weeks for 6-8 days. Week-old female Balb / C mice were mixed three times (50 μg / mouse, mixed with the same amount of Freund's complete adj...

Embodiment 3

[0113] Preparation and detection method of embodiment 3 kit

[0114] 3.1 Preparation of the kit

[0115] Antigen-coated plate: use CB buffer to separate the single antigen prepared by purification 4, purification 6, and purification 8, the mixed antigen prepared by purification 4 and 6, the mixed antigen prepared by purification 1 and 5, and the gE recombinant protein antigen respectively Dilute to a final concentration of 1 μg / ml for coating, 100 μl / well at 2-8°C for 16-24 hours, add 150 μl / well with 3% skimmed milk powder PBS solution, block at 2-8°C for 16-24 hours or at 37°C for 2 hours, discard the blocking solution and store at 2-8°C for later use.

[0116] Enzyme-labeled reagent: Take PBS solution, 200ml of fetal bovine serum, 4mg of AM dye, mix well, and add PBS to make the volume 1L. Dilute the enzyme-labeled antibody with the above solution at a final titer of 1:10000, filter through 0.22 μm, and aseptically dispense.

[0117] Sample diluent: take PBS solution, 20...

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Abstract

The invention relates to a blocking method kit for detecting porcine pseudorabies virus gE antibody, which contains a support medium coated with the porcine pseudorabies virus mutant strain whole virus inactivated antigen of the present invention or coated pig The support medium for the whole virus inactivated antigen of the variant strain of pseudorabies virus and the inactivated antigen of the whole virus of the classic strain of pseudorabies virus, the enzyme-labeled monoclonal antibody 11H1, and the antigen-antibody reaction for the porcine pseudorabies virus Detection reagents, negative control, positive control for detection. The kit of the invention can broadly detect the gE antibody in the serum of pigs infected with porcine pseudorabies virus, has high detection sensitivity and good specificity, has few gray areas after detection, i.e. suspicious samples, and is easy to purify pseudorabies.

Description

technical field [0001] The invention relates to a blocking method detection kit for detecting porcine pseudorabies virus gE antibody, a coating antigen contained therein, a method for preparing the coating antigen, and a detection antibody, belonging to the field of biomedicine. Background technique [0002] Pseudorabies (PR; also known as Aojeszky's disease, Aujeszky's disease), is caused by pseudorabies virus (Pseudorabies Virus, PRV), including domestic animals and a variety of wild animals with fever, itching and cerebral spinal cord disease. Inflammation is an important infectious disease with symptoms, which has caused huge economic losses to the healthy development of the pig industry in my country and even the world, and has become one of the major infectious diseases that seriously endanger the healthy development of the pig industry. Pigs are the source of infection, natural host and reservoir of the disease, and healthy pigs can be infected with the disease through...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/032
Inventor 田克恭晋育丹昌静峰
Owner LUOYANG PULIKE WANTAI BIOTECH
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