A blocking ELISA method for detecting avian hepatitis E virus specific antibodies

A hepatitis E virus and antigen-coated technology, which is applied in the field of serological diagnosis, can solve the problems of no detection of avian hepatitis E virus-specific antibody blocking ELISA method, no commercial combination promotion and use, etc., and achieves good application prospects, Easy to promote on a large scale and easy to operate

Active Publication Date: 2018-01-12
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Regarding serological ELISA diagnostic technology, researchers in the United States have established an indirect ELISA method for the detection of avian HEV antibody by using a local isolate of avian HEV and prokaryotically expressing the second half of the capsid protein of the isolate, but this method is limited to the laboratory On the other hand, the antigen used in the ELISA method is established with the capsid protein of the genotype 2 avian HEV isolate, while the domestic avian HEV isolate is genotype 3, and there is no antigen yet It is an ELISA method for antibody detection using the capsid protein of genotype 3 avian HEV isolates
[0007] At present, there is no report on the blocking ELISA method for detecting the specific antibody of avian hepatitis E virus at home and abroad

Method used

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  • A blocking ELISA method for detecting avian hepatitis E virus specific antibodies
  • A blocking ELISA method for detecting avian hepatitis E virus specific antibodies
  • A blocking ELISA method for detecting avian hepatitis E virus specific antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of recombinant protein with 267 amino acids at the C-terminus of poultry HEV ORF2 protein

[0052] 1.1 Amplification of the C-terminal 267 amino acid genes of poultry HEV ORF2 protein

[0053] According to the whole genome sequence of avian hepatitis E virus from China (Hepatitis E virus from China, CaHEV, Genbank number GU954430), design primers with BamHI and XholI restriction sites, the primer sequences are as SEQ ID NO.2 and SEQ ID NO.2 ID NO.3.

[0054] Using the CaHEV virus suspension as a template, the target gene was amplified by RT-PCR.

[0055] Reverse transcription system:

[0056]

[0057] Reverse transcription conditions:

[0058] 55℃ 60min

[0059] 85℃ 5min

[0060] Store at 4°C.

[0061] PCR reaction system:

[0062]

[0063] PCR reaction conditions:

[0064]

[0065] 1.2 Construction of recombinant expression plasmids

[0066] The pET-28(a) vector (Promega Company) was subjected to BamHI and XholI (TaKaRa Company) ...

Embodiment 2

[0079] The preparation of embodiment 2 monoclonal antibody 1H5

[0080] 2.1 Emulsification of protein

[0081] The purified target protein was emulsified by mixing complete and incomplete Freund's adjuvant 1:1 (V / V).

[0082] 2.2 Immunization procedure

[0083] BABL / c mice aged 6-8 weeks were selected as immunized animals. Blood was collected from the tail vein before each immunization. The interval between two immunizations was 2 weeks. For the first time, complete Freund's adjuvant was used, the protein dose was 75 μg / monkey, intraperitoneal injection, and the total dose was 200 μl / bird. For the second time, Freund's incomplete adjuvant was used, and the protein dose was still 75 μg per mouse. Refer to the same dosage and method as the second time for the third immunization. Immunization was boosted once before fusion.

[0084] 2.3 Cell Fusion

[0085] The immunized BALB / c mice were killed by neck dislocation, and the immunized splenocytes and SP2 / 0 myeloma cells wer...

Embodiment 3

[0090] Embodiment 3 detects the blocking ELISA method of avian hepatitis E virus specific antibody

[0091] 3.1 Optimization of optimal reaction conditions for blocking ELISA method

[0092] 3.1.1 Preparation of solution

[0093] (1) Coating buffer: 0.01M PBS (500ml, pH=7.2±0.1): NaCl 4.25g, NaH 2 PO 4 2H 2 O0.178g, Na 2 HPO 4 12H 2 O 1.386g, dissolve to 500ml, measure the pH at 7.1-7.3 (if it exceeds this range, it cannot be used), and it can be stored at room temperature for one week;

[0094] (2) Washing solution (PBST) (1L): add Tween-205ml for every 1L of 0.01M PBS (pH=7.2±0.1), and mix well.

[0095] (3) Blocking solution: Dissolve 2.5g skimmed milk powder in 100ml PBS’T, store at 4°C for short-term and -20°C for long-term;

[0096] (4) Substrate TMB: Solution A (1L): Weigh 66.5063g of potassium citrate (potassium citrate), dissolve it in 800ml of four-distilled water and adjust the pH value to 4.0 with concentrated hydrochloric acid, add 314μl H 2 o 2 , dilute...

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Abstract

The invention discloses a blocking ELISA method for detecting the specific antibody of avian hepatitis E virus. The invention uses the 267 amino acids (aHEV‑ORF2‑268) at the C-terminal of the ORF2 protein of the prokaryotic expression and purification of the avian hepatitis E virus as the coating For the antigen, the monoclonal antibody 1H5 purified by affinity chromatography for this region is a blocking antibody. Through a large number of experiments, the detection conditions of blocking ELISA are optimized, so that the method has good repeatability, specificity and sensitivity.

Description

technical field [0001] The invention belongs to the field of serological diagnosis, and relates to a bird hepatitis E virus ORF2 recombinant protein (aHEV-ORF2-268), a monoclonal antibody 1H5 for recognizing the protein and a blocking ELISA method for detecting a bird hepatitis E virus specific antibody. Background technique [0002] Avian hepatitis E virus (Hepatitis E virus, HEV) is the main pathogen of chicken big liver and spleen disease (BLS) or hepatitis-splenomegaly (Hepatitis-Splenomegaly, HS) syndrome, mainly infecting 30-72 Week-old laying hens and broiler breeders lead to an increase in the death rate of chickens and a decrease in egg production rate. Some chickens have abdominal congestion, ovarian degeneration, and occasional hepatosplenomegaly. [0003] In 2001, scholars such as Haqshenas described the genome of avian HEV for the first time, and then Europe and Australia also described the gene sequence of domestic isolates of avian HEV. Through sequence analy...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/08C07K16/10G01N33/68G01N33/577
Inventor 赵钦周恩民刘宝元孙亚妮李爽
Owner NORTHWEST A & F UNIV
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