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Blocking ELISA (Enzyme-Linked Immunosorbent Assay) method for detecting special avian HEV (Hepatitis E Virus) antibody

A hepatitis E virus, specific technology, applied in the field of serological diagnosis, can solve the problems of no blocking ELISA method for detection of avian hepatitis E virus specific antibody, no commercial combination, popularization and use, etc., achieves good application prospects, easy to use Large-scale promotion, good repeatability effect

Active Publication Date: 2013-10-09
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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  • Blocking ELISA (Enzyme-Linked Immunosorbent Assay) method for detecting special avian HEV (Hepatitis E Virus) antibody
  • Blocking ELISA (Enzyme-Linked Immunosorbent Assay) method for detecting special avian HEV (Hepatitis E Virus) antibody
  • Blocking ELISA (Enzyme-Linked Immunosorbent Assay) method for detecting special avian HEV (Hepatitis E Virus) antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1 Preparation of recombinant protein with 268 amino acids at the C-terminus of poultry HEV ORF2 protein

[0052] 1.1 Amplification of 268 amino acid genes at the C-terminal of poultry HEV ORF2 protein

[0053] Based on the whole genome sequence of the Chinese isolate of avian hepatitis E virus (Hepatitis E virus from China, CaHEV, Genbank number GU954430), primers with BamHI and XholI restriction sites were designed. The primer sequences are as shown in 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] 5×First-strand buffer 4μl

[0057] dNTP (10mM) 1μl

[0058] 0.1M DTT 0.5ul

[0059] Primer (20pm / μl) 1μl

[0060] RNA 8μl

[0061] RNase inhibitor 1 μl

[0062] Superscript II Reverse Transcriptase 0.5μl

[0063] Total volume 20μl

[0064] Reverse transcription conditions:

[0065] 55℃ 60min

[0066] 85℃ 5min

[0067] S...

Embodiment 2

[0094] The preparation of embodiment 2 monoclonal antibody 1H5

[0095] 2.1 Emulsification of protein

[0096] The purified target protein was emulsified by mixing complete and incomplete Freund's adjuvant 1:1 (V / V).

[0097] 2.2 Immunization procedure

[0098] 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.

[0099] 2.3 Cell Fusion

[0100] The immunized BALB / c mice were sacrificed, and the splenocytes and SP2 / 0 myeloma cells of the immunized mice were as...

Embodiment 3

[0105] Embodiment 3 detects the blocking ELISA method of avian hepatitis E virus specific antibody

[0106] 3.1 Optimization of optimal reaction conditions for blocking ELISA method

[0107] 3.1.11. Solution preparation

[0108] a) Coating buffer: 0.01M PBS (500ml, pH=7.2±0.1): NaCl4.25g, NaH 2 PO 4 2H 2 O0.178g, Na 2 HPO 4 12H 2 O1.386g, 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;

[0109] b) Washing solution (PBST) (1L): add Tween-205ml to every 1L of 0.01M PBS (pH=7.2±0.1), and mix thoroughly.

[0110] c) 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;

[0111]d) 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 to...

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Abstract

The invention discloses a blocking ELISA (Enzyme-Linked Immunosorbent Assay) method for detecting a special avian HEV (Hepatitis E Virus) antibody. According to the invention, 268 amino acids (aHEV-ORF2-268) at the end C of an avian HEV ORF2 protein purified through prokaryotic expression are used as coating antigens, a monoclonal antibody 1H5 purified through affinity chromatography in the region is used as a blocking antibody, and blocking ELISA detection conditions are optimized through a great number of experiments, so that the method has good repeatability, specificity and sensibility.

Description

technical field [0001] The invention belongs to the field of serological diagnosis, and relates to avian hepatitis E virus ORF2 recombinant protein (aHEV-ORF2-268), a monoclonal antibody 1H5 for recognizing the protein and a blocking ELISA method for detecting avian hepatitis E virus specific antibody. Background technique [0002] Avian hepatitis E virus (HEV) is the main pathogen of big liver and spleen disease (BLS) or hepatitis-splenomegaly (HS) syndrome in chickens, mainly infecting 30- 72-week-old laying hens and broiler breeders caused an increase in the death rate of the chickens and a decrease in the egg production rate. Some chickens had 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 analysis, it is found that the homology...

Claims

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Application Information

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Patent Type & Authority Applications(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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