ELISA kit for quantitative detection of dermatophagoides pteronyssinus allergens

An enzyme-linked immunosorbent reagent and quantitative detection technology, applied in the biological field, can solve the problems of high cost, insufficient detection sensitivity, and inconvenient promotion of nano-magnetic particle enzyme-linked immunoassay, and achieve high sensitivity, less time-consuming, and low cost. low effect

Active Publication Date: 2016-02-10
RAYBIOTECH INC GUANGZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Among the existing related detection technologies, capillary electrophoresis electrochemical detection technology requires very professional and relatively expensive detectors, which is not very convenient to promote, and the cost of nano-magnetic particle enzyme-linked immunoassay is relatively high, and alkaline phosphatase...

Method used

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  • ELISA kit for quantitative detection of dermatophagoides pteronyssinus allergens
  • ELISA kit for quantitative detection of dermatophagoides pteronyssinus allergens
  • ELISA kit for quantitative detection of dermatophagoides pteronyssinus allergens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Allergen DerP2 gene cloning expression and protein purification

[0028] 1. Nucleotide sequence of allergen DerP2 gene and amino acid sequence of recombinant expression.

[0029] According to the DerP2 gene sequence (Genbank accession number is AM263560), primers were designed,

[0030] The primer sequences are:

[0031] Upstream primer: GATCAAGTCGATGTCAAA

[0032] Downstream primer: GCTAAAATCCGCGATTAA

[0033] The DerP2 gene was cloned into the expression vector pET28a, and the sequencing method was used to verify whether the gene was cloned successfully.

[0034] 2. Construction of expression plasmids and acquisition of high-expression engineered strains

[0035] Expression vector construction method: obtain the target DerP2 gene by PCR amplification, use NdeI+XhoI double cutase PCR product and pET28a plasmid to connect the two fragments with T4DNA ligase, and transform the ligated product into Escherichia coli top10. Single clones were selected on the...

Embodiment 2

[0040] Embodiment 2: Preparation of allergen DerP2 monoclonal antibody

[0041] 1. Preparation of DerP2 monoclonal antibody cell line

[0042] Immunize BalB / c mice with the above-mentioned purified DerP2 protein: mix Freund's complete adjuvant with antigen 150 μl (50 μg) in an equal volume for the first immunization, fully emulsify, and inject subcutaneously at multiple points. Two weeks later, the mice were immunized again, and the Freund's incomplete adjuvant was used instead, and the injection volume and method remained unchanged. After that, the mice were continuously immunized with the same method every 2 weeks, and immunized three times in total. After the serum titer of the mice reached the requirement, cell fusion was prepared, and 50 μg antigen was injected intraperitoneally into the mice for shock immunization three days before the fusion.

[0043] Mouse myeloma cells SP2 / 0 were prepared at the same time as the mice were immunized.

[0044] The sensitized B lymphocyt...

Embodiment 3

[0050] Example 3: Preparation of allergen DerP2 polyclonal antibody

[0051] 1. Preparation of DerP2 polyclonal antibody

[0052] Immune SPF grade New Zealand rabbits with the above-mentioned purified DerP2 protein: emulsify the antigen with Freund's adjuvant, and immunize animals by multi-point injection from the back of SPF grade rabbits. After three immunizations, a small amount of serum was collected from the marginal ear vein for ELISA detection. If the titer reaches the requirement, the animal is given a final booster immunization, and a large amount of serum is collected from the heart three days later.

[0053] 2. Detection of serum titer by ELISA enzyme-linked immunosorbent assay

[0054] Coat each well with 100ng of immunogen on the ELISA plate, add 5% skimmed milk powder, different concentrations of polyclonal antibodies, biotin-labeled species secondary antibody goat anti-rabbit, HRP-streptavidin and TMB substrate for color development. Read on a Bio-Tek micropl...

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Abstract

The invention provides a monoclonal antibody capable of recognizing an allergen of DerP2, wherein the monoclonal antibody is secreted by a hybridoma cell strain DerP2 with the accession number being CCTCC NO:C2014112. The invention further provides an ELISA kit for quantitative detection of dermatophagoides pteronyssinus allergens of DerP2 and Derf2. The ELISA kit includes an enzyme label plate, a specimen diluent, a coating antibody, a detecting antibody, a detecting antibody diluents, a standard substance, a washing liquid, a substrate chromogenic reagent, and a stopping solution, wherein the coating antibody is the monoclonal antibody related in the claim 1, and the detecting antibody is an anti-DerP2 polyclonal antibody marked with HRP. The anti-DerP2 monoclonal antibody is excellent in specificity and produces no cross reaction with a DerP1 protein. Therefore, based on the anti-DerP2 monoclonal antibody, the fast, specific, and sensitive in-vitro diagnostic kit for detecting the allergens is established.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to an ELISA kit for detecting dust mite allergens, in particular to an ELISA kit for quantitatively detecting dust mite allergens DerP2 and Derf2. Background technique [0002] Dust mite is a strong allergen that causes many allergic diseases such as asthma, atopic dermatitis and infantile eczema. DerP2 is a major specific allergen from dust mite (Dermatophagoides spteronyssinus), and 80%-90% of patients allergic to dust mite have a strong reaction to this allergen. The detection of DerP2 index in dust mite lysate can calculate the content of dust mite in the air, so as to prevent the occurrence of allergies in time, and also provide data support for environmental testing. [0003] The currently developed technologies for detecting allergens in mite lysates include capillary electrophoresis electrochemical detection technology and nano-magnetic particle enzyme-linked immunoassay technolo...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/535
Inventor 罗树红方建民吕志强张文姬张玉明黄若磐
Owner RAYBIOTECH INC GUANGZHOU
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