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Enzyme-linked immunoabsorbent kit for detecting serum amyloid protein A as well as preparation method and application of kit

An enzyme-linked immunosorbent assay and serum amyloid technology, applied in the field of analysis and testing, can solve the problem that serum amyloid A does not have an effective method, and achieve the effects of stable results and good detection specificity.

Inactive Publication Date: 2017-05-31
BEIJING MEIZHENG BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0015] There is currently no effective method for the detection of serum amyloid A (SAA) in milk

Method used

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  • Enzyme-linked immunoabsorbent kit for detecting serum amyloid protein A as well as preparation method and application of kit
  • Enzyme-linked immunoabsorbent kit for detecting serum amyloid protein A as well as preparation method and application of kit
  • Enzyme-linked immunoabsorbent kit for detecting serum amyloid protein A as well as preparation method and application of kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1: Recombinant expression and purification of recombinant bovine serum amyloid A

[0094] 1. Cloning of bovine serum amyloid A (HP) gene

[0095] Sequence analysis of the bovine serum amyloid A (SAA) gene (GeneBank ID NM_001040470.2) was performed using software, PCR amplification primers were designed to amplify the SAA gene, and restriction sites NdeI and BamHI were added at both ends.

[0096] 2. Cloning of HP gene into expression vector

[0097] The amplified SAA gene was double digested and inserted into the expression vector pET28a to construct the recombinant plasmid pET28a / SAA. Identification of Recombinant Plasmid by Enzyme Digestion and Sequence Determination

[0098] 3. Induced expression of HP gene

[0099] The recombinant plasmid pET28a / HP was transformed into Escherichia coli BL21 (DE3), and positive clones were screened. Pick positive single clones to inoculate LB kan+ medium, and culture overnight at 37°C and 200rpm. The next day, according ...

Embodiment 2

[0104] Embodiment 2: the preparation of the preparation of recombinant bovine serum amyloid A monoclonal antibody

[0105] 1. Establishment of bovine serum amyloid A hybridoma cell line:

[0106] The purified recombinant HP protein was used to immunize BALB / C mice, the first immunization was injected with Freund's complete adjuvant in the back of the neck and under the skin at multiple points, and the booster immunization was injected with Freund's incomplete adjuvant in the back of the neck, the underarm and the foot pad . Blood was collected every two weeks to detect antibody titers. Eight weeks later, the mouse spleen was taken for cell fusion with mouse myeloma cell SP2 / 0. Indirect ELISA screening was performed with recombinant HP and inactivated LM. Select strong positive cells to continue culturing, and use the limited dilution method to obtain monoclonals that can secrete antibodies. The monoclonal cells obtained after screening were expanded and stored in liquid ni...

Embodiment 3

[0111] Example 3: Preparation of Bovine Serum Amyloid A ELISA Kit

[0112] 1. Preparation of ELISA plate coated with bovine serum amyloid A antibody

[0113] w coated

[0114] The bovine serum amyloid A antibody was diluted with 0.01-0.05M PBS buffer solution of pH 6-8 to a final concentration of 1-50ug / ml, and added to the microwells of the microtiter plate in an amount of 100-300ul per well. Incubate for 1-4 hours at 37°C or overnight at 4°C

[0115] w washing

[0116] Wash the plate 3-5 times with 0.01-0.05M PBS buffer and add 0.1% Tween20

[0117] w closed

[0118] Add 300ul blocking solution (10% calf serum or 3% skimmed milk powder) to each well of the above-mentioned microtiter plate to block at room temperature for 1-3 hours

[0119] w washing

[0120] Use 0.01-0.05M, PH6-8 PBS buffer to add 0.1% Tween20 buffer to wash the microtiter plate 3-5 times

[0121] w dry

[0122] Dry at 20-37°C for 5-10 hours to dry the plate

[0123] After drying, the microtiter plate...

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Abstract

The invention discloses an enzyme-linked immunoabsorbent kit for detecting serum amyloid protein A as well as a preparation method and an application of the kit and belongs to the field of analytical test. The kit comprises an ELISA plate coated by a monoclonal antibody of a cow serum amyloid protein A, a cow serum amyloid protein A antigen standard substance, a cow serum amyloid protein A monoclonal antibody, an enzyme labeled goat anti-rat antibody, a diluent, a scrubbing solution, a developing solution and a stop buffer. The content of the serum amyloid protein A in milk is measured by a double-antibody sandwiched indirect enzyme-linked immunosorbent assay method so as to diagnose bovine subclinical mastitis. The kit disclosed by the invention is easy and convenient to operate, has the characteristics of high specificity, high sensitivity and high reproducibility and provides a novel testing tool for detection of the bovine subclinical mastitis.

Description

technical field [0001] The invention describes an enzyme-linked immunosorbent assay kit for detecting bovine serum amyloid A and its preparation method and application, which are used for detecting the concentration of serum amyloid A in milk and diagnosing bovine recessive mastitis . It belongs to the field of analytical testing. Background technique [0002] Mastitis (Mastitis) is one of the most common diseases of cows, the most frequent, and the most serious economic loss, and it is harmful to human health, because the milk of cows with mastitis often contains a large number of pathogenic microorganisms, toxins or residues after treatment. Antibiotics can cause adverse reactions such as fever, vomiting, diarrhea and allergies after consumption. With the development of my country's dairy industry and dairy industry and human beings' emphasis on public health, the hazards of dairy cow mastitis have aroused widespread concern and great attention. [0003] Mastitis not on...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/543
CPCG01N33/6893G01N33/54306
Inventor 张彦明
Owner BEIJING MEIZHENG BIOTECHNOLOGY CO LTD
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