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ELISA test kit of human TFF3

A technology for detection kits and reagents, which can be used in biological tests, measuring devices, material inspection products, etc., and can solve problems such as increased content

Inactive Publication Date: 2010-05-12
WUHAN SANYING BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although some studies have pointed out that the content of TFFs is increased in various cancer cases, the mechanism of these proteins' function remains to be further studied

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1: Expression of TFF3 protein in Escherichia coli

[0054] (1) Prokaryotic expression of the 8kDa TFF3 protein fragment: BC017859 DNA (purchased from the American Type Culture Collection, ATCC) was used as a template, and a pair of specific forward and reverse primers (primer 1: TTTT GAATTC ATG CTG GGG CTGGTC CTG GCC; primer 2: TTTT CTCGAG TCA GAA GGT GCA TTC TGC TTC), the TFF3 DNA fragment was amplified by PCR, and the resulting fragment was cloned into the vector pET28a (purchased from Merck, Germany), and transformed into BL21(DE3) (purchased from Merck) competent Escherichia coli, Positive clones were screened based on kanamycin resistance. The plasmid DNA of the positive clone was extracted for identification, and a fragment with the same molecular weight as the target fragment was obtained, which was initially judged as positive. Plasmid DNA was extracted from PCR-positive clones and sequenced. The results showed that the TFF3 cDNA fragment was correctly ...

Embodiment 2

[0057] Embodiment 2: Preparation of monoclonal antibody

[0058] Taking the TFF3 obtained in Example 1 as an example, mice were immunized with TFF3 as an antigen to obtain lymphoid B cells secreting specific monoclonal antibodies, which were fused with mouse myeloma cells to obtain hybridoma cells, and directly obtained from the supernatant after cell culture The anti-TFF3 monoclonal antibody was isolated, or the hybridoma cells were directly inoculated into the peritoneal cavity of mice to obtain the monoclonal antibody. The latter procedure is exemplified as follows:

[0059] (4) Animal immunization of TFF3 protein: After mixing the TFF3 protein fragment of about 8kDa with complete Freund's adjuvant and fully emulsifying it, immunize BALB / c healthy mice aged 8-12 weeks (purchased from Hubei Provincial Laboratory Animal Research Center) 3 to 4 rats were injected subcutaneously, with an interval of 2 weeks between each immunization, and the second and subsequent immunizations...

Embodiment 3

[0062] Embodiment 3: the preparation of polyclonal

[0063] (1) Animal immunization: Anesthetize 3 to 4 healthy Japanese big-eared white rabbits (purchased from Hubei Experimental Animal Research Center) 3-4 months old and weighing more than 1.7Kg, use the code on the 0th day and the 28th day The DNA expression vector of human TFF3 was immunized. For the expression vector, refer to the "Expression System in Mammalian Cells" in the "Host Cell" above. Specifically, for the first and second immunizations, each rabbit was intramuscularly injected with 0.6 mg of DNA expression vector, and briefly apply a mild electric current to the injection site to stimulate the muscle cells in the site to take up DNA. Then, intramuscular injection of recombinant human TFF3 protein such as SEQ 1 is given to rabbits every month to boost immunity. Fully emulsified in complete Freund's adjuvant.

[0064] (2) Serum acquisition: 14 days after each immunization, a blood sample was collected from the m...

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Abstract

The invention relates to a new ELISA test kit of human TFF3, and the key technique is characterized in that the specific anti-TFF3 antibody is prepared by a gene engineering method, an elisa plate coated by the antibody is used as an important part of the kit, and then is assembled with TFF3 multi-antibody, secondary antibody marked by horse radish peroxidase, TFF3 standard, TFF3 positive control, sample diluent, washing liquor, coloration solution and stopping solution into the ELISA test kit of human TFF3; ELISA is carried out by a bi-anti sandwich method to establish a measurement method which can sensitively and rapidly capture TFF3 concentration in human serum or cell and tissue culture, and has the characteristics of high specificity and high stability. The kit is mainly used for clinical research use such as general laboratory or gastrointestinal tract pathology.

Description

technical field [0001] The present invention relates to a variety of biological preparations - human TFF3 protein, anti-TFF3 specific polyclonal antibody, monoclonal antibody, and an ELISA detection kit for human TFF3 content detection, suitable for human tissue, cell culture or serum Quantitative detection of TFF3 in . Background technique [0002] TFFs (The trefoil factor families), the trefoil factor family, is a secreted polypeptide on the surface of a class of mucin-producing epithelial cells, because its proteins contain one or more amino acid residues from 38 to 39 through internal 6 It is named for the trefoil structure formed by three disulfide bonds formed by cysteine ​​residues. [0003] The genes of TFFs are located on chromosome 21q22.3, and the genes of all three proteins in the family occupy a total of 55kb. TFFs are expressed in multiple tissues in the human body, but the highest expression level is in the gastrointestinal tract. [0004] In the serum of p...

Claims

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Application Information

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IPC IPC(8): G01N33/53G01N33/68G01N33/577
Inventor 彭进洪兰萍
Owner WUHAN SANYING BIOTECH
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