Antibody detection kit (OmpC-IgA)

A technology for detecting kits and antibodies, which is applied in biological testing, measuring devices, material inspection products, etc., and can solve the problems of not detecting OmpC antibody ELISA kits, etc.

Inactive Publication Date: 2013-05-08
苏州和锐生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] There is a double-antibody sandwich ELISA kit for detecting

Method used

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  • Antibody detection kit (OmpC-IgA)
  • Antibody detection kit (OmpC-IgA)
  • Antibody detection kit (OmpC-IgA)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0014] Example 1 Preparation of Escherichia coli OmpC

[0015] Reagents and instruments

[0016] 1) Reagent

[0017] Escherichia coli host bacteria DH5α, BL21(DE3), cloning vector pCR2.1T-vector, expression plasmid pET28a(+), DNA polymerase rTaq, T4 DNA ligase, DNA polymerase rTaq, LATaq and restriction endonuclease BamH I, Hind III and EcoR I, BamH I, DL2000 DNA Marker, T4 DNA ligase, low molecular weight standard protein, DNA gel recovery kit, IPTG, etc.

[0018] 2) Instrument

[0019] Ordinary shaker SCS-24; water-proof constant temperature electric heating incubator; Biophotometer spectrophotometer, desktop refrigerated centrifuge Centrifuge 5810R, desktop centrifuge MiniSpin; high-speed refrigerated centrifuge; protein electrophoresis and gel imaging system; PCR instrument; ultrasound Lysis instrument; constant temperature metal bath; HIS protein purification column, etc.

[0020] 3) Experimental method

[0021] vector construction

[0022] Primers ATTG GATC CGCT CC...

Embodiment 2

[0027] Example 2 Preparation of enzyme-labeled anti-human IgA antibody

[0028] Horseradish peroxidase-labeled anti-antibody: The enzyme-labeled anti-human IgA antibody was obtained by coupling the anti-human IgA antibody with horseradish peroxidase by the glutaraldehyde method. The method is to dissolve 10mg of HRP in 0.2ml of PH6.80.mol / LPBS containing 1.25% glutaraldehyde,

[0029] Dissolve 10mg of HRP in 1ml of 0.2mol / L pH5.6 acetate buffer, dissolve and mix well, then add 1ml of 0.1mol / L NaI04 solution, mix well, put it in the refrigerator for 20min, take it out and add 0.4mol / L ethylene glycol solution 0.5ml, after standing at room temperature for 30min, add 1ml of aqueous solution (or PBS) containing 5mg of anti-human IgA antibody, mix well and put in a dialysis bag, slowly stir and dialyze with 0.05mol / L, pH value 9.6 sodium carbonate buffer for 6h (or overnight ) to combine, then take out and add 0.2ml of NaBH4 solution (5mg / ml), and put it in the refrigerator for 2h...

Embodiment 3

[0030] Example 3 microwell plate coating

[0031] Coating buffer: pH9.60.5mol / L sodium bicarbonate buffer

[0032] Blocking solution: 1% BSA (bovine serum albumin) in PBS

[0033] Dilute OmpC with coating buffer, add 100ul to each well, incubate at 37°C for 2h or overnight at 4°C; pour off the coating solution, wash with washing solution 5 times, each time for 1min; pat dry, then add to each well 200ul blocking solution, incubate at 37°C for 2h; pour off the liquid in the well, dry and store in a vacuum-sealed aluminum film.

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PUM

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Abstract

The invention relates to a detection kit and detection method for detecting Escherichia coli outer membrane protein C (OmpC) IgA antibody, belonging to the field of immunoassay detection. The OmpC antibody kit provided by the invention comprises a OmpC-coated microporous plate and an enzyme-labeled anti-human IgA antibody. The detection kit provided by the invention has the characteristics of high accuracy, high specificity, high sensitivity and the like, can be used for detecting human body tissue OmpC IgA antibody, and has important functions in diagnosing certain autoimmune diseases.

Description

technical field [0001] The invention relates to the field of immunoassay detection. Specifically, the present invention relates to an immunoassay detection kit for detecting anti-Escherichia coli outer membrane porin C IgA antibody. technical background [0002] Escherichia coli is one of the normal bacteria in the human intestinal tract and generally does not cause disease. However, in some special cases, when the body overreacts to a certain protein of Escherichia coli, enteritis symptoms such as abdominal pain and diarrhea may occur. Studies have shown that a non-specific inflammatory bowel disease (Inflammatory Bowel Disease, IBD) with complex etiology is related to the body's excessive response to Escherichia coli outer membrane porin C, and about 50% of IBD patients have serum anti-OmpC IgA antibodies It is more than 2 times higher than that of normal people. Therefore, the detection of anti-OmpC IgA in serum can assist in the diagnosis of IBD. [0003] There is a d...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/569G01N33/535G01N21/76
Inventor 朱凡唐太清陈菲霍如松
Owner 苏州和锐生物科技有限公司
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