A kit for measuring anti-cyclic citrullinated peptide antibody and its application
A technology against cyclic citrullinated peptides and citrullined peptides, which is applied in the direction of biological testing, general structural detail detection of gas analyzers, and measuring devices, and can solve the problems of low negative coincidence rate, improved detection sensitivity, and long detection time, etc. problem, to achieve the effect of improving sensitivity, avoiding result deviation, and saving labor costs
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Embodiment 1
[0048] Example 1: Preparation of biotinylated antigen
[0049] Synthesis of citrulline 16 branched peptide:
[0050] According to the structural characteristics of citrulline and arginine, bioinformatics-related software is used to predict its spatial structure changes, and the known citrullined antigens are analyzed, classified, and screened out peptide libraries containing citrulline, which will contain citrulline A group of citrulline peptides was obtained by screening the polypeptide library of acid and rheumatoid arthritis serum, and four citrulline peptides with the highest combined detection sensitivity were screened. Using the characteristics of two amino groups of lysine, each citrulline peptide was citrulline peptide chains are coupled to form a molecule with four identical branched peptide chains, and finally four citrulline tetrabranched peptides are obtained, and then these four different branched peptide molecules are coupled into a molecule with lysine, which is...
Embodiment 2
[0065] Example 2: Preparation of alkaline phosphatase-labeled anti-human IgG antibody:
[0066] Materials and Instruments:
[0067] Materials: anti-human IgG antibody, alkaline phosphatase, coupling agent 2-iminosulfane hydrochloride, glycine, tris buffer.
[0068] Instruments: G-25 gel column, reagent low temperature storage box, Supperdex200 gel purification column, analytical balance, biochemical incubator;
[0069] Steps:
[0070] Step 1: Add 3mg of anti-human IgG antibody to 40mL of 2-iminosulfane hydrochloride coupling agent with a concentration of 10mg / mL, and let stand at 20°C for 20min;
[0071] Step 2: Add 2mL of 0.08mol / L glycine solution, let it stand at 20°C for 4min, use G-25 gel column to desalt, collect the activated anti-human IgG antibody, and store it at 5°C for later use;
[0072] Step 3: Add 3 mg alkaline phosphatase solution to 4 mg / mL 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid succinimide ester solution at 25°C Let it stand for 30 minutes, us...
Embodiment 3
[0075] Example 3: Preparation of calibrator: used for calibration curve.
[0076] 1. Materials and instruments:
[0077] Materials: anti-citrullinated peptide antibody, phosphate buffer saline, standard;
[0078] 2. Preparation steps:
[0079] Select an anti-citrullinated peptide antibody, dilute it in a certain ratio with a phosphate buffer solution with a pH of 7.5 and a substance concentration of 0.01 mol / L, and prepare 20RU / ml and 200RU / ml with reference to the standard. calibrator.
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