CN1343887A

CN1343887A - Process for preparing antigen microarray based on self antibody repertoire

Info

Publication number: CN1343887A
Application number: CN 01127073
Authority: CN
Inventors: 虞伟; 顾宁; 武建国; 廖建辉; 张海黔
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2001-08-07
Filing date: 2001-08-07
Publication date: 2002-04-10
Anticipated expiration: 2021-08-07
Also published as: CN1142433C

Abstract

A process for preparing an antigen microarray based on self antibody repertoire includes pre-treating substrate for adapting molecular self-assembly, generating a layer of agarose gel moleculae, chemical modifying it, designing antigen microarray, preparing antigen microarray by applying different biomacromolecular antigens on the surface of said substrate, and testint the chip by use of microscope or fluorescent scan to acquire the information about specimen. Its test method includes culturing the said microarray chip and specimen to be tested in warmhouse, hybridization with the second antigen probe of three fluorescein labels, and acquiring the information about specimen by multi-channel fluorescent scan.

Description

Method for making based on the autoantibody repertoire antigen microarray

One, technical field

The present invention is a kind of method for making of biotechnology microarray, especially a kind of method for making based on the autoantibody repertoire antigen microarray.

Two, background technology

Biochip is to combine with the self-assembling technique of biomolecule by the plane microfabrication, is assembled into numerous different biological molecules microarray on a small slide, to realize the extensive detection to target dna or protein molecule information.This technology is considered to the efficient tool in the life science, thereby becomes the focus of at present domestic and international scientific technological advance.

Protein immune detection cake core is different from general DNA chip detection technology, and its principle is based on mainly that the specific binding reaction of antigen and antibody designs, and the molecules of interest of being surveyed only has structural selectivity, and does not have the sequence specificity.The detection method of at present relevant autoantibody has the colloidal gold immunity percolation technology, enzyme linked immunosorbent assay (ELISA) and radiommunoassay (RIA) etc., the former is because method susceptibility is not good enough, the latter then has characteristics such as isotopic contamination and instability, total difficult satisfactory in the use, and the elisa technique of present clinical labororatory widespread use, its method repeatability is subjected to the interference of multiple mensuration factor again easily, and said method is only applicable to the mensuration (being that a kind of experiment can only detect a kind of autoantibody at every turn) to single project, is necessary to seek a kind of sensitivity for this reason, stable high-throughout detection method.

The leaders' such as Angelika Lueking of Germany Max planck molecule genetics research institute research group has reported the protein molecule microarray that is used for gene expression and antibody screening in 1999, they with the molten born of the same parents' thing of bacterium of 92 people cDNA clonal expression gained as antigen, with the mechanical arm method protein site is made antigen microarray to pvdf membrane, (the array dot spacing is 4.5mm can to detect the protein of 10pg with this method, spot diameter is 250 μ m), then see and identify that protein expressioning product (sees Analytical Biochemistry.1999 for details with monoclonal antibody; 270:103-11), the Mendoza LG. etc. of the U.S. then made on the polystyrene reactant plate and " based on the antigen microarray (144 points/hole) of enzyme-linked immunosorbent assay, adopted the ELISA method of standard to detect and (sees Biotechniques.1999 the same year; 27 (4) .778-788).This manufacturing technology based on protein microarray has been carried out good try for the high throughput testing that realizes antigen and antibody and even part and acceptor.But be assembled in same solid phase carrier (as microslide) simultaneously for some micromolecule haptens (as phosphatide), recombinant polypeptide, DNA and high molecular weight protein etc., then also have many require further improvement and perfect work will be done.

Three, summary of the invention

Purpose of the present invention just provides and a kind ofly combines with the group of molecules packing technique with the gel cementing layer, make the antigen molecule after the solid phase still keep original steric configuration, thereby reach the method for making of guaranteeing follow-up autoantibody testing result authenticity based on the autoantibody repertoire antigen microarray.

The method that the present invention makes is: the 1. pre-service of substrate: the surface of solid phase carriers that will intend selecting carries out the processing of cleanliness and flatness, makes the single characteristic of substrate surface trend, to adapt to the molecule self assembly;

2. the making of substrate surface Ago-Gel tack coat and chemical modification; Forming the orderly bifunctional molecule film of one deck at substrate surface is the Ago-Gel molecular layer, makes bifunctional molecule one end by covalent bond and substrate surface strong bonded, the other end and antigen molecule coupling to be assembled;

3. antigen microarray design: 4 points of every kind of antigen point system, spot diameter is 0.1mm～0.5mm, and dot spacing is 0.5mm～1.0mm, and four jiaos of array is the positive and negative reference mark of autoantibody, middle known autoantigen microarray for the some system;

4. make antigen microarray: by point sample equipment with the substrate surface of different biomacromolecule antigen points to the above-mentioned modification, the antigen molecule of making can with the epitaxial end generation chemistry of bifunctional molecule layer or the bonding of physics, make required chip;

5. chip signal is detected, gather sample information with microscope or fluorescent scanning.

After the method that detects is the antigen microarray chip and tested sample incubation that will make, hybridize with three kinds of fluorescein-labeled second antibody probes of difference again, adopt the hyperchannel fluorescent scanning to gather sample information, in order to determine the different immunoglobulin classes of autoantibody.Detect three kinds of fluoresceins of usefulness and use FIFC, TRITC and AMCA mark goat anti-human igg, IgA and IgM antibody molecule respectively.

The invention has the advantages that:

1. adopt gel cementing layer group of molecules packing technique to make the antigen molecule after the solid phase still keep original steric configuration, guarantee the subsequent detection real result thereby reach.

2. practical, possess skills reasonable in design, experiment flow is clear and implement characteristics such as facility, has stronger practicality and industry development to be worth.Use this kind pattern and also can expand to other laboratory diagnosis field, as: the mensuration of infectious disease, tumor markers and micromolecule hormone etc. (based on antigen/or the microarray of antibody).

3. our antigen microarray of making: include different biomacromolecule (as: phosphatide, recombinant polypeptide, DNA and protein etc.), be different from general simple protein/or dna microarray, (by the bonding of physics with chemistry) still keeps original molecular configuration after the antigen solid phase, and helps and the reaction of follow-up antibodies.

4. chip signal collection: can adopt the fluorescent microscope microscopy or make multichannel fluorescent scanning (3 look fluorescence: FITC, TRITC and AMCA) by the fluorescent scanning instrument, can measure the different immunoglobulin classes of multiple autoantibody simultaneously, also can select for use common observation by light microscope (signal show to adopt the solid phase enzyme-substrate/or immunogold silver staining).

Four, embodiment

1. the pre-service of substrate (microslide): reach the requirement (comprising the cleaning requirement, roughness requirement etc.) that is suitable for the molecule assembling.

2. with the hydroxylation surface-treated microslide of cleaning, place certain density APTES (2%) acetone soln, behind the immersion adaptation time, take out and use acetone and ether rinse successively, standby after drying naturally.

3. substrate surface is made gel molecular layer (agarose) → NalO again ₄Oxidation 20min → distilled water washing 30min → drying back makes aldehyde radical surface, drying for standby in baking oven with certain density GA.

4. use point sample equipment that different antigen molecule (condition is groped in advance) is put to microslide, point sample concentration 100 μ g/ml, point sample amount 0.1 μ l establishes anti-people's gamma Globulin and bovine serum albumin(BSA) (BSA) simultaneously as positive and negative reference mark; 37 ℃ of rearmounted 4 ℃ of refrigerator overnight of incubation 4h; Use 37 ℃ of 1h of BSA (containing 1% glycocoll) sealing of 1.Omg/ml again, dry after the PBS washing.

5. add the second antibody probe of certain dilution tested sample and mark respectively, incubation conditions is 37 ℃ of 30min.(when the antibody labeling thing was HRP, follow-up reaction then needed enzyme-added effect substrate).

6. microscopy or fluorescent scanning: when the labelled antibody molecule was selected enzyme (HRP) or nm of gold (10nm) for use, then available ordinary optical microscope was observed, and the result is pale brown look to occur or the black splotch person is positive; Labelled antibody is that fluorescein then adopts fluorescence microscopy (band CCD camera) or fluorescent scanning to carry out signals collecting, the result is with the spot positive reaction of person that fluorescence occurs: the fluorescence of 3 kinds of different colours of red, green, blue is arranged, and they represent the autoantibody of different immunoglobulin classes respectively.

Claims

1. method for making based on the autoantibody repertoire antigen microarray is characterized in that the method for making is:

1. the pre-service of substrate: the surface of solid phase carriers that will intend selecting carries out the processing of cleanliness and flatness, makes the single characteristic of substrate surface trend, to adapt to the molecule self assembly;

2. the method for making based on the autoantibody repertoire antigen microarray according to claim 1, after it is characterized in that the method that detects is the antigen microarray chip and tested sample incubation that will make, hybridize with three kinds of fluorescein-labeled second antibody probes of difference again, adopt the hyperchannel fluorescent scanning to gather sample information, in order to determine the different immunoglobulin classes of autoantibody.

3. the method for making based on the autoantibody repertoire antigen microarray according to claim 1 and 2 is characterized in that three kinds of fluoresceins that detect usefulness use FIFC, TRITC and AMCA mark goat anti-human igg, IgA and IgM antibody molecule respectively.