CN101000350B - Construction method of antibody microarray based on double-antibody sandwich immunanalysis - Google Patents
Construction method of antibody microarray based on double-antibody sandwich immunanalysis Download PDFInfo
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- CN101000350B CN101000350B CN2006101616675A CN200610161667A CN101000350B CN 101000350 B CN101000350 B CN 101000350B CN 2006101616675 A CN2006101616675 A CN 2006101616675A CN 200610161667 A CN200610161667 A CN 200610161667A CN 101000350 B CN101000350 B CN 101000350B
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Abstract
The invention relates to a method for setting-up antibody micro-array based on double-antibody sandwich immunological analysis. The invention is characterized in the setting-up steps which comprises: preparing carrier, activating carrier, fixing sample application of capture antibody and immunological analysis based on micro-array. The antibody micro-array constructed by the optimized operational process has better repeatability (variation with in group and between groups < 10%) and quantitative analysis ability (standard curve relativity of antigen concentration and signal strength > 0.95) and provides reliable technology platform for parallelism, multiple-factor protein quantitative examination. Besides, if the chip is stably saved after the sample application, the activity can maintain at least 2 months, so as to provide a important condition for industrialization and scale of the chip.
Description
One, technical field
The invention belongs to protein-chip manufacturing technology field, particularly a kind of construction method of the antibody microarray based on double-antibody sandwich immunoassay.
Two, background technology
Proteomics is the research to the quantity of all proteins of a biosome, organ, organelle, structure, cell function, and their variation under different time, space, physiological status.At present, proteomic techniques mainly is divided into non-bias proteomics (unbiased proteomic) and bias proteomics.Wherein non-bias proteomics is the science of " finding to drive (discovery-oriented) ", and all proteins in the sample is analyzed, and can obtain lot of data, be used for finding the unknown, and mainly be two-dimensional gel electrophoresis connexus spectral technology.And the bias proteomics; Be called system's guiding protein matter group (system-oriented proteomic) again; Be that the researcher is having on the basis of certain understanding a certain specific pathophysiological process; Presuppose in the sample to be measured some composition and under normal and morbid state, express and there are differences, the biomarker that can go to illustrate disease incidence mechanism, discern disease from polyfactorial angle.Antibody microarray (antibodymicroarray; AbM) promptly be important bias proteomic techniques; Developed into the important platform of protein molecule in the qualitative of collimation and the detection by quantitative biological sample gradually; Can accurately analyze a plurality of related protein content in the sample simultaneously, significant to Biomedical Development.
Yet, because the complicacy of protein structure is prone to lose native conformation; In the single biological specimen, the protein concentration variation range is big; Many important protein matter molecules exist with low abundance, and therefore, protein microarray is compared with genetic chip, and indexs such as its susceptibility, repeatability, detection dynamics range and quantitation capabilities then face more challenges if reach the needs of clinical practice.At present; The research of protein microarray mainly concentrates on the introducing of carrier that seek to optimize and the fixedly approach of capture molecules, various signal detecting mode and signal amplifying system and to the research of the committed step in the micro-array construction process in the world, the influence of the chip activity being kept like the influence of point sample damping fluid glycerol concentration array point form, preservation condition behind the point sample etc.In a word, protein microarray technology prematurity still at present, its structure still need further develop and be perfect.
Three, summary of the invention
The present invention is directed to agarose and encapsulate the high power capacity property of slide carrier, high specific, the biotin-streptavidin-hypersensitivity of luciferin detection system and the high flux property of microarray analysis of double-antibody sandwich immunoassay; Through optimization, a kind of repeatable height is provided, preserves antibody microarray stable and that have the quantitative test ability each committed step of micro-array construction.
Technical solution of the present invention is: a kind of construction method of the antibody microarray based on double-antibody sandwich immunoassay; Construction step is: the preparation of carrier: ultrapure water preparation 0.5-1.5% agarose solution; Heated and boiled is to dissolving fully; Agarose solution is covered to the cleaning slide of preheating, at room temperature solidify rearmounted 37 ℃ of baking boxs oven dry, it is subsequent use to put room temperature preservation; The activation of carrier: with activation 20-30min in the NaIO4 solution of the prepared carrier immersion of step a 20mmol/L, 0.1M pH7.4 PBS washing dries up the carrier after the activation with nitrogen stream; The point sample of capture antibody is fixed: at first prepare pH9.0; Add after the 0.1mol/LNa2HPO4 point sample damping fluid that contains glycerine 200ml/L, capture antibody dilute by best titration concentration with the point sample damping fluid and put in the point sample plate hole, positive control (streptavidin-cy3 is set on the point sample plate hole then; Biological markers detection antibody) and negative control (200ug/ml BSA) point sample; As indication of microarray direction and quality control, the variant concentration of capture antibody repeats point sample 4 times, presses array of designs (Fig. 2) point sample on step b gained carrier in advance at last; Carrier is put wet box sealing behind the point sample, put spend the night in 4 ℃ of environment fixing.Immunoassay based on microarray: in 37 ℃ of waters bath with thermostatic control, use concentration the nonspecific binding site of antibody on the carrier to be sealed and handled 1-2 hour as the PBS solution of 1-5%BSA; Dry up with nitrogen after PBST (PBS that the contains 0.05%tween20) wash vehicle; The different inferior arrays of identical carrier are covered antigenic solution or sample to be measured, and overlay capacity is the 2-5ul/ array, after the covering carrier is put in the airtight wet box, in 37 ℃ of water bath with thermostatic control reactions 1-2 hour; Dry up with nitrogen after the PBST wash vehicle; The different inferior arrays of identical carrier are covered detection antibody, and overlay capacity is the 2-5ul/ array, puts airtight wet box, and 37 ℃ of waters bath with thermostatic control were reacted 1-2 hour; Dry up with nitrogen after the PBST wash vehicle; The different inferior arrays of identical carrier are covered streptavidin-cy3, and overlay capacity is the 2-5ul/ array, puts airtight wet box; 37 ℃ of waters bath with thermostatic control were reacted 20-30 minute; PBST washing back dries up with nitrogen, with laser confocal scanning appearance scanning carrier, picture is carried out the fluorescence intensity data analysis at last.The present invention has combined the principle of double-antibody sandwich immunoassay and " analysis theories (ambient analysis theory) on every side " of microarray analysis; The high-affinity reaction that utilizes biotin-streptavidin is as detection system; Through research, set up a whole set of construction method (flow process is seen accompanying drawing 1) based on the antibody microarray of double-antibody sandwich immunoassay to crucial building process.The course of work mainly comprises the point sample of preparation and activation, capture molecules of carrier and fixing and based on immunoreactive analysis.
According to the constructed antibody microarray of the operating process after this optimization, have preferably repeatable (in the group and between-group variation < 10%), the glycerol concentration of selected point sample damping fluid and interpolation has guaranteed the form homogeneous of array each point; Constructed AbM has than accurate quantitative analysis ability (the typical curve correlativity of antigen concentration and signal intensity>0.95, see accompanying drawing 12), for collimation, the multiple-factor quantification of protein detects that the reliable technique platform is provided; Individual slide is 10 inferior arrays of point sample at least, therefore can realize that a plurality of samples detect simultaneously, have increased the comparability between the different specimens data, realize the various article of AbM, polyfactorial high throughput testing, have improved detection efficiency greatly; It is few to detect required sample size, and each array only needs 2-5ul; Positive control streptavidin-cy3, biotin labeling detect antibody and the setting of negative control BSA and the repetition point sample of capture antibody, have increased the reliability of experimental result; Point sample in the time of a plurality of capture antibody concentration, the selection of data in the time of can helping signal analysis is being analyzed under the high concentration antigen situation, and high capture antibody concentration point signal occurs saturated, can select low antibody point, can select low concentration antibody point on the contrary; In addition, chip is preserved stablely behind the point sample, and activity can be kept 2 months (seeing accompanying drawing 13-16) at least, for the industrialization and the scale of this chip provides important condition.This system realizes that above advantage is relevant with following four aspects: 1. the slide that encapsulates with agarose of native system is a carrier; The high power capacity property that not only has three-dimensional carrier; And can realize matrix, and be that the preservation of chip provides hydrated environment behind antigen-antibody reaction and the point sample with quantitative mode fixed trapped antibody; 2. the agarose three-dimensional carrier can effectively adsorb institute and add sample, has avoided the fusion of sample between different arrays, is beneficial to the detection that realizes the various article of individual slide; 3. the PBS before the capture antibody point sample handles, for fixedly providing of antibody suitable buffered environment, help fixing with active keep (the seeing accompanying drawing 5,6) of antibody; 4. to the optimization of whole antibody micro-array construction process; (see accompanying drawing 3 like the capture antibody optium concentration; 4), slide set time and the comparison of condition after the vehicle treated before the point sample, point sample, comparison, the kind of lavation buffer solution and relatively the waiting of time of confining liquid, realized the stability and the optimization of system.
Four, description of drawings
Fig. 1 is the schematic flow sheet of the construction method of antibody microarray.(1) capture antibody is fixing; (2) the antigen application of sample is hatched; (3) detecting antibody (biotin labeling antibody) application of sample hatches; (4) streptavidin-cy3 input; (5) scanning of image, signal strength analysis.
Fig. 2 is the point sample schematic layout pattern of antibody microarray.
Fig. 3,4 is the influence figure of capture antibody concentration to signal intensity.
Fig. 5 is the fluorescent scanning figure after ultrapure water is handled before the slide point sample.
Fig. 6 is the fluorescent scanning figure after PBS handles before the slide point sample.
Fig. 7～11 are antibody microarray antigen concentration-signal reaction fluorescent scanning figure.
Figure 12 is the antibody microarray antigen concentration-signal reaction curve of Fig. 6～10.
Figure 13,14 is respectively the antibody microarray and preserves the fluorescent scanning figure when placing 0 month, 2 months.
Figure 15,16 is respectively the antibody microarray and preserves the signal strength map when placing 0 month, 2 months.
Five, embodiment
Embodiment 1: be example with MCP-1, make up double-antibody sandwich antibody microarray: 1. the preparation of carrier: ultrapure water preparation mass ratio is 1% agarose solution, and micro-wave oven boils to dissolving fully; The 2ml agarose solution is covered to the cleaning slide of preheating, treat that the agarose solution room temperature solidifies rearmounted 37 ℃ of baking boxs oven dry; 2. the activation of carrier: carrier is immersed 20mmol/L NaIO
4Solution activation 30min, 0.1M pH7.4 PBS washs 5min
*3 times, point sample immediately after nitrogen stream dries up; 3. the point sample of capture antibody is fixed (the 1st step of accompanying drawing 1): point sample damping fluid preparation: 0.1mol/L Na
2HPO
4Solution, pH9.0 contains glycerine 200ml/L; Capture antibody is diluted to 125ug/ml, 62.5ug/ml, 31.25ug/ml, and positive control (streptavidin-cy3, biological markers detection antibody) and negative control (200ug/ml BSA) are set simultaneously, as indication of microarray direction and quality control; Put the point sample plate hole, press array of designs point sample in advance, 4*6*10/ opens (row1 SA-cy3, row2 BSA; Row3 biotin-antibody, row4,5; 6capture Ab), dot spacing 500um, point sample; Slide is put wet box sealing behind the point sample, and 4 ℃ are spent the night fixing; 4. based on the immunoassay (accompanying drawing 1 2-4 step) of microarray: the sealing of nonspecific binding site: 2%BSA/PBS30ul, 37 ℃ of waters bath with thermostatic control sealings 2 hours; PBST washing 5min*3 time, nitrogen dries up; The different inferior arrays of same slide are covered an amount of variable concentrations antigenic solution or sample to be measured (4ul/ array), note avoiding the liquid between different arrays to merge, put airtight wet box, 37 ℃ of waters bath with thermostatic control were reacted 1 hour; PBST washing 5min*4 time, nitrogen dries up; The different inferior arrays of same slide are covered an amount of 3.125ug/ml detect antibody (dosage and points for attention are with (3)), put airtight wet box, 37 ℃ of waters bath with thermostatic control were reacted 1 hour; PBST washing 5min*5 time, nitrogen dries up; The different inferior arrays of same slide are covered an amount of streptavidin-cy3, put airtight wet box, 37 ℃ of waters bath with thermostatic control were reacted 20 minutes, and PBST washs 5min
*6 times, nitrogen dries up; 5. picture is preserved in laser confocal scanning appearance scanning (sweep parameter: laser power is sent out intensity 90%, photomultiplier transit FACTOR P MT80%), fluorescence intensity data analysis (the 5th step of accompanying drawing 1).
Embodiment 2: the preparation of carrier: ultrapure water preparation 0.5～1.5% agarose solution, and heated and boiled covers agarose solution to the cleaning slide of preheating to dissolving fully, at room temperature solidifies rearmounted 37 ℃ of baking boxs oven dry, and it is subsequent use to put room temperature preservation; The activation of carrier: it is the NaIO of 20mmol/L that the prepared carrier of step a is immersed concentration
4Activation 20～30min in the solution, 0.1M pH7.4 PBS washing dries up the carrier after the activation with nitrogen stream; The point sample of capture antibody is fixed: at first prepare pH9.0, contain the 0.1mol/LNa of glycerine 200ml/L
2HPO
4The point sample damping fluid; Add after capture antibody dilutes by best titration concentration with the point sample damping fluid and put in the point sample plate hole, positive control (streptavidin-cy3, biological markers detection antibody) and negative control (200ug/ml BSA) point sample are set on the point sample plate hole then; As indication of microarray direction and quality control; By array of designs point sample on step b gained carrier in advance, carrier is put wet box sealing behind the point sample at last, put spend the night in 4 ℃ of environment fixing.Immunoassay based on microarray: in 37 ℃ of waters bath with thermostatic control, use concentration the nonspecific binding site of antibody on the carrier to be sealed and handled 2 hours as the PBS solution of 2%BSA; Dry up with nitrogen after PBST (PBS that the contains 0.05%tween20) wash vehicle; The different inferior arrays of identical carrier are covered antigenic solution or sample to be measured, and overlay capacity is the 4ul/ array, after the covering carrier is put in the airtight wet box, in 37 ℃ of water bath with thermostatic control reactions 1-2 hour; Dry up with nitrogen after the PBST wash vehicle; The different inferior arrays of identical carrier are covered detection antibody, and overlay capacity is the 4ul/ array, puts airtight wet box, and 37 ℃ of waters bath with thermostatic control were reacted 1-2 hour; Dry up with nitrogen after the PBST wash vehicle; The different inferior arrays of identical carrier are covered streptavidin cy3, and overlay capacity is the 4ul/ array, puts airtight wet box; 37 ℃ of waters bath with thermostatic control were reacted 20-30 minute; PBST washing back dries up with nitrogen, with laser confocal scanning appearance scanning carrier, picture is carried out the fluorescence intensity data analysis at last.
Claims (1)
1. construction method based on the antibody microarray of double-antibody sandwich immunoassay is characterized in that construction step is:
A. the preparation of carrier: ultrapure water preparation 0.5-1.5% agarose solution, heated and boiled covers agarose solution to the cleaning slide of preheating to dissolving fully, at room temperature solidifies rearmounted 37 ℃ of baking boxs oven dry, and it is subsequent use to put room temperature preservation;
B. the activation of carrier: it is the NaIO of 20mmol/L that the prepared carrier of step a is immersed concentration
4Activation 20-30min in the solution, 0.1M pH7.4 PBS washing dries up the carrier after the activation with nitrogen stream;
C. the point sample of capture antibody is fixed: at first prepare pH9.0, contain the 0.1mol/LNa of glycerine 200ml/L
2HPO
4Add after point sample damping fluid, capture antibody dilute by best titration concentration with the point sample damping fluid and put in the point sample plate hole, positive control and the negative point sample of controlling are set on the point sample plate hole then; As indication of microarray direction and quality control; The described positive is controlled to be streptavidin-cy3, and feminine gender is controlled to be the BSA solution that concentration is 200ug/ml, presses array of designs point sample on step b gained carrier in advance at last; Carrier is put wet box sealing behind the point sample, put spend the night in 4 ℃ of environment fixing;
D. based on the immunoassay of microarray: in 37 ℃ of waters bath with thermostatic control, use to contain concentration and as the PBS solution of 1-5%BSA the nonspecific binding site of antibody on the carrier is sealed and handled 1-2 hour; Dry up with nitrogen after the PBST wash vehicle; The different inferior arrays of identical carrier are covered antigenic solution or sample to be measured, and overlay capacity is the 4ul/ array, after the covering carrier is put in the airtight wet box, in 37 ℃ of water bath with thermostatic control reactions 1-2 hour; Dry up with nitrogen after the PBST wash vehicle; The different inferior arrays of identical carrier are covered detection antibody, and overlay capacity is the 4ul/ array, puts airtight wet box, and 37 ℃ of waters bath with thermostatic control were reacted 1-2 hour; Dry up with nitrogen after the PBST wash vehicle; The different inferior arrays of identical carrier are covered streptavidin-cy3, and overlay capacity is the 4ul/ array, puts airtight wet box; 37 ℃ of waters bath with thermostatic control were reacted 20-30 minute; PBST washing back dries up with nitrogen, with laser confocal scanning appearance scanning carrier, picture is carried out the fluorescence intensity data analysis at last.
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| 金世辉等.蛋白质微阵列生产用琼脂糖修饰玻片制备的条件优化.《中国生物工程杂志》.2005,第25卷(第2期),57-60. * |
| 金世辉等.蛋白质微阵列载体的选择及表面修饰方法.《国外医学生物医学工程分册》.2005,第28卷(第4期),209-213. * |
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