Fabrication And Application Of Glass Slide Protein Microarray For Quantitative Detection Of Human Serum IgG

With the genome sequences of several organisms now in public databases,the scientific community has realized that it is time to prepare for the next step:the understanding of biological systems or systems biology.Gene expression microarray technology has exploded in the last five years with completion of the Human Genome Project.Its use has been shown across a wide range of fields including but not limited to biomarker discovery,predicting disease outcomes and response to treatment, assessing coregulation via time course and/or dose-response experiments,and detecting molecular mechanisms and/or pathways associated with a particular disease state.Whereas genes contain the information for life,the encoded proteins and RNAs fulfill nearly all the functions,from replication to regulation.At present,there is a perceived demand for high-throughput and parallel analytical devices as research tools in systems biology,and,in addition,for new concepts to extract knowledge and value from these data.For this reason,a new technology called antibody microarrays has been developed to assess differential expression directly at the protein level. Antibody microarrays can be used for expression profiling of hundreds of thousands of proteins simultaneously,with the goal of identifying disease/protein or protein/protein relationshipsProtein quantificati on is an indispensable tool in clinical and research laboratories.Classic ELISA is a useful tool for this quantification,but problem arise when limited sample is available and quantification of multiple antigens are desired.The protein microarray is one tool that would potentially fill this need for simultaneous multi parameter analysis of biologic samples.Objectives:An antibody based glass microarray for detecting human serum IgG has been developed based on the similar technology as the traditional ELISA approaches.The purpose of this study was to compare results obtained with the traditional ELISA method and those from the glass array technology,to establish the protein microarray platform.The investigation were divided into two chapters(1) Comparison of two different surface modifications for the preparation of protein microarray(2)Comparison of glass slide protein microarray and traditional ELISA for quantitative detection of human serum IgG.Methods:To fabricate protein microarray for evaluating immobilization efficacy,Cy3-1abeled goat-anti human IgG was dissolved in PBS with 20%glycerol included to prevent evaporation of nanodroplets.Cy3-labeled goat-anti-human IgG was spotted on glass slide modified with aldehyde,GTPS and poly-L-lysine at 7 concentrations of 0.5,0.75,1.0,1.25,1.5,1.75,2.0 mg/L,Each concentration in triplicate formed 3×7 arrays.To fabricate protein microarray for evaluating Human serum IgG standard samples were dissolved in PBS with 20%glycerol and were spotted on glass slide modified with aldehyde,GTPS and poly-L-lysine at 6 concentrations of 3.125,6.25, 12.5,25,50,100 mg/L.Each concentration in triplicate formed 3×7 arrays.After spoting,the slides were incubated for in box at 37℃for 3 hours.The slides were immersed in PBS containing 1%boyine serum albumin(BSA)for 1 hour,quenching the un-reacted aldehyde and poly-L-lysine groups on the slide and reducing nonspecific binding of other protein in subsequent steps. Then washed twice in PBST.The slides were rinsed twice with PBST(PBS with 0.1%Tween-20)and once with PBS at 25℃for 10 min each time.The slides were scanned and analyzed by Genepix 4000B.Immobilization efficacy(%)and responsive activity of protein on microarray were evaluated.After pro-10 spot to order,the fluorescence measured value of each protein sample is tend in stability0 Then We can spot formally.After spotting the fixed time take 24-48h as proper. Both long and short time will cause the fluorescence measured value low.It's the best choice using EA solution to remove unbound protein.The best antigen-antibody particularity combines temperature is 37 0C,time is 2h,choosing the 1 mg/L dose the optimal spotting concentration.To fabricate protein microarray for detecting human serum IgG,we use a robot (pixsys5500,from Cartesians INS.)to deliver nanoliter volumes of protein samples to the slides.Protein samples were dissolved in PBS with 20%glycerol included to prevent evaporation of nanodroplets.Goat-anti-human IgG was spotted on glass slide at a concentration of 0.5 mg/L,human serum albumin(HAS)from SIGMA was as negative control.After a 3-hour incubation,the slides were immersed in PBS containing 1%bovine serum albumin(BSA)for 1 hour,quenching the un-reacted aldehyde groups on the slide and reducing nonspecific binding of other protein in subsequent steps.Then washed twice in PBST.We examined specificity and sensitivity while determined the effectiveness by statistics.IgG concentrations for 10 human serum samples were obtained with protein microarray assay and traditional ELISA method,respectively.Results:The purified goat anti-human IgG sample was dissolved in PBS with 30% glycerol for preventing evaporation of droplets,the antibody was subsequently printed on commercial glass slides which coated with a nitrocellulose polymer at a concentration of 0.5 mg/mL.Meanwhile,human serum albumin(HSA)was a negative control.The slides were incubated first with 10 serum samples and subsequently with fluorescently labeled secondary antibody.Human serum IgG bound to the printed antigen were detected by confocal scanning microscopy and quantified with internal calibration curves.The software SPSS 10.0 for windows was used for statistics analysis.The results were compared between the two methods by using one way ANOVA test,P＜0.05 was regarded as significant.R~2 values generated by protein mieroarray and ELISA for IgG calibration curves were 0.996 and 0.994, respectively.The detection limit of protein microarray was 1.56μg/100μL,which was as sensitive as ELISA.IgG concentrations for 10 human serum samples were compatible with those levels by protein microarray and ELISA.There wasn't significant difference compared protein microarray assay and ELISA(f=0.188, P=0.670＞0.05).The results indicated that protein microarray assay performs equivalently to ELISA for protein quantitation.Conclusion:The glass slides modified with aldehyde is selected for protein microarray preparation the glass protein microarray is an alternative for miniaturizing the ELISA technology with un-compromised detection rate sensitivity and applicability.