Preparation Of Protein Microarray For Detection Of Antibodies Induced By Vaccines Of Immunization Program

ObjectiveVaccination is the most economical, effective, and convenient means in preventing and controlling the spread of disease. Currently, the vaccine technology has achieved rapidly development, many vaccines have been used for preventing infectious diseases. In February 2008 the Ministry of Health announced that China's national immunization program had expanded to 15 diseases, corresponding vaccines were also included. However, at present, the effect of vaccine inoculation, antibody levels of ordinary people and other indicators cannot make an effective evaluation at large scale. The main problems of the regular immunoassays are the low level of sensitivity, the limited types of antibodies'parallel detection, which may unable to meet current needs. Protein microarray which with many advantages such as high throughput, parallel detection, high sensitivity, high specificity, miniaturization and automation has been successfully applied in the pathogen antigen and antibody detection, and called an increasingly attention from the medical inspection field. In this paper, for the purpose of screening vaccine antibodies with a way of high throughput, high sensitivity and diversity, we took protective antigens of these pathogens as probes and prepared a protein microarray for detection of antibodies induced by vaccines of immunization program, and made it possible to detect lots of vaccine antibodies through a single experimental, shorten the detection cycle and improve detection efficiency greatly.Methods1. Saturated sodium carbonate solution, the mixed liquor of potassium dichromate and diluted hydrochloric acid, 80%(v/v) ethanol were used for pretreating group A meningococcal polysaccharide vaccine separately. Saturated sodium carbonate solution, the mixed liquor of potassium dichromate and diluted hydrochloric acid were used for pretreating absorbed diphtheria and tetanus combined vaccine, live measles vaccine, group A and C meningococcal polysaccharide vaccine, inactivated Hepatitis A vaccine, BCG and live MMR combined vaccine separately. 75%(v/v) ethanol were used for pretreating live poliomyelitis vaccine in dragee candy. PAGE was used for purifying protective antigen(PA) from anthracic vaccine strain lysate. Then the antigenicity of each vaccine was detected by protein microarray.2. With the principle of ensureing the detection sensitivity and economy, the selected antigens were gradient diluted to determine the optimal printing concentration of each antigen probe.3. Prepared protein microarray for detection of antibodies induced by vaccines of immunization program, then a large number of serum samples were detected to determine the value of cutoff of each probe. The protein microarray were used to detect children serum samples and compare with the results that detected by ELISA kit to observe the results of statistics differences between the two detection methods.4. Three different signal detecting methods--fluorescence, one-step GLSS, two-step GLSS--were used to detect serum samples and their detection effect and sensitivity were contrasted.Results1. With saturated sodium carbonate solution pretreating group A meningococcal polysaccharide vaccine and adsorbed diphtheria and tetanus combined vaccine, with the mixed liquor of potassium dichromate and diluted hydrochloric acid pretreating live measles vaccine and group A and C meningococcal polysaccharide vaccine, with 75% (v/v) ethanol pretreating live poliomyelitis vaccine in dragee candy, and PAGE for purifying PA from anthracic vaccine strain lysate, the antigenicity of these probes were better than these with untreated or pretreated in other ways. However, there was not significant change in the antigenicity of inactivated Hepatitis A vaccine, BCG and live MMR combined vaccine after pretreatment.2. Determined the optimal printing concentration of each antigen probe: measles virus H protein 4000μg/mL, rubella virus E1 protein 500μg/mL, mumps virus NP protein 1000μg/mL, HAV vp1 protein 1000μg/mL, HAV vp3 protein 1000μg/mL, HBsAg 500μg/mL, JEV E protein 1000μg/mL, live poliomyelitis vaccine in dragee candy pretreated solution 1:10 dilution, pertussis FHA 125μg/mL, diphtheria toxin 50μg/mL, tetanus toxin 250μg/mL, group A and C meningococcal polysaccharide vaccine pretreated solution, bacillus anthracis PA 250μg/mL, hemorrhagic fevers virus NP protein 330μg/mL, leptospira antigen 500μg/mL. 3. Analyzed the detecting results of a large number of serum samples, determined the value of cutoff of each probe: measles virus H protein 1.41, rubella virus E1 protein 1.76, mumps virus NP protein 1.51, HAV vp1 protein 2.03, HAV vp3 protein 1.99, HBsAg 1.10, JEV E protein 1.87, poliomyelitis antigen 4.88, pertussis FHA 1.77, diphtheria toxin 1.34, tetanus toxin 1.41, meningococcal capsular polysaccharide antigen 2.95, hemorrhagic fevers virus NP protein 3.82, bacillus anthracis PA 3.10, leptospira antigen 2.77.4. The accumulative total coincidence rate of result detected by protein microarray and ELISA was 91.59%. Numeration data of statistical matching pairs were detected:χ~2=0.17, P>0.05, which showed that there was no statistical difference between the two methods in the detecting results.5. The detection effect and sensitivity of three different signal detecting methods were contrasted, the result suggested that the three methods had favourable consistency, and the detection sensitivity of two-step GLSS was 10 times higher than fluorescence, 2 times higher than one-step GLSS.ConclusionsIn this study, the effects of different vaccines pretreated by different methods were contrasted and presented favourable antigenicity after proper pretreatment, which may lay a foundation for making use of vaccines as probes to detect corresponding antibodies. This study prepared a protein microarray for detection of antibodies induced by vaccines of immunization program, and with which we could simultaneously detect 14 species of antibodies that induced by vaccines (expect BCG) of national immunization program in a rapid, accurate, high throughput and high sensitivity way. Based on the high sensitive new Nanogold-DAB substrate signal amplification technology to achieve the visualization of the detection signal, improved the sensitivity of the detection and reduced the costs that could meet the need of on-site inspection and the basic units, and provided the nation and army with a reliable evaluation tool for carrying out immunization program smoothly.