21821580 major of phamacologyGroup EPark seong-yeonTitleIdentification of the level of antibody present in the given serum by ELISAMaterials15 well plate, serum, TBST buffer, Anti IgG antibody, 2% FBS, stop solution, pipettes, glove, TMS solution.IntroductionELISA is an experiment to determine how many antigens are present in a solution by using the reaction between an antigen and an antibody.We proceed with the sandwich ELISA. The reaction is confirmed by attaching a substrate to the antigen bound to the antibody and adding an enzyme to change color.Method1. Wash the plate with TBST for 3 times. and shake off the liquid.2. Add 100レl 2% FBS buffer into each well3. Mix 100レl serum (IgG antibody) at 1:1 well of A4. Take 100レl from thd mixed well and mix in 1 : 2 well5. Proceed to 1:4,1:8,1:16 wells in same way6. proceed to B, C line in same way7. Put at 37” for an hour8. Wash the plate 3 times with TBST9. Put 100レl 2nd antibody into all 15 wells10. Put at 37” for 30 minutes.11. Wash the plate 3 times with TBST12. Add TMB solution(substrate) 100レl to all wells.1213. Put the plate in a dark place for 15 minutes14. Add Stop solution(2N H��SO��) 100レl to all wells15. Measure the absorption of 450nm wavelength14ResultABC1 : 10.72900.84100.70801 : 20.72400.70500.67901 : 40.81700.72500.78801 : 80.81000.75600.71001 : 160.81800.78500.7510DIscussionThe results of the experiment were very different from what was expected.Prior to the experiment, we expected that as the solution was diluted, the amount of antigen would decrease. However, All of line¨s result were worng.This can be expected that there was a mistake in the pipetting process during the experiment or that the solution was not properly mixed during the dilution process.Also, when pipetting, we had to reduce the difference in the reaction time between antibodies and antigens by using the fastest speed possible, but I think the poor use of pipettes and taking a long time is also the cause of poor results.conclusionWe didn't get the right absorbance values, but we found that performing ELISA requires a lot of effort in every step. We must carefully use tools such as pipettes and observe the set time for experimentation.
