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the Using Elisa Technique and Blood Typing to describe antibody-antigen reactions Introduction This experiment aims to investigate the hypothesis that the absorbance read from the spectrophotometer will decrease as the dilution increases from the 1 st well to the 11th .The ELISA and blood typing experiment is worth investigating because it can be performed to evaluate either the presence of antigen or the presence of antibody in a sample, it is a useful tool both for determining serum antibody concentrations (such as HIV) and also for detecting the presence of antigen. (Wikipedia, 2007). The ELISA (Enzyme-linked immunosorbent assay) technique can be used to diagnose diseases by 2 methods--direct and indirect. The direct ELISA method detects antigens while the indirect method (which is the technique we used in our experiment) detects antibodies. It does this by agglutination reactions that occur when antibodies and antigens interact. ELISA uses enzyme labeled antibodies to detect an antigen or antibody (Lum, 2006). This diagram shows the steps taken to prepare an indirect ELISA technique: A patients blood type can be detected by its antibody-antigen interaction. When foreign antigens are found, red blood cells either burst or clump together by agglutination (Lum, 2006). If agglutination in your blood occurs, this indicates the presence of one of the ABO blood types. Type A blood contains antigen A and anti-B antibodies, while Type B is the opposite of this. Type AB (also known as the universal receiver) contains both antigens A and B and neither anti-A or B antibodies. Type O (also known as the universal donor) contains neither A or B antigens but both Anti-A and B antibodies. This can be applied by testing a patients blood sample. If the blood agglutinates after the addition of anti A serum, this shows the person has Type A blood. If no agglutination happens in after the addition of anti A or anti B serum-this person has Type O blood. We performed this experiment by using the indirect ELISA technique by diluting the patients serum from the first well to the last. This is the reason why I predict that the absorbance will decrease, since the color of the enzyme substrate will be progressively lighter. Materials and Methods The antigen we used to detect the presence of antibodies is Salmonella Typhimurium. We received our microtiter plate that had 100 l of coating buffer and the antigen S. Typhimurium in each of the 12 wells that had been refrigerated for 1-7 days at 5 C. Once we received our plate, we ,,shook the plate by inverting its contents into a waste bowl. We then proceeded to wash the wells by adding washing buffer and disposing its contents, we repeated this 3 times. After the wells were washed out, we added 100 l of 3% BSA/PBC blocking buffer to the 12 wells and left it for 45 minutes. After waiting for the instructed time, we performed the dilution of the patients serum by adding 100 l of it to the first well, mixing it using the micropipette and transferring 100 l of this liquid to the next well. We continued doing this until we reached the 11 th well and discarded the 100 l from that well into the waste. We left the 12 th well without the patients serum so it could act as a control. Next, we incubated the plate for an hour at 30 C. After it was incubated, we shook out its contents and washed it using washing buffer (as mentioned above) 3 more times. We then added the enzyme alkaline phosphotase-labeled antiantibody to wells 1-12 and incubated the plates till the following week. After removing the incubated well we added the alkaline phosphate substrate (BCIP/NBT) to each of the 12 wells and waited for the color to develop. We then recorded the absorbance using the spectrophotometer of each of the 12 wells by mixing the contents of the well with water in a cuvette. Results Using Table 1 and Figure 1 in the appendix, we can compare the absorbances for each of the dilution factors. The dilution factor is the amount of serum that was diluted in each of the wells. The first well contained half the patients serum, the second well contained a quarter of the patients serum, and it continued like this until we reached the 11th well which was diluted 1:2048 or 211. It can be seen that the absorbances did not decrease as we anticipated it would in the hypothesis. The absorbances taken from the spectrophotometer fluctuated greatly from one well to the next, and did not follow a smooth decrease as the dilution increased. factor Using Table 1, we can see that the absorbance of the 2 nd well is 0.001, according to our hypothesis we would expect it to decrease in the 3rd well as the dilution factor increases. However, instead of decreasing the absorbance read 0.008 which makes no sense. The strangest abnormality of the absorbance readings was well 11. Well 11 had the highest amount of diluted serum because it was the last well. In this well, the absorbance reading was 0.012 which was the highest absorbance reading of any of the 12 wells. Discussion In the introduction I stated my hypothesis to be that the absorbance reading would decrease as the dilution factor increases. However, my hypothesis was not supported according to my results. The reason the absorbance should decrease as the dilution factor increases is because the color of the substrate added to the enzyme should be very strong and dark for the first well. However, as we transfer and dilute the contents from one well to another, the color should turn lighter which causes it to have a lower absorbance reading. The absorbance results we obtained for each well did not follow a certain pattern, but were fluctuating instead. In Well 11, we expected it to have the lowest absorbance because its dilution factor was 1:2048, but it was in fact the highest absorbance. The main reason for the fluctuating absorbances was due to the fact that we washed out and ,,shook the contents of the container several times. Shaking the contents refers to inverting the plate and disposing out the liquids in each well. When we flip the plate back, it is obvious that many of the wells could have been contaminated with the contents of the other wells. Since we inverted the plate at least 6 times to get rid of its contents, it is almost for certain that each of the wells did not contain what it was supposed to due to high levels of contamination. Other problems that arose while performing this experiment were that we were told to use the same micropipette tip for washing each well with the washing buffer as long as it did not touch the walls of the well. Most people were careful with washing the wells, but this could also be a very easy source of contamination of the contents of each of the wells as it is impossible to perfectly wash each well. Ways that we could improve this lab to ensure us more accurate results is by firstly, repeating the experiment more than one time. The next way is to ensure that each well gets its own micropipette tip to make sure no contamination occurs while washing the wells with washing buffer. Another major way to improve this lab regarding the inverting of the plate is using a different micropipette tip for each of the 12 wells and manually removing the contents of each well into a waste container. This process will take much longer since the wells need to be washed 3 times, but it should ensure more accurate results. Elisa is used as a diagnostic tool to determine whether a certain antibody or antigen is present in a patients serum to determine if they have a certain disease or not. This shows ELISA is extremely useful in clinical and medical applications Conclusion The conclusion of this experiment is that the absorbance and color of each of the 12 wells should progressively decrease as its dilution factors increase. However, due to the contamination of the wells, the hypothesis and prediction for this experiment was not supported. Appendix Table 1:Table to show the absorbance's for the different dilution factors from well 1-12. Well Number 1 2 3 4 5 6 7 8 9 10 11 12 Dilution Factor 1:2 1:4 1:8 1:16 1:32 1:64 1:128 1:256 1:512 1:1024 1:2048 Control Absorbance 0.010 0.001 0.008 0.007 0.006 0.005 -0.001 0.008 -0.002 0.000 0.012 -0.003 Figure1: Graph to show the absorbance's of the 12 wells that had different dilution factors Results of ELISA method 0.014 0.012 0.01 Absorbance 0.008 0.006 Absorbance 0.004 0.002 0 -0.002 -0.004 2^1 1 2^2 2 2^3 3 2^4 4 2^5 5 2^6 6 2^7 7 2^8 8 2^9 2^10 2^11 2^12 9 10 11 12 Dilution Factor Sources Cited: 1)"ELISA." Wikipedia, The Free Encyclopedia. 16 Apr 2007, 08:49 UTC. Wikimedia Foundation, Inc. 18 Apr 2007 <http://en.wikipedia.org/w/index.php?title=ELISA&oldid=123201349>. 2) Decker, Janet M "ELISA" http://microvet.arizona.edu/Courses/MIC419/ToolBox/elisa.html, February 1, 2006, Date Accessed 15th April 2007. 3) Lum, Pam, 2006. "General Biology Laboratory Manual", USC. E.d. Shakhbandaryan, Arpine.

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