Background Information:

The ELISA (Enzyme Linked ImmunoSorbent Assay)(as shown above) Test is the first test used to test for HIV (Human Immunodeficiency Virus). It specifically detects and tests for the antibody specific to the antigen for HIV. Antigens are anything foreign to the body that cause the production of antibodies. Antibodies attach to the antigen and enable the body to destroy it.

There are ELISA Tests used for many other viruses as well. For example, the ELISA for Salmonella is the fastest, safest, and mot documented test for Salmonella. It is done the same way as the ELISA for HIV, but uses the Salmonella antibody. There are also ELISA tests for Herpes, Hepatitis, and even pregnancy.

The ELISA Test is done using a plate that contains many wells. A solution containing the HIV antigen is put into each well. The antigen sticks to the surface of the plate. The solution containing the serum (part of the blood that contains antibodies) is added to each well. Specific antibodies in the serum bind to the antigen, and remain in the well after they are washed. A series of dilutions of the serum has to be done, and each one tested. For human serum, the ratio is 1:400. The dilutions are done, because if the serum is tested without being diluted, it will always test positive. Every person has some level of the antibody in their blood, because everyone has been exposed to some level of the virus. Those who continue to test positive through the series of dilutions have a higher level of the antibody in the blood, and have a higher level of the virus in their blood. These are the ones who have HIV.

This is an example of human serum that actually contains the HIV antibody.

The ELISA Test we are going to perform is going to use horse, cow, rabbit and chicken serum, not human. We will be using goat-anti-rabbit IgG antibody instead of HIV to test the antibody-antigen reaction. The effects of the test will be the same as a real HIV test. The dilutions we will be doing are 1:10, instead of 1:400.

MATERIALS:

This is a picture of horse serum with enzymes attached to it.

1. Chicken Serum (1%)

2. Horse Serum (1%)

3. Cow Serum (1%)

4. Rabbit Serum (1%)

5. TBS (This-Buffer Saline pH 8.0)

6. TBS + gelatin (TBS + 2% gelatin)

7. TBS + NP-40 (TBS + 0.05% Nonidet P-40

*Steps 5-7 made as per Experiment IND-3 Instructor Guide

8. Goat anti-rabbit IgG - Peroxidase - The antibody was prepared by injecting goats with rabbit Ig G. After preparation, the antibody was linked to horse radish peroxidase. The antibody should be diluted immediately before use by adding the antibody concentrate to 25 ml of TBS - Gelatin.

9. Color Development Solution - This solution should be prepared immediately before use by adding 0.9ml of H2O2 and 1 ml of the substrate TMB (Tetramethy Benzidine) to 25ml of water.

10. One microtitration plate

11. 40 Large transfer pipets

This is a large transfer pipet.

12. Microliter dispensers

13. Small beakers or test tubes

14. 0.1 N HCL

15. Spectrophotometer

16. Micropipet tips for p20/200, and p10.

This is what a pipet looks like with a tip on it.

PROCEDURE

* Pre-Experiment Preperation

1. One microtitration plate.

2. Black water proof marking pen

3. Microliter dispenser

4. Small beakers or test tubes containing the indicated amounts of the following solution.

0.1 HCL ~ 10mL

TBS ~ 5ml

TBS - gelatin ~ 13 ml

TBS-NP40 ~ 20ml

Distilled water ~ 10ml

This is what your four solutions should look like.

This picture is showing the TBS gelatin, the HCL, the gelatin, and the TBS NP40.

5. Empty 200-500ml beaker - To be used for discarded liquids

6. One 250-500 ml beaker containing water. - To be used for rinsing pipets

7. micropipets (One p20/200, One p10).

This is an example of a pipet.

*Absorbtion of the Antigen

This is a picture of an antigen that has been coated with the Elisa immunoassay. (or the Elisa test)

1. Using a mircroliter dispenser, place 50 ml of TBS into wells A2-6, B2-6, C2-6, and D2-6.

2. Place 55 microliters of chicken serum into well A-1, 55 microliters of cow serum into well B-1, 55 microliters of horse serum into well C-1, and 55 microliters of rabbit serum into well D-1.

3. Dilution of the Sera:

A. Perform serial ten-fold dilutions so that the concentrations of sera in the wells after dilution should be as follows:

Wells 1: 1%

Wells 2: 0.1%

Wells 3: 0.01%

Wells 4: 0.001%

Wells 5: 0.0001%

Wells 6: No serum

B. To perform the dilutions, transfer 5 microliters of the serum from well A-1 to well A-2. Mix the contents in well A-2 thoroughly by drawing the sample intlo the pipet and expelling it back into the well a few times. Transfer 5 ml of the sample from well A-2 to A-3,k A-3 to A-4, and A-4 to A-5. Mix between each transfer as described above.

C. Using the above procedure perform 1:10 serial dilutions in wells B1 - B5, C1 - C5, and D1-D5.

D. The plate should be left undisturbed for about 20 minutes in order to provide time for the proteins to be absorbed to the well surfaces.

4. After 20 minutes, add two drops of TBS-gelatin to each well using transfer pipet. The gelatin is added to block sites on the plastic that are not bound to serum proteins.

5. Using a transfer pipet, remove the liquid from the wells in the following order: A6 to A1, B6 - B1, C6 to C1, and D6 to D1. The discarded liquid should be placed in your discard beaker. Discard this pipet when these steps are complete.

6. Using a transfer pipet, add 3-4 drops of TBS-gelatin to each well. After about 3 minutes, remove the solutions as described in the preceding step.

*THE ANTIBODY REACTION

1. Obtain one tube of goat anti-rabbit IgG peroxidase. Add 50 microliters of the antibody to each well and rotate the plate to ensure that all surfaces at the bottoms of each well are in contact with the antibody solution. Allow about 20 minutes for the antibody to bind to the immobilized IgG.

2. Add two drops of TBS-gelatin to each well and then remove and discard the solutions in the wells as described in Step 5.

3. Add 3-4 drops of the following solutions and then immediately remove and discard the solutions as described in Step5. A single pipet should be used for these well washes and it should be rinsed with water between solutions.

A. TBS-gelatin

B. TBS-NP40

C. TBS-NP40

D. TBS-NP40

E. H2O

*Color Development and Data Analysis

1. Obtain one tube (~1.5ml) of color development solution. Add 50 microliters of the solutions to each well.

2. After about 15 minutes, place the plate over a white sheet of paper and examine the intensity of the blue color. The blue product is the insoluble product of the peroxidase reaction.

3. At low pH, the insoluble blue product is converted to a soluble yellow product which should be easier to detect. To enhance the sensitivity of the ELISA, add .25ml of .1HCL to each well. Place the plate over a white sheet of paper and record the intensity of yellow product.

This is a picture of a negative ELISA test. Notice that there is no color, there is no pigment.

This is an example of a positive ELISA test. Notice that there IS color, there IS pigment.

DATA/ANALYSIS

EXPECTED RESULTS

("O" for no color, "+" for low color, "++" for high color)

Well Number	Blue	Yellow	Absorbance at 450 nm
A1	0	0	0
A2	0	0	0
A3	0	0	0
A4	0	0	0
A5	0	0	0
A6	0	0	0
B1	0	0	0
B2	0	0	0
B3	0	0	0
B4	0	0	0
B5	0	0	0
B6	0	0	0
C1	0	0	0
C2	0	0	0
C3	0	0	0
C4	0	0	0
C5	0	0	0
C6	0	0	0
D1	++	++	0.163
D2	++	++	0.155
D3	++	++	0.122
D4	+	+	0.048
D5	0	0	0.02
D6	0	0	0

OUR RESULTS

("O" for no color, "+" for low color, "++" for high color)

Well Number	Blue	Yellow	Absorbance at 450mm
A1	+	+	0.112
A2	0	0	0.414
A3	0	0	0.490
A4	0	0	0.431
A5	0	0	0.533
A6	+	+	0.547
B1	+	+	0.684
B2	0	0	0.597
B3	0	0	0.538
B4	0	0	0.488
B5	0	0	0.524
B6	0	0	0.485
C1	0	0	0.681
C2	0	0	0.489
C3	0	0	0.645
C4	0	0	0.615
C5	0	0	0.515
C6	0	0	0.465
D1	+	++	1.052
D2	+	++	0.466
D3	++	++	0.488
D4	++	++	0.581
D5	0	0	0.468
D6	0	0	0.521

The above graph shows exactly what we discovered in the test. You'll notice that well A1 does not have any color compared with D1. THis shows that D1-4 tested positive for the mock HIV antibody, and the rest tested negative.

This is an example of how judge color results. As you can see, the negative test result still appears to have color.

CONCLUSION

In conclusion, our test showed that only the rabbit serum tested positive for the mock HIV antibody. We concluded this from our qualitative results that were taken as soon as the test was finished. We recorded "O" for no color, "+" for little color, and "++" for high color. The more color there was, the more antibody was present. In the above picture all the wells appear to have color, however, based on the standard ELISA Test, the lightest yellow is considered to be no color, or negative for the presence of the antibody. We also took results from a spectrophotometer (a machine that reads absorbance of light) reading. This reading was done two days after the experiment was complete, and the color solution was added to the samples. The results of this test weren't accurate as shown in the comparison between the expected results, and the results that we obtained.

There are many factors that could have caused our spec. analysis results to not be what was expected. The first is that it was left out for two days before running the test. The color had darkened enormously, and there wasn't much of a difference in color between each of the wells as there was when we first recorded our qualitative results. Dr. Anderson, a biologist for Modern Biology Inc. said leaving the sample out, though it was covered with parafilm, may have allowed it to become contaminated, which would have altered the results. There was also enough time for other reactions to take place in the solutions within the wells which may have affected the absorbance reading as well.

To obtain our qualitative results, we first observed the blue color change by placing the plate against a white peice of paper, and tilting it to try and eliminate any reflection that may be cast from one well to another. We then recorded the appropriate symbol for the color obsereved. We then repeated this process after the yellow color change, which is a double check on the results of the blue color change. The results of the qualitative data has many posibilities for errors and limitations. The first being that it is observed, and the differences in color can be very slight, therefore, it was often a judgment call, and people could have seen it differently. Another possibility for error is the reflection of color from one well to the other. For example, as the picture of our results shows, well B1 appears to be darker than the others. This could be because there is a reflection of color from another well, or because we had cross contamination in a step of our procedure.

We were fairly accurate in this experiment. We took many precautions not to contaminate equipment or samples. For example, when liquid had to be removed from each well during the series of dilutions and well washes, and different sterile tip was used on the micropipetter to make sure there wasn't cross contamination between the wells. We did this every time we had to make a series of dilutions. Micropipets were used to measure the amounts of most of the solutions that had to be added. They are extremely accurate for measuring microliters, which is mostly the units we were dealing with. Mistakes could have been made in the pre-experiment procedure when we were diluting and mixing the solutions we needed to use such as the TBS buffer and distilled water. Measuring amounts for these was done by using graduated cylinders, which are less accurate that the pipets. We were also very precise while conducting the experiment. We used the same pipet for a step we were on to make sure the measurments in that step would be the same. We also used the same mixed solution throughout the experiment instead of mixing a new batch each day.

If we were to conduct the experiment again, we would try to do it all in one day. Unfortunatly it took us about a week and a half to conduct the experiment, and we had to try to find the best stopping points or breaks in the procedure. This was hard to do, and sometimes our sample had to be left for up to two days or longer over a weekend. It was kept covered in the refirgerator through most of the process, as were the solutions, but during the last couple of days when there was gel in the wells, we had to leave it at room temperature so the gel didn't solidify. We got lucky, and nothing grew in it, but just the fact that it was left out at room temperature may have had an effect on our results.

Overall, our experiment was successful. We got the expected results, even without the spectrophotometer results, and the fact that it took so long to execute it. We didn't have ideal conditions, but the test still turned out fairly accurate. This shows how accurate the test itself is, and why it is the main test done for HIV and other diseases.

BIBLIOGRAPHY

Anderson, John N. IND-3. The ELISA Immunoassay pgs. 1-10. copyright 1992.

Michailides, George. Jury Selection. 07-Jan-1998. [www.stat.ucla.edu/cases/elisa/]

Grimes, William. Chambers, Linda. Narro, Martha. Immunology and AIDS. [www.blc.arizona.edu/aidscopy/] Wednesday, November 13, 1996.

Grimes, William. Chambers, Linda. Narro, Martha. Immunology and AIDS. [http://www.blc.arizona.edu/aids] © 1996. All rights reserved.

Giraldo, Roberto. Everybody Reacts Positive on the ELISA test for HIV. [www.virusmyth.com/aids/data/rgelisa.htm]. 1996

Practical Applications unit 2. Continuum Midwinter 1998/9

[www.biology.lsa.umich.;edu/courses/bio311/applications/pa.unit2.html] February 17, 1996.

Immunity Information Center / Information Section. [www.supercolostrum.com/colostrum/information/information9.htm]

Copyright © April 03, 2000 10:28 PM by Vibrant Life, ALL RIGHTS RESERVED. Permission is granted for non-commercial downloading.

Interview: Dr. Anderson at Modern Biology Inc.