Derek Wong
Biology 265L-03
Lab 6
Enzyme-Linked
Immunsorbent Assays Tests and Applications
Introduction:
This experiment is about Enzyme-Linked Immunosorbent Assays,
or ELISA for short. ELISA uses enzymes conjugated to antibody molecules
to detect an antigen in a sample. There are different types of ELISA,
Direct, Indirect, and
This experiment will use alkaline phosphatase as the enzyme, p-nitrophenyl
phosphate as the substrate and peptide 579 serves as the unknown concentration
to be determined by use of a standard curve. Four unknowns were given and by
using the standard curve it can be determine their concentrations. Application
of ELISA will also be examined such as a pregnancy test and a cholesterol test.
Results:
ELISA: See Figure 1: Standard Curve for ELISA test on the last page.
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Table 1:
Raw Data from ELISA showing the absorptions (nm) for the known and unknown concentrations. |
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Wells: |
1 |
2 |
3 |
|
|
A |
2.581 |
2.423 |
0.366 |
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B |
2.44 |
2.175 |
0.127 |
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C |
2.115 |
2.064 |
2.234 |
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D |
1.525 |
1.035 |
2.364 |
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E |
0.681 |
0.748 |
1.021 |
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F |
0.417 |
0.284 |
0.537 |
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G |
0.609 |
0.17 |
0.232 |
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H |
0.128 |
0.115 |
0.324 |
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Table 2:
Average of the blanks in H1 and H2 wells. |
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Blanks
(nm) |
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H1 and H2 |
0.1215 |
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Table 3:
Concentrations in each well and their absorption with the blanks subtracted
from it. |
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Wells |
Concentration
(ug/mL) |
Absorptions
– Blanks (nm) |
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A1 and A2 |
100 |
|
2.381 |
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B1 and B2 |
25 |
|
2.186 |
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C1 and C2 |
6.25 |
|
1.968 |
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D1 and D2 |
1.56 |
|
1.1585 |
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E1 and E2 |
0.4 |
|
0.593 |
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F1 and F2 |
0.1 |
|
0.268 |
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G1 and G2 |
0.025 |
|
0.229 |
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Table 4:
Unknown Absorptions (nm) with the blank readings subtracted. |
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Unknown 1 |
A3 and B3 |
0.125 |
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Unknown 2 |
C3 and D3 |
2.1776 |
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Unknown 3 |
E3 and F3 |
0.6575 |
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Unknown 4 |
G3 and H3 |
0.1565 |
Pregnancy: Urine Sample #3 - 27 year old recently engaged
teacher. It was observed that only one pink band was seen.
Cholesterol test: Plasma Sample #1 - 21 year old male single college student.
Total Cholesterol was found to be 254 mg/dL using the Cholestech LDX System,
which registers as high cholesterol.
Discussion:
In this lab, seven known concentrations of a solution were given to create a standard curve and then determine the concentrations of four unknown concentrations. However, when the ELISA was ran, three of the unknown concentrations did not fall on the standard curve and therefore can not be determined. Unknown #1 and Unknown #4 could not be determined since they did not fall on the sigmoidal standard curve. Unknown #2 was found to be around 30 ug/ml, and Unknown #3 was found to be around 0.4 ug/ml. Possible errors in the experiment could be contamination, fingerprints on the bottom of the ELISA plates, and/or mispippeting of solutions into the plates. This could cause the absorptions reading to be very different since ELISA is a very sensitive process. Also if the beginning parts of the experiment when running the known concentrations had more enzymes or some factor that would cause a higher absorption reading then the curve would be shifted upwards. Once the curve is shifted upwards, the rest of the values would also be higher and could cause the unknown values to fall off the graph.
In the pregnancy test only one pink line was seen indicating
that the female was not pregnant at the time of the test. The test is ran on
the basis that when pregnant the human female releases human chorionic
gonadotropin in their urine. If the
female is pregnant then the hormone should be present in the urine. The
pregnancy test tested for the presence of human chorionic gonadotropin (hCG) by
use of a colored reaction with goad alpha-mouse IgG substrate conjugate (hence
the pink color). The first band seen (closest to the test end) is the control
and if it turns pink it means that the test is working. If the second band appears (not seen in this
experiment) then it means that the person does have hCG and is hence pregnant.
The total cholesterol was found to be 254 mg/dL which is
considered to be on the high end. The
cholesterol was found by using Cholestech LDX system which uses a series of
filtration and enzyme-catalyzed reaction.
The reaction then undergoes a color change which is then read by a
micro-spectrophotometer and then amount of cholesterol can be derived from
that.
The use of ELISA has benefited both the medical and the
research community by allowing us to develop innovative ways to test for the
detection of antibodies. ELISA is a
relatively quick and simple way of determining if a person has a particular
antigen. The applications of ELISA are
almost infinite since the concept can be applicable in testing for anything that
could be conjugated to an antibody, for example the pregnancy and the
cholesterol test does not require antibodies but are based on the same
principles that ELISA are based on.