BI 106 A&P-2 HYBRID LAB ASSIGNMENT
An ELISA test is an examination method that incorporates different aspects of the immune system in order to identify different responses that occur in the body. The test involves both an enzyme and an antigen or antibody. ELISA stands for the Enzyme-linked Immunosorbent Assay. The test is usually used in numerous laboratories in order to determine whether a specific antibody exists in a particular patient’s blood sample.
The process of the ELISA test begins with the removal of the white and red blood cells. The removal of the white and red blood cells leads to the formation of a watery fluid called the serum. A sample of the serum, which may contain the antibody, is allowed to react with the desired antigen. If there is a correct match, the antigen and antibody bind together. However, the detection is possible once a second antibody is added. The latter antibody is gotten from the serum of an animal which had previously been injected with an human antibodies. In such a case, the human antibody acts as an antigen in the animal’s blood and therefore the animal produces antibodies to fight against the human antibodies. The second is capable of being chemically linked to a system that may bring out a detectable signal. The system of signaling entails an enzyme which is attached to the second antibody. When the requisite chemical is afterwards added it is changed into a colored substance by an enzyme. One can tell the amount of enzyme present by measuring the amount of color produced. The amount of the secondary antibody is dependent on the first antibody present. Since the primary antibody binds with the antigen, the higher the amount of antigen the higher the amount of antibody retention. For this reason, the amount of color can represent the quantity of antigen which was initially present.
There are a couple of problems and limitations that may be encountered during the particular test. The experimental problems that may arise during the ELISA test are as a result of the variables that play a significant role during the procedure. Some of the primary variables include temperature, time, reagent selection and volume measurement. If these variables are not adjusted to the requisite standards, the subsequent steps will be affected and consequently the final outcome.
In addition to the experimental problems, the test has some limitations. Although the test may produce a positive result, indicating the presence of antibodies, it may not signify that the patient is ill. This is because the body can continue producing antibodies even after the particular person had recovered from a previous disease.
The second limitation involves the variance in the production of antibodies in different bodies. The amount of antibodies may be too small or even go undetected. This may result to a false negative result. The final limitation is whereby an unrelated anybody reacts with the target antigen. This reaction may give a positive result. However, the positive reaction is false since the particular antibody is not detected. For this reason, scientists or physicians use more than two samples in the testing process in order to obtain the most accurate results and avoid the simple experimental mistakes
.SLE (Systemic Lupus Erythematosus) is an autoimmune disease whereby the body’s immune system loses the ability to distinguish between its own body cells and antigens. Therefore it fights its own body cells as opposed to fighting antigens. Since it is systemic it affects several parts of the body such as the kidneys, lungs, heart, nervous system and even the skin.
During the first step the blood samples are centrifuged in order to precipitate the blood cells. After the blood cells have been precipitated, serum, the clear fluid, remains. In the second step, the dilutions that are made to the sera (serial dilutions), are for the purpose of ascertaining the quantity of antibodies in the particular sample. A highly diluted sample will not give positive results if thr level of titer of the antibody is low in the sera. The most common solution that is used is PBS. This solution contains the same concentration of salt as that found in normal blood.
In the third step, the ELISA plate has to be prepared since antigens, and other biological materials are capable of binding onto the plastic material consisting the wells of the ELISA plate. For this reason, the coating process must be done with caution. If a small amount of antigen is used a false positive result will be achieved and if a lot of antigen is used, a false negative result will be achieved. A new pipette tip is used for each dilution in order to avoid the unnecessary mixing of the sera with antigens, which may bring about inaccurate results.
An ELISA test may be prone to errors. One of the major errors that may occur is as a result of the various biological or chemical reagents that are used. Another factor that contributes to the errors is that the ELISA test is not always conducted in the appropriate condition. In order to curb these problems, two controls are used. The first control should always produce a positive result if all the conditions are met while the second control should not produce a positive result. If the control samples do not react as anticipated, the test is considered a failure and hence is to be done over again.
The incubation of the serum is usually done in order to ascertain that the antibodies interact as expected with the antigens. The temperature of incubation is at 37 degrees Celsius since SLE is a disease that affects the human body, which is normally at 37 degrees. THE timer is set for fifteen minutes so that adequate binding can occur, failure to which the final measurement will be artificially low.
THE wells are usually washed in order to eliminate any of the antibodies that did not react with the SLE antigen. The unbound antibodies may also remain in the well after the fluid is removed from the well. It must therefore be removed since the anti-human antibody in the next step may react with these antibodies consequently giving a false positive result.
THE second antibody (the anti-human antibody from the rabbit) recognizes the human antibody and binds with it. It does not recognize SLE antigen, as was the case with the first antibody.
Washing with PBS helps in removing any antibodies that did not react with the SLE antigen. After the fluid has been removed, unreacted/ unbound antibodies may still remain in the wells. These antibodies must be removed since the anti-human antibody that will be added during the subsequent step may react with any antibodies which will have remained in the wells, hence producing a false positive result.
Horseradish Peroxidase is one of the most used enzymes that are attached to antibodies. Hydrogen Peroxide, together with the latter enzyme reacts with the ABTS resulting in a yellow solution. The amount of this solution may be measured with the naked eye or with the use of a spectrometer. If duration of less than fifteen minutes is set on the timer, the appropriate reaction will not occur and there will be no color evident at the end of the experiment.
Pulmonary or Respiratory function tests are a group of tests that are done to establish the proper functioning of the lungs.
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