Detection of Antiviral Antibodies in Serum Using an Elisa Technique - Essay Example

? DETECTION OF ANTIVIRAL ANTIBODIES IN SERUM USING AN ELISA TECHNIQUE Introduction: Immunity derives from the Latin word immunis, which means “free from the burden”. The branch of science that deals with the immune system is called immunology. There are two types of immunity, innate immunity and adaptive immunity. Innate immunity is the immunity that occurs right from birth. Acquired or adaptive immunity is the type which is obtained based on the induction with response to foreign substances (Edwards 1999). The foreign substance called the antigen enters into the living body and the body recognizes it and evokes an immune response. Antibody is produced by the body in response to the stimulus. This antibody is specific for a particular antigen and will react with the antigens and neutralize them. The antibodies are a large group of protein molecules present in the serum and tissue fluids. They are also called immunoglobulins (Edwards 1999). Clinical immunologists use the specific technique for the identification of the antibodies present in the human body. Rapid diagnosis of the antigen is performed using the technique called ELISA (Enzyme linked Immune sorbent assay). In ELISA technique, the antiviral antibodies are immobilized on a membrane or on a plastic plate. The test sample is then added to it (Sheehan 1997). If the viral antigen is present in the sample, then the antigen – antibody interaction occurs through immobilization technique. The unbound side of the antigen is then bound with another antiviral antibody. The binding of the second antiviral antibody is measured by using the calorimetric reading. The concentration of the antigen is detected by the intensity of the color formed (Kemeny 1991). Measles, Mumps, Rubella and Cytomegalovirus are serious viral infections. Measles and Mumps belong to the Paramyxoviruses group and they cause infection at the upper respiratory tract (Kemeny 1991). Cytomegalovirus belongs to the herpes viruses’ family and infects the lymphatic system. The last infection is Rubella from the Togaviridae family which causes infection in children. These viruses have the great danger of becoming fatal (Wild 2005). In this experiment, Measles, Mumps, Rubella and Cytomegalovirus viral antigens were immobilized on the micro titer plate and the obtained patient’s sera was added to the wells for immobilization. The plate was then checked for the presence of the particular disease by using the conjugate. The intensity of the color produced by the wells indicates the presence of the disease in the patient. If no color change is observed, it indicates the absence of the disease in the patient (Crowther 2001). Materials: Micro titer plate previously coated with viral antigens 2 x 5 ml Incubation buffer (IB) for diluting patient's serum 0.5 ml 2 patient’s serum samples labeled P1 and P2 8.0 ml Incubation buffer labeled 'conj' for diluting conjugate (made to volume). 2 ml Positive control serum for all 4 viruses labeled '+ve' 2 ml Negative control serum for all 4 viruses labeled '-ve' Blocking buffer (bovine serum albumin) in wash bottle (Buffer) Anti-human IgG-alkaline phosphates’ conjugate (Sigma) 8 ml 1 mg/ ml p-nitrophenyl phosphate in glycine buffer (pH 10.4) labeled 'Substrate' 5 ml 3M NaOH labeled 'Stop' P200 and P20 Automatic pipettes + tips Wad of paper towels positioned next to sink Method: At the outset, the plates were washed well and initials were marked. Following it, 200 µl of diluted viral antigens were added to the wells of the micro titer plates and the plates were incubated at room temperature for thirty minutes and at 4oC overnight. Since only 32 wells were used for the experiment, the remaining wells were blocked by using the blocking buffer. The wells are emptied again and then refilled with the blocking buffer, except 32 wells, and were incubated for three minutes and tipped off. After this process, the plates were drained. As a next course of action, the patients’ sera from the bottles P1 and P2 were diluted at a ratio of 1/100, and then 200 µl of the samples were added to each of the four wells. The test was duplicated for accuracy of the results. Then, 200 µl of the positive control serum was also added to the eight wells present in the column three and 200 µl negative control serum to the eight wells in the column four. After that was done, the plate was incubated at 37oC for 45 minutes in the incubator. After the incubation, the plate was removed and washed three times as previously, after which 200 µl of the conjugate was added to all the 32 wells in the plate and incubated for 30 minutes at 37oC in the incubator. The plates were then rinsed two times and refilled with the blocking buffer for 3 minutes. The plate was then emptied and 200 µl of the substrate was added to all the 32 wells. The plates were incubated at 37 degree Celsius for 15 minutes for the color development. The intensity of the color was measured using the Elisa reader. Result: P1 – patient 1 P2 – patient 2. From the figure, it was observed that the positive controls showed yellow color and the negative controls were clear. Color development was present in the wells B1, B2 and C1, C2 for the patient 1 and the other wells were clear. This indicates that the patient had mumps and measles. In the second patient’s sera, the color development was present in the wells E1, E2 and G1, G2. This indicates that the Patient 2 had measles and rubella. Discussion: Patient 1 showed positive results for mumps and rubella and negative results for measles and cytomegalovirus. Patient 2 showed positive results for measles and rubella and negative results for mumps and cytomegalovirus. Measles, Mumps and Rubella are childhood diseases and the patients who have the antibody for the disease in their body must have had the infection before or may be immune to it. The first patient was found to have the mumps and rubella. Measles, mumps and rubella are treated at the earlier stage by the MMR vaccine. If there are antibodies in the body even months after the infection, the results may be negative for the patients, so care must be taken to test the suspects after a month or so on. The second patient was found to show positive results for Measles and Rubella. This indicates that the patient has the antigens of the corresponding disease in his body. Both the patients show negative results for cytomegalovirus and positive for rubella. Conclusion: It was observed from the results that the patient 1 has measles and rubella antibodies in their body and patient 2 has mumps and rubella antibodies in their body. We don’t have the previous history of the patient regarding the previous occurrence and the duration of occurrence of the disease. Knowledge about the patient’s history will be an added advantage in knowing the series consequences of disease on the patient. On pregnant women, the test for rubella and cytomegalovirus has become compulsory in many countries. Early diagnosis can prevent the fetus from being affected as the infection rate is greater than 70% (Wild 2005). Thus, Elisa tests prove to be very helpful for the rapid diagnosis of Mumps, Measles, Rubella and Cytomegalovirus. Q1: The ELISA technique proves to be more accurate and effective for the identification of the antigens, there are some discrepancies related to it. The results are viewed by our naked eye and the intensity of the color developed is used to confirm the results. Sometimes the presence of little color may not be predicted by our eyes and can cause errors in the results. Though repetition of the tests gives accurate results, the time consumed by these tests is quite high. Similarly careful pippeting must be done to avoid errors. Non-specific binding of the antigen may also lead to false results (Wreghitt and Morgan-Capner 1990). Q2: Figure 2: Antibody is absorbed to the well Figure 3: patients sera were added to the well. The antibody which is complimentary to the antigen binds to it. Figure 4: the enzyme specific antibody binding antigen is added to the well and they form a sandwich. Figure 5: when the enzyme specific substrate is added to the well, the colorless substrate is converted into a colored product. The intensity of color formation gives the details about the concentration of the antibody present in the patient (Kemeny 1991). Q3: Competitive Elisa technique is used in the analysis of Anti-HIV antibody in the human body. It also measures the amount of antigen or antibody present in the sample. In this competitive Elisa test, the patient’s sera and the enzyme – labeled antibody are added simultaneously in to the plate. The antibodies present in the patient’s sera will compete with the enzyme labeled antibody for binding to the antigen. So if the patient’s sera contain antibodies, then the enzyme labeled anti-HIV antibody cannot bind to the antigen and during the washing stage, they will be removed from the plate. When the substrate for the enzyme labeled antibody is added, they will have less or no enzyme to get converted into product. So this is quite different from the other Elisa methods. In competitive Elisa for Anti-HIV antibody, if the color develops, then the result is negative and if color does not develop, the result is positive (Wild 2005). Figure 6: Competitive binding Elisa (Wild 2005). References: Crowther, J., 2001. The ELISA Guidebook, Humana Press. Edwards, R., 1999. Immunodiagnostics: A practical approach, Oxford. Kemeny, D., 1991. A Practical Guide to ELISA, Pergamon Press. Sheehan, C., 1997. Clinical immunology: Principles and Laboratory Diagnosis, Lippencott. Wild, D., 2005. The Immunoassay Handbook, Oxford. Wreghitt, T & Morgan-Capner, P., 1990. ELISA in the Clinical Microbiology Laboratory, PHLS. Read More