Clinical immunologists use a particular technique to detect certain antigens bound to antibodies in serum. It is called the ELISA (enzyme-linked immunosorbent assays) technique. The ELISA technique has many different stages that differ depending on the system used. There are three identified systems of ELISA technique, each form the foundation of competitive and inhibition ELISAs; direct, indirect and sandwich ELISA technique. For this experiment we will be using an indirect ELISA technique involving numerous different stages. A microtitre plate with different viral antigen extracts already attached to the surfaces of the plastics well acknowledged by Crowther 2009 as solid phase is supplied for the experiment. Once the diluted patients sera is added it is absorbed in to the wells and the antibodies in the patients sera bind to the viral antigens attached to the wells of the microtitre plate. Separation of bound and unbound antibodies are carried out by a washing method, incubation and increase of antihuman antibody conjugated to an enzyme and a substrate. Once the process is completed, detection of viral infections in patients is noticeable by development of colour in the wells.
Paramyxoviruses are one kind of family of viruses, consisting of a helical nucleocaspid, genetic material encircled by an envelope and glycoproteins. (Wagner et al…1999). Measles (morbillivirus) and mumps (rubulavirus) virus belong to the paramyxovirus family and are known to be infectious viral diseases. They mainly infect the upper respiratory tract, in some cases spread to other organs causing serious illness. Another infectious disease is rubella belonging to the Togaviridae family of viruses. It has an icosahedral nucleocaspid encircled by an envelope and glycoproteins that bind to the Fc region of the antiviral antigen in the microtitre plate. Rubella usually occurs in children, one of its symptoms being a distinctive rash. However, if rubella occurs in pregnant woman it is proven to be quite fatal. The human cytomegalovirus (CMV) is another virus belonging to the family of herpesviruses, infecting the lymphatic system and causing infectious mononucleosis. Measles, mumps, rubella and cytomegalovirus are serious viral infections. They have the potential to cause serious illness and death. Therefore, it is crucial that the ELISA technique is carried out precisely so that patients can be diagnosed correctly and undergo designated treatment.
Requirements and Method
As per schedule
Results
From the diagram we can see that the positive control showed a yellow colour and the negative control was clear. Colour development in wells B1, B2, C1, C2 and D1, D3 indicates that patient 1 can be diagnosed with mumps rubella and cytomegalovirus. Patient 2 can be diagnosed with measles, rubella and cytomegalovirus as colour developed in wells E1, E2, G1, G2 and H1, H2. Wells A1, A2 and F2 showed faint yellow colour which could have been an error of contaminating the wells.
Q2
Below is an illustration of how indirect ELISA technique is carried out.
Need to draw diagram
The wells of the microtitre already have antigens attached it to it so when the patients diluted serum is added the required viral antibodies attach to those antigens. All those unbound proteins are removed by being washed away and we are left with bound antibodies-antigen. Once the incubation period and the washing procedure are completed the anti-human IgG-alkaline phosphatase conjugate (sigma) is added and binds to the antigen-antibody. Final stage is added the substrate to the wells allowing the development of colour to those bound viral antigen-antibodies, leading to identification of viral infections. (World Health Organisation 1999).
Q1
Although the use of indirect ELISA technique proved to be a successful test some discrepancies were highlighted. To determine whether a patient had any viral infections we were made to diagnose them by reading the microtitre plate with our eyes. This in itself is an error as the determination of positive and negative control differs depending on the persons reading. Repetition of the test would have given us a more accurate result also allowing, if, any non- specific binding with the viral antigens that interfere with the results to be rectified. The need to be accurate and precise whilst pipetting is essential as they may result in mis- diagnosis of patients.
Q3
Competitive ELISA technique is used in the detection of anti-HIV antibody and measurement of quantity of antigen or antibody in a substance. Its method matches the same as the one in normal ELISA technique except that pretitration system is used to compete with the antibodies added. One major difference between competitive and normal ELISA technique would be that of colour development. If the colour is evidently seen the HIV antibody is less present and if the colour is less evident or not noticeable the higher quantity of HIV antibody, as the conjugate added did not bind successfully to produce any colour.
Although we tested for viral infections on patient one and two no background knowledge of the two patients is known. It is important to know background of patients as some viral infections can lead to serious consequences. For example, patient one is diagnosed with mumps, rubella and cytomegalovirus. Now if patient one was in early stages of pregnancy the chance at which the mumps and rubella virus can affect the foetus is higher than 70%. The possibility of miscarrying is very rare. This also applies to the patients with measles however measles tend to affect young children and their symptoms are more subtle than those that affect adults. Patients who contract cytomegalovirus whilst pregnant have a one in two hundred chance of passing the virus on to the unborn foetus. Abortion is available for those patients who are under eight weeks pregnant however if they wish to proceed the full length of pregnancy and give birth the child may have congenital abnormalities. Hence, patients who are pregnant and are experiencing symptoms associated to rubella and measles an ELISA procedure should be carried out to confirm and rule out any viruses. The ELISA procedure which would be carried out should be that of the one that uses IgM with the enzyme conjugate, (Best, 2007) as this procedure is more affective. Vaccination of rubella has been available since 1969 and has shown a decrease in births related to rubella. The vaccine has become so uncommon that children with rubella in 2009 were only 0.6% according to the Health Protection Agency.
Overall, the indirect ELISA technique helped us diagnosis patients one and two with both rubella and cytomegalovirus. Patient one was also diagnosed with mumps and patient two with measles. As the procedure was only carried out once a few errors occurred which can be rectified by repeating the experiment, read end results with a spectrophotometer and to be more precise and accurate whilst pipetting which in affect will reduce contamination as seen in the above results.
