Immunology techniques are based on the compatibility of the antigen and antibody reactions that are used in clinical diagnosis of viral infections. The case at hand is the screening of the villages with an aim to determine the infection. Blood samples of individuals were used. ELIZA and dot blot techniques were employed to detect the presence of specific antigens in the blood stream of suspected individuals.
Immune Blot Analysis
Detection of Avian Flu Antigens
Nitrocellulose membrane, with the use of a sterile micropipette, is placed on a clean towel, and the 3ul of the samples is dispensed onto the membrane in the following order: positive control obtained from known infected case, negative control from confirmed uninfected case, and a patient sample.
The membrane is divided into two parts and two plates are labeled 1 and 2. One piece is put into the first plate and soaked in 5ml of buffer. The membrane is allowed to dry. It is kept into a plate and put into the oven for 20 minutes at room temperature. Later, the membrane is rinsed and dried. This is followed by the washing of the membrane in a plate using 5ml of the buffer (Maqi, 2008).
The first antibody is put into plate 2 with the membrane and allowed to soak. This is removed by the use of forceps. The membrane is incubated at room temperature with frequent shaking. The plate is then washed and rinsed. Later, it is filled with 5ml of buffer.
The second antibody, which is an enzyme conjugate, is added, followed by the addition of 4ml of 1g horse radish peroxidase into the plate. The membrane is allowed to soak for a while before it is washed with buffer solution, and then it is incubated at room temperature for 3 minutes.
After rinsing the membrane in the plate 2, 4ml of peroxidase substrate is added to the plate and allowed to soak for 20 minutes at 370C. Excess solution is drained from the membrane, which is done by putting the membrane onto a paper towel.
The spots of precipitate substrate on the patient’s sample are compared with the spots on the controls. When the patient’s spots sample corresponds with the positive control, the inference is that the patient is infected with avian flu virus.
Patient record card #2 diagnosed with avian flu virus.
The dots on the membrane are developed after incubation and are compared to the dots on the controls samples.
Micro-plates with wells are used in ELISA. 100eul of the antigen is added into the wells and incubated for five minutes at 370C. This is discarded by one rapid movement. Washing is done on the wells by the use of buffer solution. It is done three times and blotted to dry by the use of blotting material.
In order to determine the amount of the antibodies in the samples, serial dilution should be done, with five tubes labeled 1-5 having the dilutions of 1:400, 1:1600, 1:6400, 1:25600, and 1:102400 respectively. 150ul of the PBS buffer is added to each of these tubes. 150ul of the antibody is added to the tubes. Primary antibody solution is added by use of micro pipette. Besides, by its utilization, the antibody dilutions are added to the wells: 75ul of the PBS is added into the tubes, 75ul of patient sample is added into the D7 and D8 wells. The preparation is then incubated at 370C for 30 minutes (Anon, 2000).
The contents of the wells are discarded, wells are washed using PBS buffer, and then dried. Secondary antibody preparation is incubated at 370C using the same criteria followed by washing and drying. Substrate is added. The time is allowed for color development. The intensity of the color developed is directly proportional to the amount of the antibodies in the system of the patient.
- Patient card number 1 with avian flu virus with the antibody titers of 1:100.
- Patient card number 2 with 1:1600 antibody titers.
The above shows the appearance of the micro plates after the ELISA test.
The intensity of color developed is directly proportion to the concentration of the antibody tested.
Avian flu is a viral infection that can infect both birds and the human beings; it is of the type h5n1. In human beings, the diagnosis is done in cases of suspected outbreaks and it is based on the presence of antibodies in the blood of the patient.
PCR is used to detect the presence of the DNA segments in the samples of the suspected organism. DNA amplification is applied in the test, after which it is then stained.
ELISA is used to quantify the amount of antibodies in the blood of the patients. The color developed is directly proportional to the amount of antibodies. This is determined by observing the last tube with intense color.
PCR is the fastest and the most reliable method in the diagnosis of viral infection. It is accurate, sensitive, and highly specific. Although polymerase chain reaction (PCR) is expensive, its employment is more effective and preferable when urgent diagnosis is required.