1. OBJECTIVE
In this experiment, you will use the third-generation enzyme-linked immunosorbent assay (ELISA) to diagnose the HIV virus. This assay can detect, with high specificity and sensitivity, IgM, IgG and IgA antibodies present in serum or plasma samples, which were produced against various viral antigens.


At the end of this experiment, you should be able to:

apply the standard methodology used in various immunoenzymatic assays;

interpret and understand the results obtained individually from sample analysis;

organize the immunoenzymatic assay procedure for the analysis of different numbers of samples;

identify the biosafety measures necessary for diagnosis by immunoenzymatic assays.

2. WHERE TO USE THESE CONCEPTS?
HIV diagnosis by enzyme-linked immunosorbent assay (ELISA) is a useful and dynamic tool used in the surveillance of global AIDS epidemiology. This type of test has a series of advantages compared to other diagnostic tests, as it allows the analysis of a large number of samples simultaneously, as well as being reproducible in periodic tests and capable of detecting antibodies in recent infections.

ELISA for detecting HIV can be used in clinical laboratories, in the diagnosis of patients with suspected infection, in addition to serological research in tests recommended in the prenatal care of pregnant women. Also, this type of assay is used in serological screening in blood banks, ensuring safety in the transfusion of blood and blood products, and can be used for epidemiological studies in specific regions or populations.


3. THE EXPERIMENT
In the experiment, a third-generation commercial ELISA kit will be used for HIV diagnosis, which allows the detection of IgM, IgG and IgA antibodies produced against HIV antigens. In the procedure, serum or plasma samples from patients will be analyzed using the components of the commercial kit, such as polystyrene polycuvettes sensitized with recombinant antigens, the conjugate of recombinant antigens linked to the peroxidase enzyme and the revealing solution containing hydrogen peroxide. The experiment consists of three incubation periods and two periods of washing the test plates, concluding with the addition of the enzymatic reaction stopping solution. Finally, the results will be obtained by reading the optical density of the enzymatic reaction using a spectrophotometer.


4. SECURITY
For all stages of the experiment, personal protective equipment (jacket, gloves and glasses) must be used, which prevents the handler from coming into contact with irritating or biohazardous materials. The handling of serum or plasma samples must be carried out with care, and they must be considered as potentially contaminated, in order to respect biosafety measures. Finally, all material used, including plastics and solutions, must be incubated in a 5% hypochlorite solution for 60 minutes before being discarded. The bench where the test will be carried out must be cleaned before and after the procedure.


5. SCENARIO
The experiment will be carried out on the laboratory bench, with the aid of micropipettes, disposable tips, paper towels and a container for disposing of material containing a 5% hypochlorite solution. The components of the commercial kit and the plasma or serum samples are stored in a refrigerator (2° to 10°C) and must be placed at room temperature for 15 minutes before starting the experiment. An oven at 37°C will also be used for incubations and a stopwatch to control time. The spectrophotometer must be programmed to read at a wavelength of 450 nm (or 450/600-650 in equipment that performs bichromic reading). Washing of polycuvettes will be carried out in automatic washing equipment or by manual washing with a pissette.