Post by Admin on Mar 23, 2016 19:07:05 GMT -8
Like the ELISA procedure, the western blot is an antibody detection test. However, unlike the ELISA method, the viral proteins are separated first and immobilized. In subsequent steps, the binding of serum antibodies to specific HIV proteins is visualized.
Specifically, cells that may be HIV-infected are opened and the proteins within are placed into a slab of gel, to which an electric current is applied. Different proteins will move with different speeds in this field, depending on their size, while their electrical charge is leveled by a surfactant called sodium lauryl sulfate. Some commercially prepared Western blot test kits contain the HIV proteins already on a cellulose acetate strip. Once the proteins are well-separated, they are transferred to a membrane and the procedure continues similar to an ELISA: the person's diluted serum is applied to the membrane and antibodies in the serum may attach to some of the HIV proteins. Antibodies that do not attach are washed away, and enzyme-linked antibodies with the capability to attach to the person's antibodies determine to which HIV proteins the person has antibodies.
There are no universal criteria for interpreting the western blot test: The number of viral bands that must be present may vary. If no viral bands are detected, the result is negative. If at least one viral band for each of the GAG, POL, and ENV gene-product groups are present, the result is positive. The three-gene-product approach to western blot interpretation has not been adopted for public health or clinical practice. Tests in which less than the required number of viral bands are detected are reported as indeterminate: a person who has an indeterminate result should be retested, as later tests may be more conclusive. Almost all HIV-infected persons with indeterminate western blot results will develop a positive result when tested in one month; persistently indeterminate results over a period of six months suggests the results are not due to HIV infection. In a generally healthy low-risk population, indeterminate results on western blot occur on the order of 1 in 5,000 patients.[19] However, for those individuals that have had high-risk exposures to individuals where HIV-2 is most prevalent, Western Africa, an inconclusive western blot test may prove infection with HIV-2.[20]
The HIV proteins used in western blotting can be produced by recombinant DNA in a technique called recombinant immunoblot assay (RIBA).[21]
Specifically, cells that may be HIV-infected are opened and the proteins within are placed into a slab of gel, to which an electric current is applied. Different proteins will move with different speeds in this field, depending on their size, while their electrical charge is leveled by a surfactant called sodium lauryl sulfate. Some commercially prepared Western blot test kits contain the HIV proteins already on a cellulose acetate strip. Once the proteins are well-separated, they are transferred to a membrane and the procedure continues similar to an ELISA: the person's diluted serum is applied to the membrane and antibodies in the serum may attach to some of the HIV proteins. Antibodies that do not attach are washed away, and enzyme-linked antibodies with the capability to attach to the person's antibodies determine to which HIV proteins the person has antibodies.
There are no universal criteria for interpreting the western blot test: The number of viral bands that must be present may vary. If no viral bands are detected, the result is negative. If at least one viral band for each of the GAG, POL, and ENV gene-product groups are present, the result is positive. The three-gene-product approach to western blot interpretation has not been adopted for public health or clinical practice. Tests in which less than the required number of viral bands are detected are reported as indeterminate: a person who has an indeterminate result should be retested, as later tests may be more conclusive. Almost all HIV-infected persons with indeterminate western blot results will develop a positive result when tested in one month; persistently indeterminate results over a period of six months suggests the results are not due to HIV infection. In a generally healthy low-risk population, indeterminate results on western blot occur on the order of 1 in 5,000 patients.[19] However, for those individuals that have had high-risk exposures to individuals where HIV-2 is most prevalent, Western Africa, an inconclusive western blot test may prove infection with HIV-2.[20]
The HIV proteins used in western blotting can be produced by recombinant DNA in a technique called recombinant immunoblot assay (RIBA).[21]