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Examples of Reactions by an HIV-1 Western Blot Introduction Enzyme assay kit arsenal of laboratory methods is available to screen blood, diagnose infection, and monitor disease progression in individuals infected by HIV.
These tests can be classified into those that: The focus of this discussion is on antibody detection, the most widely used and, in most situations, most effective way to identify HIV infection.
Accordingly, screening tests possess a high degree of sensitivity, whereas confirmatory assays have a high specificity.
Tests with high sensitivity produce few false-negative results, whereas tests with high specificity produce few false-positive results.
These classes of assays, performed in tandem, produce results that are highly accurate, reliable, and appropriate to protect the blood supply or assist in the diagnosis of HIV infection. Technical errors do occur, however, and there are biologic factors that can limit the accuracy of HIV tests.
Therefore, along with the testing process, there is the requirement for an extraordinary and dedicated quality assurance program. Early Detection and the Window Period Specific antibody to HIV is produced shortly after infection, but the exact time depends on several factors, including host and viral characteristics.
Importantly, antibody may be present at low levels during early infection but not at the detection limit of some assays.
Using the early-generation tests, antibody could be detected in most individuals by 6 to 12 weeks after infection. Newer-generation assays, including the third-generation antigen sandwich assays, can detect antibody at about weeks after infection.
Most antibody tests currently on the market have near perfect and equivalent degrees of sensitivity for detecting most individuals who are infected with HIV epidemiologic sensitivitybut they vary in their ability to detect low levels of antibody analytical sensitivitysuch as those occurring before complete seroconversion.
For the diagnosis to be correct, however, detection depends on the use of tests that are effective in identifying HIV antibodies, and not antibodies directed to other infectious agents that may be antigenically similar. Antigens used in HIV diagnostic tests must be appropriately specific, and usually are purified antigens from viral lysates, or antigens produced through recombinant or synthetic peptide technology.
The use of such antigens allows HIV screening tests to possess both sensitivity to detect infection and specificity to detect noninfection. Considerations in Choosing a Screening Test Methodology Regardless of the particular screening test used, serum or plasma samples first are tested screened using a test with high sensitivity, most often an enzyme-linked immunosorbent assay ELISA"rapid test," or "simple method" described below.
ELISA is the screening method used most commonly, with the other 2 methodologies offering more rapid results with simple procedures applicable for use in point-of-care testing and in developing countries. With the advent of new therapies to treat HIV infection and the recommendation to institute therapy as soon as possible but no later than 72 hours after exposure, 5 rapid assays may be the most appropriate for testing the source patient after exposure eg, needlestick injuries.
More recently, tests have been developed using fluids that can be obtained conveniently outside the clinical laboratory. Whole blood from fingerstick and oral fluid saliva has been shown to be as effective as serum or plasma for detecting antibodies to HIV.
Reactive Results Regardless of the screening method, a sample producing a reactive result must be screened again in duplicate, with at least 2 of the 3 results being repeatedly reactive before verifying infection with confirmatory assays.
The most common reason for nonrepeatable results by screening tests is technical error. Samples that produce repeatedly reactive results by screening tests must be further tested using confirmatory tests, or other confirmatory strategies see below.
Although screening tests are exquisitely sensitive, they lack an adequate degree of specificity. An example is their low predictive values when testing a population having a low prevalence of infection.
If 1 individual in that same population is truly infected, the test will produce 2 positive results 1 from the infected individual, and 1 false positive. Consequently, additional testing is required to differentiate between true- and false-positive results.
A complete review of screening assays and a description of the use of test indexes has been published. After a wash step to remove unbound serum components, addition of a conjugate an antihuman immunoglobulin with a bound enzyme binds to the specific antibody that is attached to the antigens on the solid phase.
Following another wash, addition of an appropriate substrate results in color development that is detected by a spectrophotometer and is proportional to specific HIV antibody concentration in the sample.
Optical density OD values are produced as the colored solution absorbs transmitted light, and provide an indication of the amount of color, which is proportional to the amount of antibody bound ie, antibody concentration. Several indirect ELISA tests incorporate polyvalent conjugates anti-IgG and anti-IgM and antigen-sandwich configurations in order to increase sensitivity for detecting early infection during seroconversion.
Alternate ELISA methodologies include a competitive format in which specific HIV antibody in the sample competes with an enzyme-bound antibody reagent for antigen sites on the solid phase. In this method, color development is inversely proportional to specific HIV antibody concentration.
A more recent addition to ELISA technology is the antigen sandwich method in which an enzyme alkaline phosphatase or horseradish peroxidase is conjugated to an HIV antigen similar to the immobilized antigen on the solid phase.
The antibody in the sample is "sandwiched" between 2 antigen molecules, 1 immobilized on the solid phase and 1 containing the enzyme. Subsequently, the addition of substrate results in color development in proportion to antibody concentration.
The antigen sandwich ELISA is considered the most sensitive screening method, given its ability to detect all isotypes of antibody including IgM.
Incidence estimates most often are calculated by testing a cohort of individuals at 2 different time periods and observing the number of new infections.Detecting active caspases in apoptotic cells using the Vybrant FAM Caspase Assay Kits. Unbound FLICA reagent diffuses out of the cell and is washed away.
The green-fluorescent signal is a direct measure of the amount of active caspase that was present at the time the inhibitor was added. Enzyme Activity Assays Screen Enzyme Modulators With Innovative Assays Our decades of experience in the design and manufacture of active enzymes and their substrates supports development of an ever-expanding portfolio of biochemical assays.
Some standard enzyme assays measure the activity of kinase, phosphatase, superoxide dismutase, and Beta-Galactosidase. Kinases are classed as phosphotransferases as they catalyze the transfer of phosphate groups from high-energy molecules to substrates.
This process is known as phosphorylation (where a substrate gains a phosphate), and is a . Enzyme immunoassays (EIAs), also known as enzyme-linked immunosorbent assays (ELISAs), combine antibody binding with enzymatic detection to quantify molecules of interest.
EIAs are easy to perform, require little specialized equipment, and both the experienced lab technician and the research lab novice can learn EIA skills quickly. The enzyme-linked immunosorbent assay (ELISA) (/ ɪ ˈ l aɪ z ə /, / ˌ iː ˈ l aɪ z ə /) is a commonly used analytical biochemistry assay, first described by Weiland in The assay uses a solid-phase enzyme immunoassay (EIA) to detect the presence of a ligand (commonly a protein) in a liquid sample using antibodies directed against the protein to be measured.
Protein and Enzyme Activity Assays These fluorogenic and chromogenic substrates and assay kits include substrates for phosphatases as well as reagents to measure the activity of enzymes such as ATPases, GTPases, and DNA and RNA polymerases, which hydrolyze phosphate esters.