Human T-Cell Lymphotropic Virus Types I and II (HTLV-I/-II) Antibody Screen with Confirmation, Serum
Method Description Describes how the test is performed and provides a method-specific reference
In the MP Diagnostics, HTLV-I/-II ELISA 4.0, the wells of the polystyrene microplate strips are coated with a mixture of 3 different human T-cell lymphotropic virus (HTLV) recombinant proteins, which correspond to the highly antigenic segments of HTLV-I and HTLV-II viruses. Human sera diluted in a diluent containing the conjugate are incubated in these coated wells. The conjugate is based on a trifusion recombinant protein, which is labeled with horseradish peroxidase. The trifusion antigen is generated by cloning 3 cDNA fragments coding for the 3 HTLV recombinant proteins into a single vector. HTLV-I/-II-specific antibodies (IgA, IgG, and IgM), if present, will bind to both the antigens immobilized on the solid phase and the trifusion antigen of the conjugate. After incubation, the wells are thoroughly washed to remove unbound materials. A colorless substrate solution containing chromogen 3,3',5,5'-tetramethylbenzidine (TMB) is then added to each well. The presence of specific antibodies is indicated by the presence of a blue color after incubation, which changes to yellow when the color reaction is terminated by the addition of sulfuric acid. The intensity of the resulting yellow product is measured at 450 nm using a spectrophotometer and is proportional to the amount of antibodies present in the specimen. Initially reactive specimens will be retested in duplicate. Specimens that do not react in either of the duplicate repeat tests are called "nonreactive" for antibodies to HTLV-I/-II. Specimens that are reactive in either of the duplicate tests are considered "repeatedly reactive." (Package insert: HTLV-I/-II ELISA 4.0, revision 4; MP Biomedicals Asia Pacific Pte. Limited. Singapore)
Line Immunoassay (LIA) Confirmation:
INNO-LIA HTLV I/II is a line immunoassay that uses well-defined antigens derived from HTLV-I and HTLV-II immunodominant proteins. The antigens used are either recombinant proteins or synthetic peptides, highly purified and fixed on a nylon membrane strip. The sequences are selected to allow the detection of antibodies with a wide specificity to all known isolates of the HTLV strains. The antigenicity exhibited by these proteins and peptides is either common to both HTLV-I and HTLV-II, or type-specific to 1 of the 2 viruses to allow confirmation and discrimination in a single assay. Two gag (p19-I/II, p24-I/II) and 2 env (gp46-I/II, gp21-I/II) bands are applied as nontype-specific antigens, which are used to confirm the presence of antibodies against HTLV-I/II. The type-specific antigens for HTLV-I (gag p19-I, env gp46-I) and for HTLV-II (env gp46-II) are applied to differentiate between HTLV-I and HTLV-II infections. In addition, 4 control lines are coated: 1 negative control (streptavidin), and 3 positive control lines, a strong (antihuman IgG), a moderate (human IgG), and a weak (human IgG) line.
This assay is based on the enzyme immunoassay principle. Specific anti-HTLV antibodies, if present in the clinical sample, will bind to the HTLV antigen lines on the strip. Subsequently, goat antihuman IgG antibodies labeled with alkaline phosphatase are added and will bind to any HTLV antigen-antibody complex previously formed. Incubation with a chromogenic substrate produces a dark brown color in proportion to the amount of specific antibodies present in the sample. The color development is stopped with sulfuric acid. If the sample contains no HTLV-specific antibodies, only a low background color develops.
Every sample is classified as negative, indeterminate, or positive, then further classified as HTLV-I, HTLV-II, or untypeable-positive according to the antibody band reactivity. (Package insert: INNO-LIA HTLV I/II Score kit 25957 v5; INNOGENETICS N.V., Gent, Belgium)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
HTLV-I/-II Antibody Screen: Monday through Friday; varies
HTLV-I/-II Antibody Confirmation: Wednesday; 3:00 p.m.