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BioPlex 2200 HSV-1 and HSV-2 kit uses multiplex flow immunoassay technology. Two different populations of dyed beads are each coated with glycoprotein G (gG)-based antigens associated with herpes simplex virus, types 1 or 2. Patient sample is combined with sample diluent and bead set reagent in a reaction vessel. The mixture is incubated at 37 degrees C. After a wash cycle, antihuman IgG antibody, conjugated to phycoerythrin (PE) is added to the mixture and incubated at 37 degrees C. Excess conjugate is removed in another wash cycle and the beads are resuspended in wash buffer. The bead mixture then passes through a detector where the identity of the dyed beads is determined by the fluorescence of the dyes, and the amount of antibody captured by the antigen is determined by the fluorescence of the attached PE. Raw data is calculated in relative fluorescence intensity. Three additional dyed beads, an internal standard bead, a serum verification bead, and a reagent blank bead are present in each reaction mixture to verify detector response, the addition of serum to the reaction vessel and the absence of significant nonspecific binding in serum.(Package insert: BioPlex 2200 System HSV-1 and HSV-2 IgG, Bio-Rad Laboratories Clinical Diagnostics Group, Hercules, CA)
Diluted samples are incubated with HSV 1 and 2 antigens bound to the solid surface of a microtiter well. If IgM antibodies against HSV are present in the sample, they will bind to the antigen, forming antigen-antibody complexes. Residual sample is eliminated by aspirating and washing. Conjugate (horseradish peroxide-labeled antihuman IgM) is added and binds to these complexes. Unbound conjugate is removed by aspiration and washing. The substrate is then added and incubated. In the presence of bound enzyme, the substrate is converted to an end product. The absorbance of this end product is read spectrophotometrically at 450 nm (reference 600-630 nm) and is directly proportional to the concentration of IgM antibodies to HSV 1 and 2 antigens present in the sample.(Package insert: HSV 1 and 2 IgM Enzyme Immunoassay Test Kit, DiaMedix Corporation, Miami, FL)
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