Further simplification of ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-I IgG using microplates and fluororeader

J Clin Lab Anal. 1995;9(3):204-11. doi: 10.1002/jcla.1860090310.

Abstract

In a previously reported ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for anti-HTLV-I IgG, polystyrene beads were handled with tweezers, and bound beta-D-galactosidase activity was measured with a fluorometer. The use of tweezers was causative of false-positivity by carryover, and testing many samples was difficult. Recently, these drawbacks have been minimized using microplates and a fluororeader. However, tweezers were still required in the initial and final steps. In the present study, the immune complex, comprising 2,4-dinitrophenyl-antigen, anti-HTLV-I IgG, and antigen-beta-D-galactosidase conjugate, was formed in and trapped onto microplate wells coated with (anti-2,4-dinitrophenyl group) IgG. Subsequently, the microplate wells were incubated with epsilon N-2,4-dinitrophenyl-L-lysine and polystyrene beads, modified by attaching to plates through cylindrical bars and coated with (antihuman IgG gamma-chain) IgG, to transfer the immune complex from the microplate wells to the modified polystyrene beads. Alternatively, modified polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG and microplate wells coated with (antihuman IgG gamma-chain) IgG were substituted for microplate wells coated with (anti-2,4-dinitrophenyl group) IgG and modified polystyrene beads coated with (antihuman IgG gamma-chain) IgG, respectively. The fluorescence intensity for bound beta-D-galactosidase activity was quickly measured with a fluororeader. Thus the modified polystyrene beads were transferred from wells to wells more quickly and easily without tweezers, eliminating false-positivity due to carryover, and it became easy to test many samples with high sensitivity and reliability, although the assay of bound beta-D-galactosidase activity became slightly more time-consuming.

MeSH terms

  • Animals
  • Antibodies, Anti-Idiotypic / blood*
  • Antigen-Antibody Complex / analysis
  • Cattle
  • Dinitrobenzenes
  • Fluorometry / methods
  • HTLV-I Antibodies / blood*
  • Immunoenzyme Techniques / instrumentation*
  • Immunoglobulin G / blood*
  • Indicators and Reagents
  • Microspheres
  • Polystyrenes
  • Sensitivity and Specificity

Substances

  • 2,4-dinitrophenyl-IgG
  • Antibodies, Anti-Idiotypic
  • Antigen-Antibody Complex
  • Dinitrobenzenes
  • HTLV-I Antibodies
  • Immunoglobulin G
  • Indicators and Reagents
  • Polystyrenes