Photometric evaluation of indirect immunofluorescence chessboard titrations for the characterization FITC-labelled antibodies

摘要

The immunofluorescent staining properties of three FITC-labelled anti-human IgG rabbit globulin preparations with very similar antibody concentration (595, 570 and 565 μg/ml), but different molar fluorescein/protein ratios (4.2, 1.4 and 0.7) were compared by means of indirect immunofluorescence chessboard titrations. A selected serum from a patient with systemic lupus erythematosus containing antinuclear factors was tested on formalinized chicken red blood cells using incident light illumination. Both visual and photometric readings were taken. The characteristic pattern of chessboard titrations with a constant titre (plateau titre) over a wide range of conjugate dilutions followed by an abrupt fall (plateau end-point) was obtained and showed good correlation by both methods of evaluation. The height of the plateau titre increased with increasing fluorescein/protein ratio, but no linear relationship could be established with the three conjugates under investigation. The plateau end-point was encountered at conjugate dilutions containing similar antibody concentrations namely 9.3, 8.9 and 8.8 μg/ml. From the good correlation between photometric and visual readings it is concluded that chessboard titrations provide an objectively-acceptable method for the evaluation of fluorescent conjugates and adoption of the procedure by manufacturers of conjugates is suggested.