12th International Symposium on Bioluminescence & Chemiluminescence

Symposium abstracts:

Applications of Lumigen PS-1 chemiluminescent substrate to microplate format immunoassay using horseradish peroxidase as label: a comparison with the colorimetric end-point using TSH as the analyte

Pazzagli, Mario¹, Pinzani, Pamela¹, Manzoni, Angelo², Salti, Sara², Zotti, Maria Grazia²

1. Dept of Clinical Physiopathology, Clinical Biochemistry Unit, University of Florence, Italy. Email: m.pazzagli@dfc.unifi.it
2 . Radim, R&D Department , Via del Mare,125, 00040 Pomezia Rome, Italy. Email: amanzoni@seacfi.com

Horseradish peroxidase (HRP) is a commonly used reporter enzyme in enzyme-linked immunoassays. Numerous chromogenic detection systems such as tetramethylbenzidine (TMB), o-phenylenediamine (OPD) and 2-2’-azino-bis(3-ethylbenzothiazolone) (ABTS) have been reported for visualization of peroxidase activity. While colorimetric assays may be adequate for many qualitative assays and for quantitation of analytes present in relatively high concentration, chemiluminescent detection offers potential advantages in sensitivity, assay speed and the ability to choose from a variety of detection format. Better sensitivity allows reduced usage of expensive antibodies and improved specificity in immunological binding. We now report the application of Lumigen PS-1, a reagent for the ultrasensitive chemiluminescent detection of HRP in immunoassay. In particular , we analyze the results obtained for the ultrasensitive TSH measurement by both a colorimetric and the PS-1 end-point.

An ELISA for TSH was performed using the components of an enzyme immunoassay kit for TSH from RADIM (KT3IW) . White 96 well plates were appositely produced by RADIM. Samples prepared from the supplied TSH standards were treated according to the manufacturer’s instructions up to the detection stage. Chemiluminescence intensities were measured in a Berthold luminometer . We obtained a linear calibration curve ranging from 0 to 50 µIU/ml, with a lowest detected quantity of 0.0015 IU/mL (n = 4) and a reduction in incubation time from 90+30 +15 (colour reaction included) to 45 + 15 minutes (negligible luminescent reaction time). The quoted analytical sensitivity of the manufacturer assay which uses a colorimetric endpoint is 0.015 µIU/ml. An excellent correlation was obtained when comparing samples TSH results obtained with the colorimetric and the luminescence assay format.

This is a preprint of an article accepted for publication in Luminescence: Copyright 2001 John
Wiley & Sons, Ltd (Wiley website)