Changes in bio-marker expression in various cardiovascular disorders can be demonstrated by mRNA expression concentration and cardiac proteins by ELISA, histology, immunohistology or immunofluorescence. The latter methods are less sensitive but are cheaper. We now report the development of enhanced immunofluorescence detection of a cardiovascular biomarker, cardiac myosin (CM), by immunofluorescence microscopy and flow cytometry using pretargeting of CM with bispecific antibody (bsAb) and targeting with multi-Rhodamine conjugated negatively charged polymers that show signal amplification, no fluorescence quenching and low background activity.
Methods: bsAb was made by covalent conjugation of monoclonal anti-myosin and anti-DTPA antibodies via thioether bonds. Rhodamine conjugated polymers were made by simultaneous covalent conjugation of 20X molar concentrations of Rhodamine-isothiocyanate (RITC) and anhydride of DTPA to 13.8 kDa polylysine polymers followed by succinylation (R-DSPL). Embryonic cardiocytes or frozen-murine cardiac tissue (10 μ) sections after pretargeting with bsAb (1, 0.1 and 0.01μg/ml) were targeted with the R-DSPL. Controls consisted of anti-myosin RITC, Anti-DTPA-RICT, rabbit anti-murine IgG-RITC or the antibodies labeled with TRITC (1, 0.1, and 0.01).
Results: The 20X R-DSPL contained 18 moles RITC and 30 moles DTPA per mole of polylysine. The distance between adjacent RITC in the 20X polymers was calculated to be approximately 33.6 Angstroms (A). CM in frozen sections still showed fluorescence signal pretargeted with 0.01μg/ml bsAb concentration and targeted with 20X R-DSPL whereas anti-myosin-RITC showed about the same fluorescence at 10 μg/ml concentration. Flow cytometry also confirmed that the pretargeting signal with 1 μg/ml of bsAb was 5 times greater than that obtained with 5 μg/ml of Rhodamine conjugated anti-myosin.
Conclusions: The use of polymer as carriers of multiple fluorophores for targeting after pretargeting with bsAb resulted in fluorescence signal amplification by at least 1000 times. This method of signal enhancement may allow higher sensitivity detection of changes in other cardiac bio-marker associated with development of various cardiovascular disorders.