The ELISAs carried out in our laboratory over the past few years have utilized 2,2'-azino-bis(3-ethylbenzthiazoline-6- sulfonic acid) (ABTS) as the horseradish peroxidase (HRP)-reactive colorimetric agent. Although ABTS performs very well for most applications, it is not the most sensitive method for quantitating bound antibody. Recently, while attempting to develop an ELISA to quantitate the level of a protein in serum, I had hit a stumbling block with the lack of sensitivity of ABTS. Since we had just acquired a fluorescence plate reader, I decided to try a fluorescent reagent for quantitation. After a bit of research, I decided on Amplex® Red from Molecular Probes (Eugene, OR, USA). Several weeks later, the choice seems to have been a good one.
The Amplex® Red reagent (10-acetyl-3,7-dihydroxyphenoxazine) reacts with
H2O2 in the presence of HRP with a 1:1 stoichiometry to form resorufin, the fluorescent product. Because resorufin also has strong absorption, it can be detected either fluorometrically or spectrophotometrically. For fluorescence, I have used 544 and 590 nm for absorption and emission, respectively, with very good results. Molecular Probes lists the absorption and emission maxima at approximately 563 nm and 587 nm, respectively. Absorption is measured at 576 nm although the fluorometric detection is quite a bit more sensitive.
The positive features of this reagent include sensitivity, stability, and low background. I have been able to detect antibody binding at a concentration of 0.5 ng/ml of total rabbit IgG. Assuming the specific IgG to be approximately 10% of the total IgG, this represents a concentration more on the order of 50 pg/ml. The same assay quantitated with absorbance, 576 nm for Amplex® Red or 405 nm for ABTS, was approximately 4-fold less sensitive. To check the stability, I read the same plate after 24 h and got identical results. Background signal was on the order of 1-2% of the maximum signal.
Molecular Probes sells a kit that contains the Amplex® Red reagent, DMSO, 5x reaction buffer, stabilized H2O2,, and anti-IgG-HRP conjugate. I have found the kit to be unnecessary as all ingredients other than the fluorescent reagent are readily available in most labs. I solubilized the whole vial of bulk reagent in DMSO to a concentration of 10 mM, aliquoted it, and stored it at –20°C. For use, I simply dilute it to 50 µM in 50 mM sodium phosphate buffer containing 0.0015% H2O2 and add 100 µl to the plate. After a 30-min incubation at RT in the dark, the plate can be read.
I highly recommend Amplex® Red from Molecular Probes for ELISA quantitation. I have switched totally from ABTS to this reagent. The one caveat is that you must have access to a fluorescence plate reader for it to be worth the switch. Absorbance detection of the reagent seems no better than ABTS.
Michael Campa, Ph.D.
Asst. Research Professor of Radiology
Duke University Medical Center