Fluorogenic Product Research Articles

Which of the commonly used marker enzymes gives the best results in colorimetric and fluorimetric enzyme immunoassays: Horseradish peroxidase, alkaline phosphatase or β-galactosidase?

Comparing the marker enzymes horseradish peroxidase (HRP), alkaline phosphatase (AP) and β-galactosidase (βGal) in IgG-coupled form with respect to their temperature-dependent kinetics over a period of 22 h the temperature of 37°C warrants highest substrate turnover for all enzymes at all reaction times using fluorogens. Also applying chromogens the optimum temperature for βGal is 37°C and depends for HRP and AP on the reaction time. The substrate turnover of HRP using ABTS as chromogen is much higher compared to the other enzymes — both related to mol enzyme (molar activity) and to gram enzyme (specific activity). The turnover decreases for all enzymes in different degrees after coupling to IgG. The turnover of fluorogenic substrates is lower for all enzymes than the turnover of chromogenic substrates but due to the more sensitive detection of fluorogenic products the detection limits for all conjugates were lowered too — especially for βGal-IgG by a factor of 333 compared to the colorimetric procedure. In a 2-site binding enzyme immunoassay for alpha-1-fetoprotein (AFP) the detection limit for AFP was reduced by a factor of 2 only by the fluorimetry compared to the colorimetry with all 3 marker enzymes. The HRP-IgG conjugates warranted lowest detection limits for AFP (0.5–1 μg/1), highest analytical sensitivity (slope of standard curves) at shortest periods of substrate reaction compared to the other enzymes.

Detection of eastern equine encephalomyelitis virus and Highlands J virus antigens within mosquito pools by enzyme immunoassay (EIA). I. A laboratory study.

Enzyme immunoassays (EIAs) producing either chromogenic or fluorogenic end products were developed and evaluated for detection of eastern equine encephalomyelitis (EEE) and Highlands J (HJ) viruses in pools of Aedes triseriatus mosquitoes. Overnight incubation of the mosquito samples in the EIA significantly enhanced the sensitivity of the test. Both the EEE and HJ EIAs were sensitive, readily detecting one infected mosquito in a pool with 99 noninfected, and specific, distinguishing homologous from the alternate alphavirus and other arboviruses. By 3 days post-infection after intrathoracic inoculation, EEE virus was isolated from 100% (30/30) of the mosquitoes examined. Concurrently, EEE virus antigen was detectable by EIA in 100% (30/30) of examined mosquitoes and by indirect fluorescent antibody (IFA) technique in 77% (23/30) of the examined mosquito head squash preparations.

Fluorogenic assays for immediate confirmation of Escherichia coli.

Rapid assays for Escherichia coli were developed by using the compound 4-methylumbelliferone glucuronide (MUG), which is hydrolyzed by glucuronidase to yield a fluorogenic product. The production of glucuronidase was limited to strains of E. coli and some Salmonella and Shigella strains in the family Enterobacteriaceae. For immediate confirmation of the presence of E. coli in most-probable-number tubes, MUG was incorporated into lauryl tryptose broth at a final concentration of 100 micrograms/ml. Results of both the presumptive test (gas production) and the confirmed test (fluorescence) for E. coli were obtained from a variety of food, water, and milk samples after incubation for only 24 h at 35 degrees C. Approximately 90% of the tubes showing both gas production and fluorescence contained fecal coliforms (they were positive in EC broth incubated at 45 degrees C). Few false-positive reactions were observed. The lauryl tryptose broth-MUG-most-probable-number assay was superior to violet red bile agar for the detection of heat- and chlorine-injured E. coli cells. Anaerogenic strains produced positive reactions, and small numbers of E. coli could be detected in the presence of large numbers of competing bacteria. The fluorogenic assay was sensitive and rapid; the presence of one viable cell was detected within 20 h. E. coli colonies could be distinguished from other coliforms on membrane filters and plates of violet red bile agar if MUG was incorporated into the culture media. A rapid confirmatory test for E. coli that is amenable to automation was developed by using microtitration plates filled with a nonselective medium containing MUG. Pure or mixed cultures containing E. coli produced fluorescence within 4 h (most strains) to 24 h (a few weakly positive strains).

Detection of La Crosse arbovirus antigen in mosquito pools: application of chromogenic and fluorogenic enzyme immunoassay systems.

An enzyme immunoassay producing either a chromogenic or fluorogenic end product was developed and evaluated for detecting La Crosse viral antigen within mosquito pools. The enzyme immunoassay was found to be sensitive, detecting one infected mosquito within a pool of 100 mosquitoes, and specific, distinguishing between closely related California group viruses. Assays were completed within 5 h after the addition of test samples. La Crosse viral antigen could be readily detected in mosquito pools after seven freeze-thaw cycles.

Semiautomated fluorometric and colorimetric methods for the determination of reserpine in single tablets

Semiautomated fluorometric and colorimetric methods for the determination of reserpine in single tablets are described for 0.25- and 0.5-mg. potency tablets. A simple practical manual method for the extraction of reserpine from a large number of tablets is outlined. The yellow-green fluorogenic product formed by the reaction of reserpine and nitrous acid is analyzed in the automatic analyzer system. Using excitation maximum of 390 mμ the fluoresence is measured at 510 mμ. In the colorimetric determination the absorbance is measured spectrophotometrically at 390 mμ. Using the colorimetric method, the effect of temperature on the reaction of reserpine and nitrous acid, and the effect of nitrogen gas on the recordings are investigated. In the fluorometric method there is no interference by the excipients and no effect of nitrogen gas on the recordings is noted. Further, this is more sensitive than the colorimetric method.