Focus: One-step Luminescent CEDIA^® Analysis

Sensitive Homogeneous CEDIA^® Analysis Using the
Beta-Glo^® Assay System

Here we present a study that compares spectrophotometric, chemiluminescent and bioluminescent technologies for performing one-step CEDIA^® analysis.

From the article: Yang, X., Janatova, J. and Andrade, J.D. (2005) Homogeneous enzyme immunoassay modified for application to luminescence-based biosensors. Anal. Biochem. 336, 102–7. Department of Bioengineering, University of Utah, 50 South Central Campus Drive, Room 2480, Salt Lake City, UT 84112-9202, USA.
 

CEDIA^® Analysis Principle

CEDIA^® (cloned enzyme donor immunoassay) analysis is a technology that can be used for on-site testing for small molecule analytes in samples. The assay is based on a bacterial enzyme, β-galactosidase, which is genetically engineered into two inactive fragments, enzyme donor (ED) and enzyme acceptor (EA). When mixed together, these two fragments can spontaneously associate to form tetramers that express β-galactosidase activity (1,2).

In CEDIA^® analysis, the ED is conjugated to the analyte without affecting the formation of the functional enzyme. An antibody directed against the analyte prevents the ED-analyte conjugate from interacting with the EA to form a functional enzyme. Analyte present in the sample competes with the ED-analyte conjugate for antibody binding sites, freeing the ED-analyte conjugate to interact with EA to form the functional β-galactosidase. Consequently, β-galactosidase enzyme activity is proportional to the amount of analyte in the sample tested (Figure 1).

Figure 1. Overview of CEDIA^® Analysis Principle. Analyte in the sample competes with analyte-conjugated enzyme donor (ED) for binding sites on an anti-analyte antibody. This allows the association of EA with ED and formation of the functional enzyme.

Yang et al. (2005) sought to design a single-step, sensitive CEDIA^® method that could be used for a “dipstick” type of on-site chemical analysis. They tested three technologies for detecting β-galactosidase activity: a spectrophotometric method, a chemiluminescent method and a bioluminescent method. For the bioluminescent method, the authors used the Beta-Glo^® Assay System (Cat.# E4720).

Beta-Glo^® Assay Principle

The Beta-Glo^® Assay can be used to measure β-galactosidase activity in a variety of biological applications. In this paper, the Beta-Glo^® Assay allows measurement of β-galactosidase activity through a coupled luciferase reaction in a homogeneous assay format (Figure 2). β-galactosidase catalyzes a reaction in which the substrate (D-luciferin-o-β-galactopyranoside) is cleaved to release luciferin. This luciferin, in turn, serves as a substrate for luciferase present in the assay reagent. The reagent is formulated so that the Beta-Glo^® Assay is a single-step procedure that involves adding an equal volume of reagent to a sample that contains the enzyme either in solution or present in cells grown in medium and serum.

Figure 2. Summary of the coupled reactions in the Beta-Glo^® Assay System. β-galactosidase activity from lysed cells or in solution catalyzes the conversion of d-luciferin-o-β-galactopyranoside to d-luciferin, which is in turn a substrate for luciferase.

Beta-Glo^® Assay Provides a Sensitive Method for On-Site CEDIA^® Analysis  

Yang et al. determined that CEDIA^® analysis using bioluminescent detection of enzyme activity (Beta-Glo^® Assay) yields about 300 times more light output than the chemiluminescent method using the same concentration of analyte in sample. Figure 3 presents data generated from a one-step valproic acid (VPA) CEDIA^® assay using the Beta-Glo^® Assay to detect β-galactosidase activity. The data show that using the Beta-Glo^® Assay, differences in VPA concentration can be detected in only four minutes. The spectrophotometric and chemiluminescent detection methods require six and 15 minutes, respectively to detect VPA concentration differences (data not shown here).

 

Figure 3. CEDIA^® analysis with bioluminescent Beta-Glo^® Assay System. CEDIA^® reagents R1 (8μl) + R2 (6μl) of different concentrations + 16μl Beta-Glo^® Assay Solution. Detection senstivity at 70.0% was the same as that for the chemiluminescent method tested. Curves from top to bottom represent VPA initial concentration as follows: 144, 72, 36, 18, 9 and 0μg/ml, respectively. Figure reprinted with permission of Analytical Biochemistry (Elesevier) and J. Andrade (University of Utah).

Figure 4 shows data comparing a small volume and a large volume CEDIA^®/Beta-Glo^® analysis. The CEDIA^®/Beta-Glo^® analysis can be conducted in as little as 2µl. The smallest practical volume for the CEDIA analysis using chemiluminescent detection was 16.2µl.

Figure 4. Calibration curves of one-step CEDIA^® analysis with the Beta-Glo^® Assay System for two different volumes. Large volumes (cross lines): 16μl CEDIA reagents (8μl R1, 6μl R2 and 2μl VPA) + 16μl Beta-Glo^® Reagent. Small volumes (open circles); 1μl CEDIA Reagents (0.5μl R1, 0.375μl R2 and 0.125μl VPA) + 1μl Beta-Glo^® Reagent. This figure represents slopes of kineteics curves in the last minute as a function of VPA initial concentration. The values obtained with large volumes are within the range of 0–40,000 RLU per minute (left side scale), while small volumes yielded signals corresponding to the right side scale (0–1600 RLU per minute). Figure reprinted with permission of Analytical Biochemistry (Elsevier) and J. Andrade (University of Utah).

Conclusions

Because of the increased sensitivity and the ability to perform the assay in a small volume (see Table 1), the authors suggest that one-step CEDIA^® Analysis using the Beta-Glo^® Assay to detect β-galactosidase activity is suitable for application in a ChemChip format for on-site testing.

References

1. Henderson, D.R. et al. (1986) CEDIA, a new homogeneous immunoassay system. Clin. Chem. 32, 1637–41.
2. Khanna, P.L. et al. (1989) A new homogeneous enzyme immunoassay using recombinant enzyme fragments. Clin. Chim. Acta. 15, 231–9.