Protein microarray: sensitive and effective immunodetection for drug residues

Abstract

Background: Veterinary drugs such as clenbuterol (CL) and sulfamethazine (SM2) are low molecular weight (<1000 Da) compounds, or haptens, that are difficult to develop immunoassays due to their low immunogenicity. In this study, we conjugated the drugs to ovalbumin to increase their immunogenicity for antiserum production in rabbits and developed a protein microarray immunoassay for detection of clenbuterol and sulfamethazine. The sensitivity of this approach was then compared to traditional ELISA technique.

Results: The artificial antigens were spotted on microarray slides. Standard concentrations of the compounds were added to compete with the spotted antigens for binding to the antisera to determine the IC50. Our microarray assay showed the IC50 were 39.6 ng/ml for CL and 48.8 ng/ml for SM2, while the traditional competitive indirect-ELISA (ci-ELISA) showed the IC50 were 190.7 ng/ml for CL and 156.7 ng/ml for SM2. We further validated the two methods with CL fortified chicken muscle tissues, and the protein microarray assay showed 90% recovery while the ci-ELISA had 76% recovery rate. When tested with CL-fed chicken muscle tissues, the protein microarray assay had higher sensitivity (0.9 ng/g) than the ci-ELISA (0.1 ng/g) for detection of CL residues.

Conclusions: The protein microarrays showed 4.5 and 3.5 times lower IC50 than the ci-ELISA detection for CL and SM2, respectively, suggesting that immunodetection of small molecules with protein microarray is a better approach than the traditional ELISA technique.

Figure 1

Schematics of the ci-ELISA and protein microarray procedures. 5 μg of the artificial antigens (CL or SM₂) are coated onto the 96-well plates in (A) ci-ELISA, or 20 ng of the artificial antigens were spotted onto the protein microarray slides in (B) protein microarray. Antibodies against the particular artificial antigens are added together with a range of concentration of the corresponding hapten. As the haptens would compete with the immobilized artificial antigens for binding to the antisera, an increasing concentration of the hapten would result in a decreasing signal. For ci-ELISA, the bound antibodies are visualized by an anti-rabbit-HRP conjugated antibody with the addition of a chromogen (A). For the protein array, the secondary antibodies are conjugated to a fluorescent dye (Cy3), and the signal can be directly measured by a fluorescence microarray scanner (B).

Figure 2

IC₅₀ of protein microarray immuntodetection and ci-ELISA. Upper panels: Dose-response curves for (A) CL and (B) SM₂ from ci-ELISA and protein microarray. The log of the hapten concentration (x-axis) was plotted against the percentage of inhibition (y-axis), which is (OD_sample/OD_control) × 100%. The control group OD was considered to be the point of 100% activity. The IC₅₀ was determined by non-linear regression analysis. Lower panels: fluorescent signals from the protein microarray slides for (C) CL and (D) SM₂. The concentration of the standard solution for each slide is: 1: 0 ng/ml; 2: 1 ng/ml; 3: 5 ng/ml; 4: 10 ng/ml; 5: 20 ng/ml; 6: 100 ng/ml; 7: 1000 ng/ml.