Anti-Biotin monoclonal antibody, clone C36 (CHO/00/7336)

Herein, a high-affinity single-stranded DNA aptamer (59 nt) against Campylobacter jejuni, defined as CJA1, was obtained using the whole-bacterium-based systemic evolution of ligands by exponential enrichment procedure. CJA1 was analyzed with a stable secondary structure and low dissociation constant (Kd) value of 1.37 +/- 0.28 nM. The potential use of CJA1 was exemplified by the construction of a hetero-sandwich platform, in which C. jejuni was bound with a biotin-tagged CJA1 to perform a colorimetric reaction that is associated with visible color changes and detectable optical responses. Dependent upon this sensing platform, C. jejuni can be detected from 1.7 x 10(1) to 1.7 x 10(6) colony-forming units (CFU)/mL. The limit of detection (LOD) is obtained as 10 CFU/mL in PBS. The specificity study showed that the sensing platform is easy to distinguish C. jejuni from other common pathogens. Moreover, the C. jejuni-contaminated milk samples can also be accurately probed (LOD = 13 CFU/mL) without sacrificing its assay abilities, indicating the promising prospect of CJA1 in the fields of biosensing and diagnostics.

Chemiluminescence-enzyme immunoassays make it possible to measure trace components with high sensitivity and selectivity due to the high specificity of the antigen-antibody reaction and the high sensitivity of chemiluminescence assays. However, using an enzyme-labeled antibody suffers from many problems such as low reproducibility due to the instability of the enzyme and inhibition of antigen-antibody reaction due to its steric effect. Therefore, herein we report an innovative non-enzymatic chemiluminescence immunoassays labeling reagent through using quinone as a signal-generating tag coupled with biotin as a binder, to overcome enzymatic labeling problems. Biotinylated-1,4-naphthoquinone (biotin-NQ) was synthesized and characterized and it could produce long-lasting chemiluminescence upon mixing with dithiothreitol and luminol based on the redox cycle of quinone. Biotin-NQ showed exceptional stability towards different stress factors that may be encountered during performing the immunoassay such as high temperatures, highly acidic and alkaline conditions, and repeated freeze-thaw cycles. On the other hand, all these conditions lead to decreased labeling enzyme reactivity due to possible denaturation of its protein structure. Finally, the measurement of the biotin-labeled antibody was successfully performed using biotin-NQ and avidin. As a result, the antibody could be detected down to 25.7 nM which is 2.5 times sensitive than biotin-HRP chemiluminescence-enzyme immunoassays. Moreover, our method was applied successfully for determination of avidin using immobilized biotinylated antibody and biotin-NQ, which simulates immunoassays. Avidin could be detected down to 23.4 nM with excellent linearity (r = 0.996). Accordingly, our developed reagent, biotin-NQ, could be used as a universal highly stable, cost-effective, and steric free non-enzymatic label for immunoassays.