In this study, we have developed bridge heterologous ELISA for the detection of 17α- Methyltestosterone by incorporating aromatic spacers between 17α-Methyltestosterone-3-Carboxymethyloxime and Horseradish peroxidase label through N-hydroxysuccinimide mediated carbodiimide reaction method. The immunogen 17α-Methyltestosterone-3-Carboxymethyloxime-Bovine serum albumin used to generate the antibody was also prepared by the N-hydroxysuccinimide mediated carbodiimide reaction without using any spacer. We have studied the impact of bridge/aromatic spacers on functional parameters i.e. sensitivity, affinity and ED50 of the bridge heterologous assay and compared it with homologous assay. The five combinations of bridge heterologous assay using 17α-Methyl testosterone-3-CMO-BSA antiserum and 17α-MT-3-CMO-4,4\'-Diaminodiphenyl sulphide-HRP, 17α MT-3-CMO-4,4\'-Oxydianiline-HRP, 17α-MT-3-CMO-Benzidine-HRP, 17α- MT-3-CMO-p-Phenylenediamine-HRP and 17α-MT-3-CMO-Dapson-HRP enzyme conjugates were evaluated. Out of these five combinations, the combination 17α-MT-3-CMO-BSA with 17α-MT-3-CMO-Benzidine-HRP showed the best results. Sensitivity, affinity and ED50 were improved and found to be 0.02 ng/mL, 0.086 × 10-8 L/mol and 2.95 ng/mL than homologous assay where Sensitivity, affinity and ED50 were 0.11 ng/mL, 0.02 × 10-8 L/mol and 5.78 ng/mL respectively. The cross-reactivity for this bridge heterologous assay combination was seen with only 4 steroids (6-hydrotestosterone- 6%, Testosterone-5.14%, Danazol-0.9% and Nandrolone-0.85%) instead of eight steroids (6-hydrotestosterone-43.75%, Testosterone-38.3%, Danazol-25.14%, Androstenediol-19.16%, Nandrolone-19%, Metandienone-5%, Androstenedione-3.52%, and 17α dimethyltestosterone-2%) as in homologous assay out of 59 structurally related steroids. Thus, the results of this study conclude that the incorporation of aromatic spacer (bridge) in enzyme conjugate has a crucial role in improving sensitivity, specificity, ED50 and affinity of the developed assay. The assay was then studied for parameters such as recovery (97.4%-108.6%), precision (Inter and Intra-assay coefficient of variation <10%), correlation coefficient (R2 = 0.96) by comparing with the commercial kit and validated by measuring levels of 17α- methyltestosterone in rat serum after administering them.

Nucleic acid lateral flow assays (NALFA) are often performed with gold nanoparticles. These are typically associated with ligand-labeled PCR amplicons via affinity interactions of adsorbed/conjugated proteins. Otherwise, they are conjugated to specific ssDNA sequences that hybridize to the target sequence. To avoid the need to generate ssDNA and to reduce the costs associated with primer labeling and antibody use, NALFA assays were developed that allow the direct detection of PCR amplicons using conjugates of a DNA binding protein with carbon nanoparticles (CNPs). The target gene encoding 16S ribosomal RNA of Escherichia coli was amplified by PCR using a single fluorophore-labeled forward primer and a reverse primer extended with the binding sequence of the bacteriophage lambda Cro repressor protein. Three different detection approaches were evaluated: (a) scCro/CNPs conjugate (black color), (b) HRP-scCro enzyme conjugate (red color), and (c) HRP-scCro/CNPs conjugate for dual color development. The limits of detection were between 6.9 and 10.4 ng of PCR product for all three approaches. These correspond to 3.0 to 4.5 × 103 CFU·mL-1. The single-step scCro/CNP approach proved to be the fastest one to perform and gave no false-positive signals. It also showed a broad dynamic range even though the signal intensities were lower compared to the enzyme-amplified tests. However, the latter ones produced some background signal. In our perception, the application of scCro in lateral flow assays to bind dsDNA appears to be an excellent alternative to the use of small tags that have to be chemically linked to synthetic primers. Finally, the approach is generic because any primer sequence can be extended with the specific scCro binding sequence. Graphical abstract Schematic presentation of the lateral flow-based fluorometric detection of DNA amplicons using conjugates of scCro DNA binding protein with (A) carbon nanoparticles, (B) HRP and (C) HRP and carbon nanoparticles.