Analysis of DDT Isomers with Enzyme-Linked Immunosorbent Assay and Optical Immunosensor Based on Rat Monoclonal Antibodies as Biological Recognition Elements

Abstract

New rat monoclonal antibodies (mAbs) for DDT [1,1,1-trichloro-2,2-bis (4-chlorophenyl) ethane], namely DDT 7C12, DDT 1C1, and DDT 1B2, were developed, characterized, and applied in ELISA both in coating antigen and in enzyme-tracer format. The latter used horseradish peroxidase (HRP) or glucose oxidase as enzymes. The lowest concentration of p,p-DDT was determined with mAb DDT 7C12 and DDT-hapten HRP, with a test midpoint (IC50) of 0.5 ± 0.2 µg/L (n = 10) in 40 mM PBS (phosphate-buffered saline). The mouse anti-rat immunoglobulin lambda-light chain mAb LA1B12 was used as capture mAb. The best IC50 for o,p´-DDT in 40 mM PBS was 1.0 ± 0.3 µg/L (n = 12) and was obtained with mAb DDT 1C1 and DDT-hapten HRP, whereas mAb DDT 1B2 was very selective for p,p-DDT with an IC50 of 4.2 ± 1.6 µg/L (in 40 mM PBS, n = 9). An optical immunosensor was optimized and applied for the analysis of DDT (or DDT equivalents). This immunosensor consists of a bench-top optical readout device and disposable sensor chips, which include the fluidic system. Evanescent field excitation and emission of the fluorophore Oyster<sup/>-645 was used. An IC50 for p,p´-DDT [in 5 (v/v) isopropanol in 40 mM PBS] of 4 µg/L was obtained using DDT 7C12-Oyster-645. ELISA and immunosensor were used for the analysis of p,p-DDT in unspiked and spiked surface water samples. Within the working ranges of these immunotechniques, recoveries ranged from 80 to 120.

FK506-Binding Protein 11 Is a Novel Plasma Cell-Specific Antibody Folding Catalyst with Increased Expression in Idiopathic Pulmonary Fibrosis

“…For determination of yield and functionality of antibodies secreted from scr siRNA, FKBP11 siRNA or PPIB siRNA-transfected hybridoma H3 cells, ELISA plates were coated with a mixture of anti-κ LC (TIB172, ATCC) and anti-λ LC (mAb LA1B12 [60], both 5 µg/mL in 0.2 M carbonate buffer pH 9.5), the cognate antigen-GST fusion protein, or the untagged cognate antigen (each 10 µg/mL in 0.2 M carbonate buffer pH 9.5, in-house generated) overnight at 4 • C. After washing one time with PBS, the wells were blocked with PBS/2% fetal calf serum for 15 min. After blocking, a serial 2-fold dilution of supernatants (starting dilution 1:10) from siRNA-transfected hybridoma cells were added for 30 min.…”

Multi-parameter determination of TNFα, PCT and CRP for point-of-care testing

“…The sensor system is very adaptable for clinical diagnostics, but also for the determination of environmentally relevant contaminants. [13][14][15]…”