Development of a creatinine ELISA and an amperometric antibody-based creatine sensor with a detection limit in the nanomolar range.

Creatinine-specific antibodies have been generated and used for highly sensitive and specific immunochemical creatinine determinations. Creatinine was derivatized at N3 and coupled to KLH carrier protein. On the basis of this immunogen, monoclonal antibodies were developed by hybridoma technology. Antibodies from various clones have been characterized with BIAcore 2000 with respect to the dissociation constant and specificity. Antibodies of clone B90-AH5 exhibited the lowest dissociation constant (0.74 microM) and the highest specificity for creatinine and were chosen for the development of a competitive ELISA and an amperometric creatinine sensor. The creatinine sensor was constructed by fixing a creatinine-modified membrane on the top of a platinum working electrode which was then incorporated into a stirred electrochemical measuring cell. For creatinine determination the creatinine-containing sample was incubated with B90-AH5 and anti-IgG(mouse)-glucose oxidase conjugate and applied to the measuring cell. After a washing step glucose was added and the produced hydrogen peroxide was registered at Eappl = +600 mV vs Ag/AgCl. The measuring range was 0.01-10 microg/mL. The highest sensitivity for creatinine was achieved at 330 ng/mL (3 microM) and the lower detection limit at 4.5 ng/mL (40 nM). This is far below the relevant clinical range, which is 5-17 microg/mL (44-150 microM) and allows a reliable determination of very low creatinine concentrations in serum, where standard methods cannot be applied. After each measurement the sensor was regenerated with 10 mM HCl without any loss in binding activity.

Enzyme kinetic assays with surface plasmon resonance (BIAcore) based on competition between enzyme and creatinine antibody.

A procedure is described which allows the characterization of enzyme by a hybrid approach using an enzyme and an antibody. The presented method is related to the affinity determination of antibodies by the 'affinity in solution' procedure for BlAcore. The antibody is used as an indicator for the concentration of substrate, which is also the antigen. A mixture of enzyme, substrate and antibody is incubated, and an aliquot of this solution is injected periodically into a flowcell containing immobilized substrate, which is bound by the antibody, but not cleaved by the enzyme. The chosen initial concentration of substrate inhibits the binding of antibody to the immobilized substrate by 90%. During the enzymatic reaction, increased amounts of antibody bind to the surface, as the substrate concentration is decreased. With this method, the cleavage of creatinine with creatinine iminohydrolase (6 mU/ml) was monitored for up to 11 h. A recently developed monoclonal antibody against creatinine was used as the indicating protein. For the calculation of enzyme activity, the signals were compared with a calibration curve for inhibition of antibody binding to the chip by creatinine in solution.

Production and characterization of monoclonal antibodies against urea derivatives.

A panel of monoclonal antibodies was generated against the urea-based hapten N-(2-N-chloroacetylaminobenzyl)-N'-4-chlorophenylurea as a tool for building up sensitive immune assays to detect urea derivatives and to screen them for catalytic antibodies (Abs). Eleven hybridomas were obtained that produced Abs reactive to the hapten. All Abs were of IgG class. Cross reactivities of the Abs to different haptens were examined, especially to a possible transition-state analog. Only four of the hybridomas (R2-DA10/F7, R2-GE7/H2, R2-HC2/A5, R2-HD6/F7) produced Abs crossreactive with the transition-state analog. From the 11 hybridomas, hybridoma B76-BF5 was chosen for further characterization. Compared to the other Abs, B76-BF5 showed the strongest binding and had a rather restricted specificity. These Abs could be used to build up a sensitive enzyme immunoassay for the detection of the hapten. All Abs were screened for crossreactivity with the pesticides monuron and diuron. No reactivity could be detected. In addition, the nucleotide sequences of the variable light and heavy chain genes of the similarly reactive Abs B76-BF5, B76-BB3, R2-DA10/F7, and R2-GA6/G3 were determined to clarify whether structure and binding specificity of these Abs showed any correlation.

Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages.

Three monoclonal antibodies (MAbs) were produced which react with epitopes of the main structural coat protein (pVIII) of filamentous fd phages as demonstrated by solid-phase fluorometric enzyme immunoassays and by immunoelectron microscopy. The antibodies are of the IgG1, IgG2a and IgG2b immunoglobulin subclasses. Since they also react with recombinant phages expressing antigen fragments in their pIII region they may be suitable reagents for the demonstration and isolation of filamentous phages used in recombinant protein technology.

Monoclonal antibodies to human chorionic gonadotropin (HCG) and their use in two-site binding enzyme immunoassays.

A panel of mouse monoclonal antibodies (MABs) was produced against human chorionic gonadotropin (HCG) and its isolated beta-subunit (beta-HCG). According to their binding specificities the antibodies could be divided into HCG-specific and cross-reactive MABs. The HCG-specific antibodies reacted with antigenic sites on holo-HCG or holo-HCG and beta-HCG, or exclusively with the non-associated beta-HCG chain. The cross-reactive antibodies reacted with either HCG and luteinizing hormone (LH) or with HCG, LH, follicle-stimulating hormone (FSH) and thyroid-stimulating hormone (TSH). According to the binding specificities of the MABs and their reciprocal inhibition detected in two-site binding enzyme immunoassays (EIA), altogether 13 epitopes (including the 3 hidden epitopes detectable only on free non-associated beta-HCG) were distinguished by the antibodies described here. Antibody combinations resulting in most effective and specific HCG- or beta-HCG-determination were used as clinical assays and proved their reliability and correctness for monitoring patients with HCG- and/or beta-HCG-producing tumors before and after therapy.

The production and radioimmunoassay application of monoclonal antibodies to fluorescein isothiocyanate (FITC).

Monoclonal antibodies (MoAbs) were produced against the fluorescence marker fluorescein isothiocyanate (FITC). FITC was used as a hapten to label different proteins and the anti-FITC MoAbs were used to identify these labelled proteins in a solid-phase radioimmunoassay and in cellular radioimmuno-binding assays for the demonstration of antigens and antibodies.