Polyclonal murine and rabbit antibodies for the bile acid isolithocholic acid.

Bile acids are relevant markers for clinical research. This study reports the production of antibodies for isolithocholic acid, the isomer of the extensively studied lithocholic acid. The IgG titer and affinity maturation were monitored during the immunizations of three mice and two rabbits. In both animal models, polyclonal antibodies with a high selectivity and affinity were produced. The development of a direct competitive ELISA with a test midpoint of 0.69 ± 0.05 µ g/L and a measurement range from 0.09-15 µg/L is reported. Additionally, the crystal structure of isolithocholic acid is described for the first time.

Comparison of antibody and albumin catalyzed hydrolysis of steroidal p-nitrophenylcarbonates.

A monoclonal antibody (MAb) was produced against the p-nitrophenylphosphate derivative of 3 alpha, 5 beta-lithocholic acid, a transition-state analog for hydrolysis of a steroidal p-nitrophenylcarbonate. The indicated reaction was catalyzed by this Ab with kinetic constants kcat = 4.0 x 10(-2)/min and K(m) = 3.3 microM at pH 9.0 and 35 degrees C. The Ab also hydrolyzed the isomeric p-nitrophenylcarbonate of 3 beta, 5 beta-lithocholic acid with kcat = 8.4 x 10(-2)/min and K(m) = 1.0 microM. Bovine serum albumin (BSA) was found to catalyze the same reactions with similar turnover rates and Michaelis constants of 15 and 14 microM, respectively. Although the BSA-catalyzed reaction was only weakly inhibited by the phosphate ester TSA (IC50 ca. 40 microM), the Ab-catalyzed reaction was completely inhibited at less than 1 microM of the TSA. The relative rates and efficiencies of the MAb-catalyzed and BSA-catalyzed reactions are discussed in the context of the hydrophobic sites and intrinsic reactivity of the protein surfaces, and the induction of groups on the Ab to enhance the enzymatic function.

[Radioimmunological characterization of anti lithocholic acid antisera elicited by [C-6] carboxylic acid N-succinimidyl esters as haptenic derivatives].

In order to investigate the antigenic effects of the carboxyl group in the side chain and the bridge length on producing anti lithocholic acid antibody, the immunogens in which bile acid is coupled with bovine serum albumin through propionyloxy or butyryloxy amide linkage at the C-6 position were prepared. The antibodies elicited by these conjugates had low titers but showed a high affinity for lithocholic acid with association constants in the range of 0.42-1.04 X 10(8)M-1. The cross-reactivities of both antisera were essentially the same as that of methyl 6 alpha-hemisuccinyloxylithocholate. These results suggested that the side chain hydrolysis product of the methylated antigen worked as a real antigen in the body.

Non-invasive monitoring of immunization progress in mice via IgG from feces.

A non-invasive method to monitor the humoral immune response in mice after immunization is described. From fecal pellets of an individual mouse, a sufficient amount of active immunoglobulins or their fragments can be extracted to perform a regular examination of the status of the immune response by immunoassay. Hapten-specific antibodies from the feces of mice from three immunization trials showed very similar characteristics to those obtained from serum at a given date. Therefore, it can be suspected that some serum IgG enters the intestinal lumen and ends up in the feces, where they appear to be considerably stable. Hapten-specific IgAs were not found in the feces. Being able to analyze antibody titers in feces could be an interesting animal welfare refinement to standard practice that does not entail repeated blood sampling.

Immunoprecipitation and MALDI-MS identification of lithocholic acid-tagged proteins in liver of bile duct-ligated rats.

Formation of covalently bound protein adducts with lithocholic acid (LCA) might explain LCA's known carcinogenic properties and hepatotoxicity. We performed studies aimed at isolating and identifying hepatic proteins tagged with LCA, presumably via the epsilon-amino group of lysine residues. Antibodies recognizing the 3alpha-hydroxy-5beta-steroid moiety of LCA were generated by immunizing rabbits with immunogens in which the carboxyl group of LCA was coupled to BSA via a 6-aminohexanoic acid and/or succinic acid spacer. The resulting antibodies reacted with N-alpha-(t-butoxycarbonyl)-l-lysine-epsilon-LCA, the amidated and nonamidated forms of LCA, as well as synthetically prepared LCA adducts with ovalbumin and lysozyme. Proteins tagged with LCA in the liver of bile duct-ligated rats were isolated by immunoprecipitation using these antibodies. Proteins were isolated by two-dimensional electrophoresis, and their structure was identified using matrix-assisted laser desorption ionization time-of-flight mass spectrometry and computer-assisted programs. Proteins labeled with LCA were Rab-3, Rab-12, Rab-16, and M-Ras. Rab proteins are Ras-like small GTP binding proteins that regulate vesicle trafficking pathways. The covalent binding of the Rab proteins with LCA may influence vesicular transport or binding of vesicles to their cognate membrane and may contribute to LCA-induced liver toxicity.

Radioimmunoassay of unsulfated lithocholates.

A simple, rapid, precise radioimmunoassay for total unsulfate species of lithocholate (lithocholyglycine, lithocholyltaurine, and lithocholate) in serum is described. Antiserum was raised in rabbits by injection of lithocholylglycine coupled to bovine serum albumin (prepared by a carbodiimide method) and emulsified in complete Freund's adjuvant; antisera capable of measuring 40-120 pmol at 1:400 dilution were obtained. The tracer was [11, 12-3[H]lithocholyglycine. The radioimmunoassay featured a 2-hr binding step at 42 decrease C and a 1-hr separation step using polyethylene glycol. The antibody had the following relative specificities: lithocholyglycine and lithocholytaurine, 1; lithocholate, 1.5; chenodeoxycholyglycine, 20; and deoxycholylglycine, 55. There was no binding of various other free or conjugated sulfated and unsulfated bile acids. The mean fasting-state level in 50 healthy subjects was 0.3 mu mol/l (0.14 microgram/ml), but 11 of the 50 subjects had level too low to measure by this technique.

Radioimmunoassay of sulfated lithocholates.

A sensitive, rapid radioimmunoassay for sulfated species of lithocholic acid (sulfolithocholyglycine, sulfolithocholyltaurine, and sulfolithocholic acid) was developed and used to measure the total concentration of sulfated lithocholates in serum from healthy human subjects. Sulfolithocholylglycine was conjugated to bovine serum albumin by a carbodiimide procedure, and the reaction product, emulsified in Freund's adjuvant, was injected into rabbits. The antiserum obtained was capable to binding 40% of [3H]sulfolithocholylglycine at 1:1000 dilution. The assay featured a 2-hr binding step at 42 degrees C followed by precipitation of bound tracer with polyethylene glycol at 4 degrees C. The assay can be used with 0.1 ml of serum and was reproducible. The antibody had little affinity for the 3-sulfate of cholic or chenodeoxycholic acid, a number of steroid sulfates, or unsulfated bile acids. In 50 healthy subjects, mean (+/-SE) fasting-state serum levels of immunoreactive sulfated lithocholyl conjugates (+sulfated lithocholate) was 1.6 +/- 0.1 micrometer; based on results with a separate radioimmunoassay for unsulfated lithocholyl conjugates, most of the lithocholate in serum in healthy man is present in sulfated form.