BackgroundBilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues.Materials and methodsMice were immunized with a multi-antigen peptide corresponding to segment 65–75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase.ResultsOn the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use.ConclusionsIn this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

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Cross-contamination and misidentification of various cell lines is a widespread problem that can lead to spurious scientific conclusions. DNA fingerprinting is a powerful identification technique, which can be effectively used for the authentication of human cell lines. In contrast to human cancer cell lines, little attention has so far been given to establishing authentication practices for hybridoma cell lines. Since the majority of hybridomas stem from inbred animals, they have high genetic uniformity, which reduces the applicability of DNA fingerprinting. In the present study, we propose antibody variable-region sequencing as a method of choice for hybridoma cell-line authentication. This method focuses on the most diverse characteristic of hybridoma cell lines and thereby achieves a very high discriminatory power. The sequencing of light-chain variable regions has proven to be especially suitable for routine use because of its high success rate. Two other possible authentication methods, random amplified polymorphic DNA analysis and two-dimensional gel electrophoresis, were also examined. Compared to these and other methods that can be used for discrimination between hybridoma cell lines, variable-region sequencing has many advantages, most notably those of a very high discriminatory power, insensitivity to changes in experimental conditions, simple data analysis, and accessibility to most laboratories.

The mammalian bilirubin transporter bilitranslocase (BTL, T.C.#2.A.65.1.1) is found in both absorptive (intestine) and excretory epithelia (liver, kidney) and in the vascular endothelium. The aim of this work was to investigate whether a BTL homologue is expressed also in fish hepatopancreas. Immunochemistry based on an antisequence antibody specific for rat liver BTL demonstrated the presence of such homologue in sea-bass (Dicentrarchus labrax) hepatopancreas. Furthermore the transport activity of such a carrier, measured as electrogenic bromosulphophthalein (BSP) uptake, was assayed in sea-bass microsomes, where it was inhibited by the same antibody. Transport activity in fish showed numerous kinetic similarities with rat, such as BSP K m (about 5 lM in both), bilirubin K i (about 0.1 lM), quercetin competitive K i (about 20 lM), and noncompetitive K i (about 85 lM). Biliverdin K i was instead nearly 10-fold higher in fish than in rat (0.97 AE 0.06 lM and 0.11 AE 0.01 lM, respectively). Fish BTL was found to exist in two different allosteric forms with different affinities for the substrate, similarly to rat liver BTL. It was found that sea-bass BTL is very sensitive to inhibition by HgCl 2 , a major water pollutant, making it reasonable to exploit fish BTL activity as an ecotoxicological biosensor.

HLA-C*05:156 allele differs from C*05:01:01:02 by a nucleotide change in exon 2 at codon 9.