High-affinity monoclonal antibodies for aflatoxins and their application to solid-phase immunoassays.

Abstract

Monoclonal antibodies specific for aflatoxin In a competitive radioimmunoassay using [3H]aflatoxin B1, 3pmol (1 ng) of aflatoxin B1, aflatoxin B2, or aflatoxin M1 caused 50% inhibition with this antibody. The antibody also had significant cross-reactivity for the major aflatoxin-DNA adducts: 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 and 2,3-dihydro-2-(N5-formyl-2',5',6'-triamino-4'oxo-N5-pyrimidyl)-3-hydroxyaflatoxin Bl. The antibody was also covalently bound to Sepharose-4B and used in a column-based solid-phase immunosorbent assay system. Aflatoxins added in vitro to phosphate buffer, human urine, human serum, or human milk at levels expected to be obtained in human samples acquired from environmentally exposed individuals were quantitatively recovered by applying the mixture to this antibody affinity column purification system. Preliminary studies using urine samples from rats injected with radiolabeled aflatoxin B1 have also indicated that aflatoxin metabolites can be isolated by these methods. Furthermore, we have found that the monoclonal antibody affinity columns can be regenerated for multiple use. Therefore, the monoclonal antibodies and their application to affinity chromatography represents a useful and rapid technique to purify environmentally occurring levels of this carcinogen and some of its metabolites for quantitative measurements.The aflatoxins are highly toxic and carcinogenic compounds, which are consistently found contaminating human food supplies in many areas of the world, and are epidemiologically linked to increased incidences of human liver cancer in Asia and Africa (1-3). Adverse biological consequences resulting from exposure to aflatoxin B1 (AFB1) are thought to be related to its ability to be oxidatively metabolized by constituative cellular enzymes. Of particular importance may be the ability of AFB1 to form covalently linked adducts with cellular macromolecules, including DNA. It is well established that AFB1 forms covalent DNA adducts following enzymatic oxidation to a highly reactive 2,3-epoxide, which nucleophil. ically attacks the N7 atom of guanine (4-6). The major DNA lesion produced by this reaction, 2,3-dihydro-2-(N7-guanyl)-3-hydroxyaflatoxin B1 (AF-N7-Gua), has been studied as an indicator compound of genotoxic damage to the cellular genetic material (7-9). A major objective of our work has been the development of noninvasive screening procedures for assessing the exposure of humans to environmentally occurring carcinogens. Useful protocols require an ability to quantify chemical carcinogens and their metabolites, especially DNA adducts, in readily accessible body fluids, such as serum and urine. We have been developing these screening methodologies by using immunoassays with monoclonal antibodies. We previously have reported production of monoclonal antibodies that recognize aflatoxin-DNA derivatives (10, 11). These early antibodies were found to be specific for AFBl-adducted DNA and in a competitive ELISA were able to detect AFB1 residues in DNA down to a...

Anticorpo monoclonal antiAFB1: produção in vitro visando desenvolvimento de bioferramentas

“…O desenvolvimento de meio de cultivo ideal visando obtenção de produto celular específico tem sido processo empírico em contínuo avanço (CHU; UENO, 1977;GROOPMAN et al, 1984;KAWAMURA et al, 1988.;CANDLISH;SMITH;STIMSON, 1989;HOLTZAPPLE et al,1996;KONDO et al, 2000;HEILMANN et al, 2007;HEUSSNER et al, 2007). A exemplo, o cultivo do hibridoma AF2 em RPMI iniciou-se tamponando o meio com bicarbonato de sódio; a adição de HEPES garantiu maior tempo de estabilidade do meio por manter pH fisiológico, mesmo sob mudança na concentração de CO 2 devido a respiração celular (BAICU;TAYLOR, 2002).…”

Development of Aflatoxin B 1 -Lysine Adduct Monoclonal Antibody for Human Exposure Studies

“…Pronase (70,000 proteolytic units/g of dry weight) was obtained from Calbiochem (La Jolla, Calif.). The immunoglobulin M (IgM) monoclonal antibody (2B11) was produced as described previously (11). All other materials and chemicals were commercially available.…”

“…Immunoaffinity resins with IIA4B3 were prepared as previously described (11). Briefly, ascites containing IIA4B3 were precipitated with saturated ammonium sulfate and dialyzed against coupling buffer (0.1 M ammonium carbonate [pH 8.0]).…”

“…The affinity of AFB and its metabolites and adducts for monoclonal antibodies IIA4B3 and 2B11 was determined by an RIA as detailed previously (11). Briefly, different concentrations of AFB and derivatives dissolved in 100 l of phosphate buffer (pH 7.0) were mixed with 100 l of diluted monoclonal antibody in 10% horse serum-PBS and 100 l of tracer ( 3 H-AFB or 3 H-AFB-lysine; 10,000 cpm; approximately 0.4 to 0.5 pmol)-1% normal mouse serum-0.1% BSA in PBS.…”

“…Since the major AFB-albumin adduct had been identified as the AFB-lysine adduct (23,25), the development of more specific monoclonal antibodies recognizing this adduct was initiated. In the study reported here, we have produced and characterized a new mouse monoclonal antibody (IIA4B3), developed using a synthetic AFB-lysinecationized bovine serum albumin (cBSA) conjugate, and have compared its affinity and specificity for AFB and its metabolites and adducts to those of a previously developed monoclonal antibody (2B11) (11). This new antibody has the requisite specificity for use in measuring AFB-lysine adduct levels in human serum samples.…”