Bisphenol A: A notorious player in the mosaic of autoimmunity.

The multifactorial etiology of autoimmune diseases has been studied at large. Genetic risk factors and environmental agents play an integral role in the pathogenesis of autoimmune processes. In recent decades, Bisphenol A (BPA), an exogenous compound found in polycarbonate plastic, has gained attention for its harmful multifocal effects on a diverse subset of systemic pathways, potentially contributing to disease onset and exacerbation. BPA is a xenoestrogen used globally in the manufacture of daily use products including plastic storage containers, water and infant bottles, and food and drink packaging. BPA exhibits immune stimulatory activity bringing into question the association between its greater global presence and the increased prevalence of autoimmune diseases. The purpose of this multi-study analysis is to assess recent research investigating the underlying role of BPA in autoreactive mechanisms. Although research at present does not directly link BPA exposure to the development of autoimmune diseases, a large body of evidence supports the pro-inflammatory effects of BPA on the immune system. Further studies are required to elucidate the role of BPA in autoimmune pathogenesis, however caution should be taken in the use of BPA containing products by those affected or genetically susceptible to developing autoimmune diseases.

Environmental estrogen bisphenol A and autoimmunity.

Over the past few years, there has been evidence of the increasing prevalence of autoimmune diseases. Autoimmune diseases consist of many complex disorders of unknown etiology resulting in immune responses to self-antigens. The immune system, and its function, is under complex and integrated control and its disruption can be triggered by multiple factors. Autoimmunity development is influenced by multiple factors and is thought to be a result of interactions between genetic and environmental factors. Here, we review the role of a specific environmental factor, bisphenol A (BPA), in the pathogenesis of autoimmune diseases. BPA belongs to the group of environmental estrogens that have been identified as risk factors involved in the development of autoimmune diseases.

Selection of diethylstilbestrol-specific single-chain antibodies from a non-immunized mouse ribosome display library.

Single chain variable fragments (scFvs) against diethylstilbestrol (DES) were selected from the splenocytes of non-immunized mice by ribosome display technology. A naive library was constructed and engineered to allow in vitro transcription and translation using an E. coli lysate system. Alternating selection in solution and immobilization in microtiter wells was used to pan mRNA-ribosome-antibody (ARM) complexes. After seven rounds of ribosome display, the expression vector pTIG-TRX containing the selected specific scFv DNAs were transformed into Escherichia coli BL21 (DE3) for expression. Twenty-six positive clones were screened and five clones had high antibody affinity and specificity to DES as evidenced by indirect competitive ELISA. Sequence analysis showed that these five DES-specific scFvs had different amino acid sequences, but the CDRs were highly similar. Surface plasmon resonance (SPR) analysis was used to determine binding kinetics of one clone (30-1). The measured K(D) was 3.79 µM. These results indicate that ribosome display technology can be used to efficiently isolate hapten-specific antibody (Ab) fragments from a naive library; this study provides a methodological framework for the development of novel immunoassays for multiple environmental pollutants with low molecular weight detection using recombinant antibodies.

Preparation of highly specific anti-zearalenone antibodies by using the cationic protein conjugate and development of an indirect competitive enzyme-linked immunosorbent assay.

Although anti-zearalenone (ZEN) antibodies have been widely prepared, these antibodies cross-react with α-zearalenol (α-ZOL), ß-zearalenol (ß-ZOL), zearalanone (ZAN), α-zearalanol (α-ZAL) and ß-zearalanol (ß-ZAL). To overcome this problem and improve the specificity of immunoassays, we produced anti-ZEN antibodies based on a ZEN-cationic protein conjugate. In this study, ZEN was coupled with cationic bovine serum albumin (cBSA) via a Mannich reaction. After BALB/c mice were immunized with ZEN-cBSA, an immunological response was rapidly induced. The titers of the polyclonal antisera and monoclonal antibody were 30,000 and 20,000, respectively. Cross-reactivity (CR) values of the anti-ZEN polyclonal antisera and monoclonal antibody with the 5 analogs were <7% and <2%, respectively. An indirect competitive enzyme-linked immunosorbent assay based on the monoclonal anti-ZEN antibody was established. The recovery rates of ZEN in spiked cereal and feed were in the range of 80%-120% with coefficients of variation <15%. The intra-assay variation and inter-assay variation in assay buffer were both <5%. Therefore, the results demonstrated a feasible approach for preparing highly specific, higher titer and more rapidly induced antibodies against ZEN by using a ZEN-cBSA conjugate as the immunogen instead of currently used immunogens.

Monoclonal-based enzyme-linked immunosorbent assay for the detection of zearalenone in cereals.

A monoclonal antibody against zearalenone (ZEA) was produced and used successfully to develop a direct competitive enzyme-linked immunosorbent assay (DC-ELISA) for the analysis of ZEA in cereals. This DC-ELISA had a limit of detection of 0.15 +/- 0.02 microg l(-1) and an IC50 value of 1.13 +/- 0.16 microg l(-1). Matrix interference was minimized by dilution of the sample extract before ELISA assays. Aqueous methanol (80%) gave good extraction efficiencies, and the recovery from spiked rice, barley, and corn samples averaged between 87 and 112%. Although ZEA was detected in seven (9%) of 80 rice samples and in eight (16%) of 50 barley samples, the concentration of ZEA in samples was around or below the limit of detection of DC-ELISA. Among 38 corn samples, ZEA was detected in nine (24%) samples in the range 41.0-909.8 microg kg(-1). Re-analysis of the ELISA-positive corn samples by high-performance liquid chromatography (HPLC) confirmed that seven (18%) corn samples were positive. The ZEA results for corn showed very good agreement between DC-ELISA and a commercial AgraQant zearalenone kit (r2 = 0.98). Thus, the monoclonal antibody-based DC-ELISA could be applied to the preliminary screening of ZEA contamination when analysis of a large sample number is needed.

Sensitive detection of estrogenic mycotoxin zearalenone by open sandwich immunoassay.

Zearalenone (ZEA) is an estrogenic mycotoxin produced by Fusarium sp., and its production on corn and small grains during storage has been of considerable concern. For sensitive ZEA detection, we applied an open sandwich (OS) immunoassay that can noncompetitively detect monovalent antigens utilizing an antigen-induced enhancement of the V(H)/V(L) interaction. We cloned the V(H) and V(L) cDNAs of anti-ZEA mAb to a split-Fv phagemid pKST2, and firstly both V(H) and V(L) fragments were displayed on M13 phage p9 and p7, respectively, using an amber suppressor, TG-1, as a host. The split-Fv phage showed specific binding to immobilized ZEA, which was well inhibited by free ZEA. Then, the V(H)/V(L) interaction and its antigen-dependency were analyzed using a non-suppressor HB2151 as a host to produce V(H)-displaying phage and his/myc-tagged soluble V(L) in the culture supernatant. By capturing V(L) with an anti-myc or -his antibody and probing bound V(H)-phage, ZEA was successfully detected with a superior detection limit as well as a wider working range than those of a competitive assay. Also, essentially the same results were reproduced with purified V(H)-alkaline phosphatase and MBP-V(L) fusion proteins.

A specificity-enhanced time-resolved fluoroimmunoassay for zeranol employing the dry reagent all-in-one-well principle.

A simple dry chemistry time-resolved fluorescence immunoassay (TR-FIA) method was developed for the measurement of zeranol in bovine urine samples. The samples were purified by immunoaffinity chromatography and a specificity-enhanced zeranol antibody was employed in the immunoassay. This resulted in a highly selective method, which had only negligible reactivity with Fusarium spp. toxins. The all-in-one-well dry chemistry concept made the assay very simple to use because all the assay-specific reagents were already present in the reaction wells in dry form. Only the addition of diluted sample extract was required to perform the competitive one-step TR-FIA and the results were available in less than 1 h. The analytical limit of detection (mean + 3s) for the immunoassay was 0.16 ng ml(-1) (n = 12) and the functional limit of detection for the whole method, estimated by the analysis of zeranol-free samples, was 1.3 ng ml(-1) (n = 20). The recovery of zeranol at the level of 2 ng ml(-1) was 99% (n = 18) and the within-assay variation ranged between 4.5 and 9.0%.

Immune alterations in geriatric mice dosed subacutely with diethylstilbestrol (DES).

Exposure to both physiological and pharmacological doses of estrogenic compounds has been reported to alter immunologic responses in humans as well as in developmental and adult murine models. Despite the current therapeutic use of potent estrogens, including diethylstilbestrol (DES), in geriatric human disorders, elucidation of the effects of these agents on the aged immune system is limited. The present report describes highly significant alterations in the thymus and bone marrow of aged mice (21 +/- 1 months) exposed subacutely to DES. Severe thymic hypocellularity developed in treated mice following five consecutive days of intraperitoneal injection with 1.5 or 6.0 mg kg(-1) DES. Cell maturation within the thymus was also affected, as indicated by a significant decrease in CD4+8+ cells and a concomitant increase in CD4-8- cells. Cellularity of the bone marow was unchanged by DES. However, significant changes were observed in percentages of bone marrow cells expressing surface antigens CD45 (common leukocyte lineage), Mac-1 (macrophage lineage) and CD45R (B-lineage lymphocytes). Both percentages and total numbers of cells in the spleen expressing Thy 1.2 (T-lineage lymphocytes) were also reduced. These immune changes in geriatric mice exposed to DES were similar in direction but more severe than those reported in either young adult or perinatal models. These data may suggest a need for considering the geriatric immune system separately from other age groups when determining effects of immunosuppressive or immunotoxic compound exposure.

Comparison and evaluation of the specificity and binding capacity of commercial and in house affinity columns used in sample preparation for analysis of growth-promoting drugs.

Immunoaffinity chromatography (IAC) and affinity chromatography (AC) are widely used for extraction of drugs from biological samples. Fifteen column types were purchased from five different manufacturers and their ability to bind specific drugs including beta-agonists and anabolic steroids over a range of analyte concentrations in fortified bovine urine samples was assessed. The performance data obtained from these columns were compared with columns produced in this laboratory (in house columns). The in house columns gave the highest recoveries, ranging from 92 to 100% at the 1 ng spiking concentration, for five of the seven analytes assessed. Forty percent (11 of 27) of all the commercial column assessments recorded recoveries of less than 50% even when the lowest spiking concentration was applied (1 ng). For one manufacturer, only one of seven different columns purchased delivered extraction efficiencies greater than 50%. The extraction efficiencies of the clenbuterol columns were the highest with all commercially prepared columns showing at least 50% binding of radiolabelled tracer. Recoveries of alpha-nortestosterone were the lowest. The variability of these products with respect to quality control requires constant monitoring.