A biocatalytic route to enantioenriched, sulfanyl aldol products

The aldol products derived from sulfur- or selenium containing acceptors were prepared by kinetic resolution in the presence of antibody 84G3 with enantiomeric excesses ranging from 56 to 70%. Much higher level of enantioselectivity was obtained (enantiomeric excesses all superior to 96%) for sulfanyl aldol products derived from thiomethoxyacetone with three different acceptors.     

Immunochromatographic flow-injection method for detection of human serum immunoglobulin G antibodies to Helicobacter pylori

This study reports on the development of an immunochromatographic flow-injection (FI) method for the quantitation of human serum IgG antibodies to Helicobacter pylori. Patients’ sera were injected into the carrier stream of a FI manifold incorporating an H. pylori immuno-adsorbent reactor. The immuno-adsorbent was prepared by covalently linking H. pylori antigens to periodate oxidized Sepharose CL-4B. Any H. pylori antibody present in the serum is bound to the immuno-sorbent. The bound antibody was quantified by injecting into the carrier stream anti-human IgG peroxidase conjugate followed by TMB/H₂O₂ as the enzyme substrate. The FI system was optimized with respect to antigen loading (1.0 mg/ml gel), bioreactor length (3.0 cm), enzyme conjugate (0.2 mg/ml) and sample loop size (250 μl). One hundred clinical samples were analyzed for their H. pylori antibody concentrations and the results evaluated with respect to their receiver-operator characteristics (ROC). When a cut-off absorbance of between 0.7 and 0.75 was used for positive and negative samples a specificity (>95%), a sensitivity (>90%)and an overall accuracy (>94%) were obtained.     

Selective alterations of the antibody response to HIV-1

HIV infection leads to progressive alterations of humoral immune functions, including B-cell hyperplasia, hypergammaglobulinemia, elevated autoantibody titers, a poor response to neoantigens and mitogens, polyclonal B-cell activation, monoclonal gammopathies, and a significant deterioration of the antigen-specific humoral response. There is also an important isotypic imbalance of the antibody (Ab) response in the systemic compartment and a profound modification of mucosal immune functions. These abnormalities may contribute to disease progression and development of opportunistic infections, despite the presence of serum-neutralizing anti-HIV Abs. Equally important are the abnormal selection mechanisms of the Ab repertoire that seem to be responsible for B-cell clonal deletions. The V_H3 gene family, which encodes for approx 50% of immunoglobulins expressed by peripheral B-cells from normal adults, is underrepresented in human monoclonal antibodies to HIV-1 and in the peripheral B-cells of AIDS patients. These abnormalities, together with features of germinal center alteration, could be responsible for the clonal elimination of a subset of B-cells, and could contribute to HIV pathogenesis.     

A simple direct enzyme immunoassay of antibodies based on the differences in diffusion rates in a gel of a synthetic antigen and of its complex with antibodies

A simple direct enzyme immunoassay for semiquantitative detec tion of antibodies is suggested. It is based on the difference in diffusion rates in a gel for a synthetic low-mol-wt antigen and of its complexes with antibodies to be detected. Sensitivity and specificity of the devel oped assay are equal to an ELISA method. The assay has been tested with antibodies against HIV protein gp41 in rabbit serum. Possible applications and limitations of the method are discussed.     

Synthesis of branched oxime-linked peptide mimetics of the MUC1 containing a universal T-helper epitope

Our goal was to develop mimics of MUC1, highly immunogenic to induce an efficient immune response against the tumor-associated form of MUC1, and sufficiently different from the natural antigen to bypass the tolerance barrier in humans. With the aim of obtaining a well-defined peptide construct as a means of evoking the precise immune responses required in immunotherapy, we synthesized artificial mimics of the MUC1 protein composed of two MUC1 repeat units of inverse orientation and a universal T-helper epitope. To synthesize these heteromeric peptide constructs, we followed a convergent approach using chemoselective ligation based on oxime chemistry. A stem peptide was first synthesized bearing two orthogonally masked aldehydes. After successive deprotection, two oxime bonds can be specifically generated. The proposed strategy proved to be concise and robust, and allowed the synthesis of the tri-branched protein in a very satisfactory yield. The different constructs were tested for their ability to generate antibodies able to recognize the MUC1 protein.     

Comparative study of three immunoassays based on monoclonal antibodies for detection of the pesticide parathion-methyl in real samples

Three immunoassay systems: indirect, direct competitive enzyme-linked immunosorbent assay (IC-ELISA and DC-ELISA), fluorescence polarization immunoassay (FPIA) based on monoclonal antibodies for the detection of parathion-methyl (PM) were developed and optimized. Several PM derivatives (haptens) were conjugated to proteins and fluoresceinthiocarbamyl ethylenediamine (EDF) to obtain immunogens and competitors. The influence of immunogen and competitor structures on the assay performance was investigated. IC-ELISA was the most sensitive of all techniques developed, with a detection limit of 0.08 ng ml⁻¹, but assay time was the longest (3.5 h per 96-well microtitre plate). DC-ELISA was easier to perform and quicker (1.5 h per 96-well microtitre plate) but less sensitive than IC-ELISA (detection limit was 0.5 ng ml⁻¹). FPIA was the fastest and simplest (7 min per 10 samples) but the least sensitive (detection limit was 15 ng ml⁻¹) technique. The methods were characterized by high specificity and reproducibility. The cross-reactivity for parathion-ethyl was around 30-40% for IC-ELISA and FPIA, but significantly higher (125%) for DC-ELISA. The immunoassays were applied to the analysis of PM residues in different food and environmental matrices. Methanol extracts of vegetable, fruit and soil samples were used for the analysis. Recoveries for most spiked samples averaged between 85 and 110%. The methods developed can be used for screening of food and environmental samples for PM residues without complicated clean-up.     

Enzyme-linked immunosorbent assay for the determination of 20(S)-protopanaxatriol

A highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) method has been developed for the determination of 20(S)-protopanaxatriol (PPT), one of the major aglycones of dammarane-type ginseng saponins. Polyclonal antibodies raised against ginsenoside F₁ (GF₁)-bovine serum albumin showed high reactivities to PPT and GF₁, whereas they exhibited minor or even no cross-reactivities to other ginsenosides and protopanaxadiol (0.19%). The working range of this method extends from 50 pg ml⁻¹ to 20 ng ml⁻¹ of PPT. The assay reported here has been validated against an HPLC technique using PPT-containing samples and was shown to correlate closely (γ=0.993). This ELISA could be a useful tool for the determination of PPT contained in biological fluids and plant materials.     

Rapid high-performance isoelectric focusing of monoclonal antibodies in uncoated fused-silica capillaries

Rapid (<5 min) high-performance isoelectric focusing was performed in uncoated fused-silica capillaries to resolve isoforms of monoclonal antibodies and to determine their isoelectric points (pI). The methodology involved the use of a 32 cm (effective length 9 cm)×50 μm I.D. uncoated capillary. (Hydroxypropyl)methyl cellulose was used as an additive to suppress analyte–wall interaction and to precisely control electroosmotic flow so that focusing and mobilization of focused zones past detector occur simultaneously. Urea was included in the separation medium to solubilize the antibodies that precipitated at their point of focusing. The methods with and without urea used ampholytes pH 5–8 to generate a demonstrable linear gradient between pH 5.4 and pH 7.2, based on the separation of various protein standards. Reproducibility [<2% (R.S.D.)] of the migration times (corresponding to the detectable isoforms of the antibodies) was obtained by using two sets of reagents and capillaries on three consecutive days. pI values determined from day-to-day with a reference standard were shown to vary by only 0.01 pH unit. The described capillary isoelectric focusing methods provided a rapid, simple and reproducible way of monitoring micro-heterogeneity and pI of the murine monoclonal antibodies investigated.     

Biocatalysis and biorecognition in nonaqueous media. Some perspectives in analytical biochemistry

Biocatalysis and, to a lesser extent, biorecognition in non-aqueous media (including organic solvents as well as supercritical fluids and gases) constitute at present an exciting research area which has already demonstrated its biotechnological potential in numerous, varied applications. Less attention, however, has been paid to its analytical possibilities, even though many advantages have been postulated and a wide range of poorly water-soluble analytes are present in samples (or waste materials) from food and drink, petrochemical, pharmaceutical, military and other industries. The main approaches, developed in recent years to exploit the use of enzymes, antibodies or antibody mimics in water-restricted environments for analytical purposes, as well as possible future directions are briefly discussed.     

Novel functional activities of anti-dna autoantibodies from sera of patients with lymphoproliferative and autoimmune diseases

DNA-hydrolyzing activity of IgG autoantibodies from sera of patients with various types of lymphoproliferative diseases was investigated. The association of DNA-hydrolyzing activity with the antibody (Ab) fraction has been proved by newly developed affinity-capture assay. Study of abzyme incidence in blood tumors and systemic lupus erythematosis (SLE) revealed linkage of anti-DNA Ab catalysts to mature B-cell tumors, and increased probability of DNA-abzymes formation on the background of autoimmune manifestations. These data suggest possible similarity between mechanisms of abzyme formation in SLE and B-cell lymphomas. A new mechanism of formation of DNA-specific catalytic Abs has been proposed based on the increased crossreactivity of polyclonal DNA-abzymes to DNA-depleted nuclear matrix proteins. The possibility of the abzyme production as Ab to the energetically destabilized ground state of the antigen has been discussed. Preliminary results were obtained that indicate the complement-independent cytotoxicity of anti-DNA autoantibodies isolated from blood of patients with SLE and chronic lymphocytic leukemia.