Chemical Elution Research Articles

Determination of the Parasitic Contamination of Irrigated Vegetables

In countries with limited water resources, the agricultural use of treated wastewater represents an interesting alternative. The recent World Health Organisation guidelines (1989) for the microbiological quality of treated wastewaters used for crop irrigation require an arithmetic mean of ≤ 1 intestinal nematode egg per litre. In this study diverse methods have been tried for recovering parasitic nematode eggs from vegetables artificially contaminated by Ascaris eggs. The vegetables examined were lettuce and tomatoes. Vegetables were seeded with Ascaris eggs at different levels from 3 to 900 eggs. Eggs elution was realized with chemical solution (distilled water, aceto-acetic buffer pH 4.5, formalin 20%, anionic detergent DDN50®, cationic detergent hyamine®) and/or mechanical treatment (brush - scrub or brush - scrub coupled with glass powder treatment). Concentration was performed by flotation technique with a 55% zinc sulphate solution (d=1.3). Recovery was low for all samples after chemical elution. When we tested the association of chemical and mechanical treatments the percent recovery were ranging from 72 to 100% for vegetables contaminated with 10 to 400 eggs. The method proposed includes an elution with a glass powder suspension in a cationic detergent (50/50), a concentration with ZnSO4 55% solution (d=1.3) and a quantification with a counting cell. The sensitivity threshold of the proposed method lies at about 10 eggs per samples.

Immunoadsorption: strategies for antigen elution and production of reusable adsorbents.

Immunoadsorption is a powerful and generalizable method for protein purification that exploits the fine specificity of antigen-antibody interactions. In spite of its potential utility, the more widespread process scale use of immunoadsorption has been limited by the high cost of the antibody and the lack of gentle elution schemes that completely preserve the activity of both the immunoadsorbent and the eluted product. In this report, we review common chemical elution strategies such as pH, ionic strength, chaotropic salts, denaturants, and organic solvents as well as physical techniques such as pressure, electrokinetics, and temperature. In general, selection of elution strategies has largely been an empirical art, balancing stability of the immunoadsorbent and the eluted product and efficiency. The limitations of the available choices demonstrate that more attention must be placed on the antibody. Techniques which assist in the identification or creation of new antibodies with improved binding properties and resistance to degradation, e.g., screening and/or rational protein engineering, are also discussed.

Nucleic acid hybridization assays employing dA-tailed capture probes: I. Multiple capture methods

A quantitative hybridization assay termed “reversible target capture” is described. The technique is designed to extensively purify the target nucleic acid from crude cell lysates in about 1 h without phenol extraction. Simple, rapid methods are described that explain how each process in the assay is optimized. The procedure involves hybridizing the target nucleic acid in solution with a dA-tailed capture probe and a labeled probe. The capture probe-target-labeled probe “ternary complex” is then captured on magnetic beads containing oligo(dT). After the excess unhybridized labeled probe, cell debris, and other sample impurities are washed away, the intact ternary complex is further purified by chemical elution from the beads and recapture on fresh beads. The ternary complex is then eluted thermally and recaptured on a third set of beads or on poly(dT) filters. This triple capture method results in a detection limit of approximately 0.2 amol (100 fg) of target with 32P-labeled riboprobes. This is approximately 1000 times more sensitive than sandwich assays employing only a single capture step. The method is illustrated by detecting Listeria cells in the presence of heterologous bacteria. With three rounds of target capture, as few as six Listeria cells have been detected in the presence of 1.25 × 10 7 control cells.

Optimized conditions for solid-phase sequencing: simultaneous chemical cleavage of a series of long DNA fragments immobilized on CCS anion-exchange paper

A solid-phase method for simultaneous sequencing often or more long DNA fragments has been developed, using as support the cellulose matrix for chemical sequencing (CCS), anion-exchange paper [Rosenthal et al., Nucl. Acids Res. 13 (1985) 1173–1184]. We optimized several of the seven steps which include: (i) immobilization; (ii) washing; (iii) modification; (iv) washing; (v) sorting of the paper segments; (vi) piperidine reaction and chemical elution, and (vii) lyophilization. During carrier-supported chemical cleavage with dimethylsulfate (DMS) (G), HCOOH (A + G), KMnO 4 (T > Pu) and NH 2OH (C), losses of immobilized DNA are very low. DNA fragments ranging in length from several hundred bp up to 6 kb can be effectively chemically eluted from CCS paper during the piperidine reaction with an efficiency of more than 90%. Because no DNA salt elution and ethanol precipitation steps are necessary the method is rapid, convenient and allows complete automation.

Solid-phase methods for sequencing of nucleic acids I. Simultaneous sequencing of different oligodeoxyribonucleotides using a new, mechanically stable anion-exchange paper.

A solid-phase method for simultaneous sequencing of large numbers of oligodeoxyribonucleotides has been developed using a new, mechanically stable anion-exchange paper. The excellent mechanical properties of the polymer allow the processing of several paper segments in one reaction vessel or to carry out all necessary operations on a larger area of the paper. In addition, DNA material can be chemically eluted from the new carrier during the piperidine reaction, thus avoiding salt elution of DNA and subsequent ethanol precipitation steps - a prerequisite for sequencing oligonucleotides. The approach involves 7 operations including: i) immobilization; ii) washing; iii) modification; iv) washing; v) sorting of the papers; vi) piperidine reaction and chemical elution and vii) lyophilization. All steps can be carried out in 4 to 5 hours independently of the number of oligonucleotides to be sequenced. It is also possible to sequence small oligonucleotides with 3 to 4 base pairs. The method can be fully automated.

Electrophoretic study of serum proteins in frog red leg disease.

Serum from frogs, Rana pipiens, was analyzed for quantitative changes in albumin and globulin fractions in red leg disease. Chemical fractionation plus dye elution and micro Kjeldahl analysis of the material obtained from the electrophoretic separations of the protein fractions, were used to determine the quantitative changes. Serum proteins significantly decreased in red leg disease, the globulin fractions showing a relatively greater decrease than that of albumin. The results are applicable to the diagnosis and management of an important and prevalent animal disease.