Biomedical subjects

Ramadan A Abuknesha

Publications and source records attributed to Ramadan A Abuknesha.

10 recordsLinked to original sources

PubMed2005

A highly efficient preparative-scale generic immunoaffinity chromatography method for the purification of antibodies to hydrophobic haptens: purification procedure and monitoring tests.

A generic affinity chromatography purification protocol for the isolation of preparative quantities of pure and stable polyclonal antibodies to hydrophobic haptenic analytes is described together with a panel of tests to monitor the purification process and assess the functional and structural purity of isolated antibodies. The purification method is based on the use of a mixture of acetonitrile and propionic acid to elute bound antibodies from Sepharose 4B-based immunoabsorbent gels. Highly specific and pure antibodies to steroid estrogens, pentachlorophenol and Irgarol 1051 were isolated in 50-150 mg quantities per preparation in a batch-wise method using appropriate ligands linked to the solid phase via a hydrophilic chemical arm, tetraethylene pentamine. The panel of ELISA tests together with SDS-PAGE enabled the monitoring of the absorption and elution steps and provided data relevant to the assessment of the degree of structural and functional purity of the isolated antibody preparations. The study demonstrates that the affinity purification procedure is practical, simple, generic for antibodies to hydrophobic haptens and suitable for scaling up. In addition, the study showed that the functional properties of the affinity-purified antibodies indicated improvements on the operational properties (specificity and assay detection limits) of the source antisera. The isolated IgG antibodies showed near 100% functional and structural purity and no deterioration of activity on storage for long periods. The method provides critical reagents for labelled-antibody immunoassays and immunosensors and antibody-dependent sample purification techniques.

Animals↗

PubMed2005

Efficient labelling of antibodies with horseradish peroxidase using cyanuric chloride.

An efficient and mild method for labelling of immunoglobulin G (IgG) with horseradish peroxidase (HRP) using cyanuric chloride (2,4,6-trichloro-1,3,5-triazine, CC) as a bridging molecule is described. The enzyme was treated first with cyanuric chloride to introduce dichloro triazine and after removal of excess reagent, the activated enzyme was mixed with the IgG preparation and incubated to effect linkages with amine groups in the antibody protein. Various amounts of coupling reagent were tested to optimise the conjugation method using commercially available enzyme and affinity-purified sheep IgG antibody preparations to three different test haptens. The conjugates were assessed by solid phase Enzyme Linked Immunosorbent Assays (ELISA) and commonly used peroxidase substrate preparations. The binding activity of the conjugates rose with increasing coupling reagent added during the enzyme activation step. Use of the conjugates prepared by the new method gave comparable sensitivity in direct competitive ELISAs for the three test haptens to assays carried out using indirect ELISA with commercial anti-sheep-HRP conjugates. No deterioration of enzyme activity or hapten-binding activity in the conjugates was observed after storage in 50% glycerol at -70 degrees C for up to 18 months. This study presents a relatively simple and efficient conjugating method for labelling antibodies with HRP and provides an additional and probably a better alternative to the periodate, glutaraldehyde and succinimide-maleimide procedures.

Animals↗

PubMed2005

Paraquat enzyme-immunoassays in biological samples: assessment of the effects of hapten-protein bridge structures on assay sensitivity.

Previously unreported paraquat derivatives were prepared and used to develop enzyme-immunoassay methods for paraquat in serum and urine matrices. The study involved comparison of the effects of novel paraquat derivatives made of methyl and ethyl-4,4'-bipyridinium and cyanuric chloride (heterologous bridges) or valeric acid (homologous bridges) on the ability of paraquat standards to inhibit binding of the antibody to adsorbed hapten-protein plate coating antigens prepared by coupling the derivatives to gelatine. The comparison showed striking differences in assay sensitivity due to the hapten bridge binding phenomenon where the heterologous bridge conjugates enabled achievement of sensitivity levels several orders of magnitude greater than the homologous structures. The constructed ELISA showed minimal detection limit in the range 4 pg mL(-1) in the buffer systems and less then 100 pg mL(-1) in charcoal-stripped human and horse sera and human urine. The study presents details of synthesis of novel paraquat derivatives and a highly sensitive ELISA. In addition the investigation demonstrates the critical importance of judicious selection of hapten-bridge structures to achieve improved levels of detection limits of paraquat immunoassays. The reported assay is suitable for use in monitoring of paraquat levels in exposed persons or animals and for emergency diagnostic tests.

Animals↗

PubMed2005

Enzyme immunoassays for the analysis of streptomycin in milk, serum and water: development and assessment of a polyclonal antiserum and assay procedures using novel streptomycin derivatives.

A polyclonal antiserum to streptomycin was generated in sheep using a streptomycin-bovine serum albumin conjugate as immunogen. Streptomycin was linked to the carrier protein with cyanuric chloride using a new two-step conjugation method. Plate coating antigen conjugates of streptomycin and gelatine were prepared using either cyanuric chloride (homologous bridge) or 1,4-butanediol diglycidyl ether to provide an heterologous complex. The reagents enabled the generation of a specific antiserum with a titre of 1/40,000 and the development of a sensitive ELISA method suitable for the measurement of streptomycin sulfate in milk, serum and water samples. A minimum detection value of 1 ng mL(-1) and a dynamic range of 1 to 200 ng mL(-1) were demonstrated in the three matrices. No detectable cross reactivity with any of the common aminoglycosides was found except the related dihydrostreptomycin which gave a 75% cross reaction value. The details of the preparation of the hapten-protein conjugates, characterisation of the antiserum and assay construction and assessment methods are presented. The introduction of new coupling methods and antibody assessment provide improved basic methodologies necessary for the advancement of immuno-analysis of streptomycin, one of the most widely used antimicrobial substances.

Animals↗

PubMed2004

Generation of antiserum to Irgarol 1051 and development of a sensitive enzyme immunoassay using a new heterologous hapten derivative.

A polyclonal antiserum to Irgarol 1051 was developed in sheep and used to construct an enzyme immunoassay method for the measurement of the antifouling compound in river and seawater samples. The antiserum was generated by a hapten derivative, 2-(tert-butylamino)-4-(cyclopropylamino)-6-(thiopropionic acid)-1,3,5-triazine, coupled to a mixture of keyhole limpet hemocyanin and bovine serum albumin, and the competitive enzyme immunoassay was constructed using a plate-coating antigen made of a heterologous new hapten derivative, 2-(tert-butylamino)-4-(cyclopropylamino)-6-(phenoxybenzoic acid)-1,3,5-triazine, linked to gelatine. The assay showed a sensitivity of about 5 ng L(-1) in river and seawater matrices with reasonable specificity with respect to commonly used triazines such as atrazine (3%), simazine (>0.1%) and desethylatrazine (>0.01%). However, high cross-reactivity levels were found with ametryn (56%) and prometryn (60%). Tests on the effects of organic solvents on assay performance indicated a high tolerance to methanol but much less so to acetonitrile. The assay was found to be highly reproducible and robust owing to the stability of the sheep antibody and the highly optimised competitive assay reagents which included the use of the new triazine-O-phenoxybenzoic acid derivative.

Haptens↗

PubMed2004

A highly sensitive and specific enzyme immunoassay for detection of beta-amanitin in biological fluids.

Polyclonal antisera to beta-amanitin were generated in sheep and used to construct a competitive ELISA for measurement of the toxin in human serum and urine. The assay had a detection limit of about 80 pg mL(-1), a dynamic range of 80-2,000 pg mL(-1), a cross reactivity of 22% with alpha-amanitin, and no cross reactivities with cyclic peptides from algal sources. Assay responses in buffer, serum, and urine were remarkably similar. Coupling of the toxin to carrier proteins was carried out by previously unreported methods. The key step that allowed the construction of the highly sensitive assay was the introduction of a novel heterologous hapten derivative made of beta-amanitin-cyanuric chloride derivative. The new derivative overcame the problems of bridge binding that was, in this case, particularly serious with the homologous hapten derivative. The study demonstrated that the developed antiserum and ELISA procedure can be used to detect beta-amanitin and related toxins from Amanita phalloides in human serum and urine samples from suspected poison cases and enable early treatment to be administered.

Amanitins↗

PubMed2004

Evaluation of a polyclonal antiserum to pentachlorothiophenol-acetic acid-KLH immunogen: binding properties and use with heterologous PCP derivatives in ELISA for pentachlorophenol.

A polyclonal antiserum to pentachlorothiophenol-acetic acid-KLH was generated in sheep and assessed by solid phase ELISA. The assessment procedure included use of double checkerboard analysis in the absence and in the presence of analyte loads, estimation of cross reactivities of chlorophenol pesticides, assessment of the effect of pH, Tween 20, and Thames water matrix. The antiserum was highly specific for pentachlorophenol and enabled minimum detection limits of less than 0.2 ng mL(-1) in river water matrix. Particularly important was the significant improvement of assay performance in the absence of Tween 20 and at pH 4 and the very low cross reactivity (less than 0.01%) for other commonly used chlorophenols-2,4,5-trichlorophenol and 2,4,6-trichlorophenol, 2-methyl-4-chlorophenoxyacetic acid, and 2,4-dichlorophenoxy acetic acid. The study re-affirms the importance of the judicious choice of hapten derivatives in the synthesis of immunogens and assay reagents for pentachlorophenol analysis by competitive immunoassays.

Acetic Acid↗

PubMed2002

Removal of detergents from protein extracts using activated charcoal prior to immunological analysis.

The use of dextran-coated activated charcoal (DCC) powder to absorb solubilising detergents from cell lysates is described. Normal embryonic epithelial cells were lysed in the presence of sodium dodecyl sulphate (SDS). The detergent was then absorbed with DCC to facilitate analysis of polycystin-1 with antibody-based methods. Polycystin-1 is a membrane protein that is involved in the pathogenesis of autosomal dominant polycystic kidney disease (ADPKD). The adverse effect of SDS on antibody-polycystin-1 binding was studied and the improvement resulting from its removal demonstrated using enzyme-linked immunosorbent assays (ELISAs). The results indicate that DCC can be used in a simple manner to remove highly reactive membrane-solubilising reagents from protein mixtures prior to immunological analysis. This procedure may be relevant to a variety of other techniques that are normally affected by detergents.

Adsorption↗

PubMed2002

Interaction of the leucine-rich repeats of polycystin-1 with extracellular matrix proteins: possible role in cell proliferation.

Polycystin-1, the product of the PKD1 gene, is a membrane-bound multidomain protein with a unique structure and a molecular weight of approximately 460 kD. The purpose of this study is to investigate the binding of the cystein-flanked leucine-rich repeats (LRR) of polycystin-1 to extracellular matrix (ECM) components. These interactions may play a role in normal renal development as well as the pathogenesis of autosomal-dominant polycystic kidney disease (ADPKD). In vitro assays were used to assess the binding of a fusion protein containing the LRR of polycystin-1 and that of affinity purified polycystin-1 to a number of ECM components. The results showed that the LRR modulate the binding of polycystin-1 to collagen I, fibronectin, laminin, and cyst fluid-derived laminin fragments. The addition of the LRR fusion protein to cells in culture resulted in a significant dose-dependent reduction in the rate of proliferation. Cyst fluid-derived laminin fragments had a stimulatory effect on cell proliferation, which was reversed by the LRR fusion protein. These results suggest that the LRR of polycystin-1 act as mediators of the polycystin-1 interaction with the ECM. The observed suppression effect of the LRR on cell proliferation suggests a functional role of the LRR-mediated polycystin-1 involvement in cell-matrix and cell-cell interactions. These interactions may result in the enhanced cell proliferation that is a characteristic feature of ADPKD.

Amino Acid Sequence↗

PubMedDate not supplied

Enzyme-immunoassay for the determination of metallothionein in human urine: application to environmental monitoring.

The objectives of this study were to develop an enzyme immunoassay for metallothioneins in human urine using a polyclonal antiserum and to demonstrate a possible relationship between the level of this biomarker and heavy metal exposure. The antiserum was raised in sheep against horse metallothionein conjugated to carboxylated bovine serum albumin. The antibody was used to construct a two-step competitive ELISA procedure. Human urine was treated with activated charcoal powder to remove traces of metallothioneins and known amounts of pure metallothioneins were added to provide standards for a standard curve. Metallothionein levels were measured in two groups of children living in areas of mild and high environmental pollution due mainly to heavy metals. A comparison was made between the biomarker levels and the levels of cadmium and lead in urine samples in the two groups. A group of children from a non-polluted area acted as controls. The results show that the detected levels of metallothioneins appear to correspond to levels of the two heavy metals studied and that there was an apparent relationship to the environmental exposure. Thus according to results of this study the increase in the metallothionein excretion seems to provide an indication of previous of exposure to metals. The ELISA procedure is sensitive and robust and can be used to screen large numbers of samples and is more rapid than the physical procedures currently used for analysis of these proteins. The assay can therefore be used as an additional tool for screening at-risk populations where either environmental or occupational exposure to divalent heavy metals is suspected.

Amino Acid Sequence↗