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Biomedical subjects

L G Bachas

Publications and source records attributed to L G Bachas.

35 records · Page 2Linked to original sources
PubMed1993

Sensitive and selective liquid chromatographic postcolumn reaction detection system for biotin and biocytin using a homogeneous fluorophore-linked assay.

A homogeneous fluorophore-linked assay was used to develop a postcolumn reaction detection system for high-performance liquid chromatography (HPLC). Biotin and biocytin were chosen as the model analytes. The effluent from the HPLC column was merged with a reagent stream containing avidin that was labeled with fluorescein isothiocyanate (avidin-FITC). The binding of the separated analytes by the labeled avidin was accompanied by an enhancement of the fluorescence intensity at 520 nm. This increase in fluorescence was proportional to the concentration of the analytes and constituted the analytical signal. The procedure was optimized with respect to the reagent concentration and the flow-rate of the reagent solution. Analytical characteristics of the method were determined. The procedure was highly selective for biotin and its derivatives. The detection limits for biotin and biocytin were 89 and 94 pg, respectively, for 20-microliters injections. The developed postcolumn reaction detection system was validated by determining biotin in a liquid vitamin preparation and a horse-feed supplement.

Avidin↗
PubMed1993

Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein.

Traditional chemical means of preparing enzyme-ligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment. A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production of conjugates for use in ELISAs. Specifically, a synthetic octapeptide was fused with bacterial alkaline phosphatase. The resulting enzyme-peptide conjugate is monosubstituted (one peptide per subunit), has a single site of attachment, and results in assays with good response characteristics. The use of such fusion proteins, which combine small analyte peptides with enzyme labels, can lead to a new approach to improved assays for numerous biomolecules, including peptide pharmaceuticals, neurotransmitters, hormones, cell surface antigens, etc.

Alkaline Phosphatase↗
PubMed1993

Observation of "hook effects" in the inhibition and dose-response curves of biotin assays based on the interaction of biotinylated glucose oxidase with (strept)avidin.

The inhibition of highly biotinylated enzymes by avidin and streptavidin has been used in the development of homogeneous assays for biotin and other analytes. Usually, this inhibition occurs in a similar fashion for both avidin and streptavidin. Specifically, the curves that relate the inhibition of the enzymatic activity with the concentration of avidin or streptavidin have a sigmoidal shape; I.e., the inhibition of the enzyme-biotin conjugates increases gradually with increasing amounts of avidin or streptavidin and arrives at a plateau at high binding protein concentrations. However, when these two biotin-specific binding proteins interact with biotinylated glucose oxidase a significant difference in their inhibitory action is observed. In particular, the inhibition curves have a sigmoidal shape for streptavidin, while those for avidin exhibit a maximum ("hook") at low avidin concentrations. This difference in the reactivity of the two proteins with biotinylated enzymes influences both the shape of the dose-response curve and the detection limits of homogeneous enzyme-linked competitive binding assays for biotin.

Avidin↗
PubMed1993

Defined analyte-enzyme conjugates as signal generators in immunoassays.

We investigated the synthesis of progesterone-horseradish peroxidase (P-HRP) conjugates and products purified by affinity chromatography. The obtained preparations were characterized with an immobilized monoclonal antibody in solid-phase immunoassays. Three homogeneous P-HRP conjugates were isolated. Two preparations were identified to contain a single progesterone ligand on the enzyme molecule. A third preparation contained two progesterone ligands. We postulate that conjugation can occur at two different positions on the enzyme, and that the different microenvironment of the protein structure surrounding the ligand contributes to different binding constants of the conjugates with immunoglobulin. By comparing the effective binding constants derived from affinity chromatography and from Scatchard analysis, we have demonstrated that the divalent conjugate binds to antibody immobilized on planar surfaces only by a single attachment due to steric restriction. Dose-response curves for progesterone using the isolated P-HRP conjugates have been investigated and compared.

Animals↗
PubMed1991

Homogeneous enzyme immunoassay for lipoic acid based on the pyruvate dehydrogenase complex: a model for an assay using a conjugate with one ligand per subunit.

A homogeneous enzyme immunoassay for lipoic acid was developed by using an enzyme-ligand conjugate containing only one ligand per enzyme subunit. Theoretical studies have shown that the traditional use of multisubstituted enzyme-ligand conjugates has limited the detection limits and sensitivity obtainable with these assays. The use of conjugates with a smaller number of ligands should allow for improved assays. The pyruvate dehydrogenase complex was chosen for this study because each polypeptide chain of dihydrolipoyl transacetylase contains one lipoic acid as a covalently attached prosthetic group. Thus, the naturally occurring enzyme can be considered as an enzyme-lipoic acid conjugate. Anti-lipoic acid antibodies were developed in New Zealand White rabbits to be used as the analyte-specific binders. Association and binder dilution curves were prepared in order to optimize the reagent concentrations and the analytical conditions. Unexpected inhibition by free lipoic acid resulted in a biphasic dose-response curve with a detection limit of 5 x 10(-6) M lipoic acid. This technique has several advantages over previous electrochemical and chromatographic techniques for lipoic acid determination.

Antibodies↗
PubMed1991

Isoelectric focusing electrophoresis of protein-ligand conjugates: effect of the degree of substitution.

The degree of substitution of protein-ligand conjugates can be determined from the change of the isoelectric point (pI) of the protein when ligand molecules are attached to its surface. Specifically, the pI values of conjugates with known degrees of substitution are obtained by isoelectric focusing electrophoresis and are used to generate a calibration curve that relates these two variables. The shape of the curve is sigmoidal and can be predicted by a theoretical model that takes into account the degree of substitution and the amino acid composition of the protein. By using such a calibration curve, one may estimate the degree of substitution of a given protein-ligand conjugate from its pI value. The applicability of the method is demonstrated with conjugates of pyridoxal 5-phosphate and avidin.

Amino Acids↗
PubMed1990

High-performance liquid chromatographic postcolumn reaction detection based on a competitive binding system.

Postcolumn reactions are typically employed to improve detection in high-performance liquid chromatography (HPLC) separation techniques. This study proposes the use of competitive binding principles in designing novel postcolumn reaction schemes. The feasibility of this approach was tested by using the HPLC determination of biotin and biocytin as a model system. The effluent from the HPLC column was merged with a reagent stream containing avidin, whose binding sites were occupied by the dye HABA (2-[4'-hydroxyphenylazo]benzoic acid). HABA was displaced by the analytes from the avidin-HABA complex and the free dye was determined with a UV-vis detector at 345 nm. The procedure was optimized with respect to reactor design, reagent concentrations, and the flow rate of reagent solution. Analytical characteristics of the developed procedure were determined and compared with the direct detection of biotin and biocytin at 220 nm. The postcolumn reaction scheme improved the selectivity and sensitivity of the detection of biotin and biocytin while maintaining similar detection limits.

Azo Compounds↗
PubMed1990

A simple method for estimating association constants between monoclonal antibodies and derivatized or native antigens.

We describe a simple method for estimating the association constant between an antibody and an antigen, based on a theoretical treatment of experimental dose-response data. We used this method to calculate the association constants of an anti-progesterone monoclonal antibody for native progesterone and for progesterone 11 alpha-hemisuccinate. These association constants provide a quantitative measure of the bridging-group recognition by the antibody.

Antibodies, Monoclonal↗
PubMed1989

Pyruvate carboxylase as a model for oligosubstituted enzyme-ligand conjugates in homogeneous enzyme immunoassays.

Theoretical models suggest that the detection capabilities of homogeneous enzyme immunoassays can be improved by the use of oligosubstituted enzyme-ligand conjugates rather than the traditionally used multisubstituted ones. The natural form of pyruvate carboxylase contains four covalently bound biotins (one per subunit) and it can be considered as an oligosubstituted enzyme-biotin conjugate. The enzyme is nearly completely inhibited in the presence of the natural binder for biotin, avidin. When the enzyme is incubated with avidin and free biotin, a competition occurs between the free biotin and the prosthetic group of the enzyme for the avidin. Steep dose-response curves are obtained by relating the observed inhibition to the free biotin concentration. By variation of the amount of avidin or enzyme in the assay, the detection limits of the system can be altered allowing for sensitive determinations over a wide range of biotin concentrations. Such data from real sample analysis of several vitamin supplements are reported.

Avidin↗
PubMed1989

Potentiometric homogeneous enzyme-linked competitive binding assays using adenosine deaminase as the label.

Homogeneous enzyme-linked competitive binding assays for biotin are described that are based on the competition between an enzyme-biotin conjugate and free biotin for a fixed number of binding sites of avidin. Unlike conventional homogeneous enzyme immunoassays, in this system the analyte (biotin) is labeled with adenosine deaminase (ADA), an ammonia-producing enzyme. Consequently, potentiometric rather than photometric methods can be used as means of detection. Several ADA-biotin conjugates were prepared and showed as high as 97% inhibition of the enzymatic activity in the presence of avidin. Addition of free biotin reverses this inhibition in an amount proportional to the concentration of analyte. Relatively steep dose-response curves were observed, leading to a precise and accurate assay for biotin. The detection limits of these curves were as low as 1 x 10(-8) M. Varying the concentration of the reagents in the assay allowed the detection limit and working range to be altered to a desired value. The proposed method was applied in the determination of biotin in a horse-feed supplement.

Adenosine Deaminase↗
PubMed1986

Theoretical models for predicting the effect of bridging group recognition and conjugate substitution on hapten enzyme immunoassay dose-response curves.

Models for predicting the effect of immunological recognition of the bridge group on the dose-response curves obtained with heterogeneous hapten enzyme immunoassays are presented. Appropriate theoretical treatment shows that the greater affinity of antibodies toward the enzyme-labeled species than for the unlabeled hapten analyte results in assays with limited detection capabilities. This problem is compounded when enzyme conjugates possessing multiple haptens are used. In equilibrium type competitive arrangements, the concentrations of binder and labeled hapten may be optimized to some extent to improve assay performance. However, the results presented show that only when assays are performed in a sequential binding mode using carefully controlled timing of reagent incubations can the detection capabilities of the assays be fully maximized for analyte measurements. Unfortunately, it is also shown that such sequential binding approaches render the assays essentially nonselective. The effect of decreasing the affinity of the binder to the enzyme-labeled hapten relative to the unlabeled analyte by using heterologous conjugates in equilibrium arrangements is shown to improve detection capabilities but also at the expense of reduced selectivity. Suggestions for reagent concentrations and conjugate substitution (degree of conjugation), which provide optimized dose-response curves at a given ED50 value, are also presented as are proposals for using different binders which do not exhibit bridging group recognition.

Antibody Affinity↗
PubMedDate not supplied

Effect of different binding proteins on the detection limits and sensitivity of assays based on biotinylated adenosine deaminase.

The properties of binding proteins that control the nature and magnitude of inhibition of the enzyme-ligand conjugates in homogeneous enzyme-linked competitive binding assays were investigated. An assay for biotin that employed adenosine deaminase as the enzyme-label was used as a model system because of the availability of several biotin-specific binders with different characteristics. It was found that the association constant between the ligand and the binding protein, as well as the depth of the binding pocket, affect the response characteristics of the assay. In addition, the reported data suggest that steric-hindrance effects also contribute toward the sensitivity and detection-limit capabilities of the assay.

Adenosine Deaminase↗
PubMedDate not supplied

Determination of the extent of protein biotinylation by fluorescence binding assay.

A method was developed to determine the total amount of biotin present in biotinylated protein conjugates. Conjugates of bovine serum albumin, alkaline phosphatase, and horseradish peroxidase were used in this case study. The extent of biotinylation was determined by complete acid hydrolysis or by enzymatic digestion using proteinase K to release biotin from the biotinylated proteins, followed by sensitive detection of biotin using a coupled HPLC-binding assay system. This detection system is based on the enhancement of the fluorescence of streptavidin-FITC by biotin. The extent of biotinylation determined by this method was compared with the values obtained by a conventional colorimetric method that is based on the displacement of the dye 4-hydroxyazobenzene-2-carboxylic acid (HABA) from the binding sites of avidin. It was found that, because the described method determines the amount of liberated biotin after hydrolysis, it does not suffer from steric hindrance problems associated with the ability of biotin on a protein surface to displace HABA from avidin. Therefore, this method can provide a more accurate determination of the extent of biotinylation. It was also determined that the acid hydrolysis of the biotinylated protein was more effective in releasing the conjugated biotin compared to enzymatic digestion by proteinase K.

Animals↗
PubMedDate not supplied

Activity studies of immobilized subtilisin on functionalized pure cellulose-based membranes.

The activity of immobilized subtilisin BPN' on pure cellulose-based membrane support was investigated using site-directed and random immobilization approaches. The catalytic activity of site-directed immobilized subtilisin on pure cellulose fiber-based materials was found to be 81% of that in homogeneous solution, while that of randomly immobilized subtilisin was 27%. Pure cellulose membrane supports provided large surface areas for high enzyme loading without diffusional limitations. The activity of immobilized subtilisin on pure cellulose support was more than twice that on a modified polyether sulfone (MPS) membrane, which was attributed to the higher hydrophilicity of cellulose. Immobilized subtilisin maintained its initial activity for 14 days at 4 degrees C and 7 days at 24 degrees C. The immobilized enzyme could resist higher temperature and operate over a wider range of pH without loss of activity. This study showed that pure cellulose fiber-based membranes are well suited for enzyme immobilization and biocatalysis.

Affinity Labels↗