Biomedical subjects

G S Sittampalam

Publications and source records attributed to G S Sittampalam.

11 recordsLinked to original sources

PubMed1998

Conceptual and statistical issues in the validation of analytic dilution assays for pharmaceutical applications.

The discovery, research, and development of a pharmaceutical product relies on the availability of validated assays for assessing product characteristics and drug effects in vivo and in vitro. Development of a validated assay is a multifaceted activity that provides many interesting challenges for bioanalytical chemists and statisticians. In this paper, the similarity condition for fundamental validity of an analytic dilution assay is reviewed as a basic concept underlying the validation of assays for pharmaceutical applications. The distinction between the validity and the acceptability of an assay is considered in terms of the characteristics evaluated during four stages of validation. Recent guidelines on the validation of analytical procedures published by the U.S. Food and Drug Administration are appraised from a statistical perspective, and statistical issues in the validation process are discussed.

Chemistry, Pharmaceutical↗

PubMed1997

Synthesis and characterization of insulin-fluorescein derivatives for bioanalytical applications.

Human insulin was labeled with fluorescein isothiocyanate (FITC) and fully characterized to yield four distinct insulin-FITC species. High-performance liquid chromatography and electrospray mass spectrometry were used to determine the extent and location of fluorescein conjugation. By changing the reaction conditions (i.e., pH, time, and FITC/insulin ratio) the selectivity of the fluorescein conjugation was altered, and all conjugates could be separated. The isolated species of insulin-FITC were labeled at the following residues: A1(Gly), B1(Phe), A1(Gly)B1(Phe), and A1(Gly)B1(Phe)B29(Lys). All four insulin-FITC conjugates were then used to develop fluorescence polarization binding assays with monoclonal and polyclonal anti-insulin antibodies. The assay sensitivity differed between the conjugates depending on the site of modification (B1 > A1 > A1B1 > A1B1B29). Also, the type of antibody used had an important role in the binding of insulin-FITC conjugates. Finally, for the first time the biological activity of the four conjugates was demonstrated by an autophosphorylation assay. The positional substitution dramatically affected the biological activity, confirming insights into the residues responsible for the insulin binding region. The B1 conjugate was found to retain almost all biological activity while the A1 and A1B1 conjugates had approximately 10 times lower activity. The trisubstituted species (labeled at A1, B1, and B29) was determined to be least active.

Amino Acid Sequence↗

PubMed1997

High-throughput screening: advances in assay technologies.

Both isotopic and nonisotopic assay methodologies are employed in high-throughput screening for drug discovery. Recent advances in cell-based and in vitro biochemical assays will be reviewed, with special emphasis on detection technologies amenable to automated 'mix and read' procedures in high-throughput screening. A major trend is the advent of homogenous assay systems which employ fluorescence resonance energy transfer, fluorescence polarization, and fluorescence correlation spectroscopy. Cell-based assay systems have also become popular in high-throughput screens in which active compounds that directly modulate the disease target are identified. Colorimetric and amperometric methods have also been described recently, but are yet to be adapted widely in high-throughput screens.

Colorimetry↗

PubMed1996

Application of experimental design techniques to optimize a competitive ELISA.

This paper describes the application of experimental design techniques to optimize a sensitive ELISA for a hapten molecule with a calibration range of 0-1000 pg/ml. Ten factors that were expected to affect the assay performance were initially screened, followed by factorial experiments to delineate the effects of the critical factors identified at the screening stage. Assay performance was evaluated using a unique rating system based on standard curve reproducibility, assay detection limits and the use of desirability functions. This rating system allowed multiple responses to be evaluated simultaneously. It was found that the substrate incubation time and enzyme label lot played an important role, while dilutions of the enzyme label and the anti-hapten antibody showed significant interaction. These observations were in good agreement with optimal assay conditions based on historical data collected over a period of two to three years. Application of experimental design techniques enabled us to confirm the significance of the factors affecting the assay within a three month period, with a minimum number of experiments. In addition, information on interaction between factors were determined.

Animals↗

PubMed1991

Separation of antibody-antigen complexes by capillary zone electrophoresis, isoelectric focusing and high-performance size-exclusion chromatography.

The separation of antibody-antigen complexes by free-solution capillary zone electrophoresis (CZE) has been demonstrated. The antigen, a monoclonal antibody specific for the antigen, and the complex were well resolved. The entire separation was achieved in less than 10 min using on-column UV detection. The pI values for the three species were estimated separately by isoelectric focusing (IEF) experiments on polyacrylamide gels. Reasonably good agreement was found between the relative migration times measured by CZE and the pI values. Both IEF and high-performance size-exclusion chromatography of the antibody-antigen mixtures confirmed the formation of the complexes observed by CZE. This study demonstrates the utility of CZE as a new and complementary technique for the characterization of antibody-antigen complexes.

Antibodies, Monoclonal↗

PubMed1989

Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides.

The application of free solution capillary electrophoresis (FSCE) to the separation of protein and peptide mixtures is presented. Both qualitative and quantitative aspects of FSCE separations are considered. In addition, a brief introduction describing the separation principle behind FSCE separations and a discussion of electrophoretic mobility are included. The applications were chosen in order to highlight the selectivity of FSCE separations and to demonstrate applications of potential practical interest to the bioanalytical chemist. Comparison of FSCE relative to traditional analytical separation alternatives is stressed throughout. The examples are presented in three broad categories: protein separations, peptide separations, and the application of both to the analysis of recombinant protein products. In the first section, FSCE separations of peptide mixtures are presented which demonstrate the suitability of FSCE for the analysis of the purity of peptide samples, the homogeneity of peptide samples prior to sequencing, the identity of peptides by using electrophoretic mobility values, and the reduction of an intrachain disulfide bridge. In the second section, protein separations are presented that show the resolution of glycoproteins having the same primary structure and the separation of immune complexes from free unreacted antibody and antigen. In the final section, highly purified and well-characterized samples of biosynthetic human insulin (BHI), biosynthetic human growth hormone (hGH), and their derivatives were used to evaluate FSCE as a complement and/or alternative to conventional analytical separation techniques for the determination of purity and identity of biosynthetic human proteins. In addition, the quantitative aspects of FSCE analysis such as linearity of response, precision, and limit of detection were examined.

Chromatography, High Pressure Liquid↗

PubMed1989

Capillary zone electrophoresis of insulin and growth hormone.

The separation power of capillary zone electrophoresis was examined using highly purified and well-characterized biosynthetic human insulin, growth hormone, their derivatives, and related proteins. Mixtures of proteins were chosen to illustrate practical applications of this technique. Proteins differing slightly in structure, but equivalent in net charge, were not completely separated. Degradation of insulin by dilute acid treatment was followed by capillary zone electrophoresis, native polyacrylamide gel electrophoresis, and reversed-phase liquid chromatography. Excellent correlation was observed between these techniques. Simple equipment requirements and analysis times on the order of 10 min make capillary zone electrophoresis attractive for analytical protein separations.

Chromatography, High Pressure Liquid↗

PubMed1988

Visualization of multiple protein bands on the same nitrocellulose membrane by double immunoblotting.

A method has been developed which allows the simultaneous immunodetection of more than one type of protein on the same nitrocellulose membrane. This procedure does not require special labeling of samples or elution of antibodies from the membrane as do the alternatives cited in the literature (1,2). Proteins are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to the membrane before specific immunostaining with either peroxidase/4-chloro-1-naphthol or immunogold/silver staining. Antigen identity is visually determined by the formation of different-colored precipitates on the membrane. This innovation in protein blotting offers a savings in time and reagents as well as permitting identification of closely spaced bands with certainty.

Bacterial Proteins↗

PubMedDate not supplied

Evaluation of amino acid analysis as reference method to quantitate highly purified proteins.

The applicability of amino acid analysis for accurate quantitation of reference standard preparations of proteins has been evaluated. This approach is very useful since, in addition to absolute quantitative information, it also provides a measure of composition, partial identity, and purity in a single experiment. Comparisons with Kjeldahl nitrogen assay and/or UV measurements shows that amino acid analysis is reliable for the quantitation of small-to-medium size proteins in the molecular weight range of 6-22 kDa. For larger proteins such as immunoglobulins (150 kDa), amino acid analysis may "underestimate" the total protein concentration. These results also show the effect of recovery of individual residues on protein quantitation. As expected, the recovery of more than one stable residue could be used to calculate total protein content of samples, which is in good agreement with the results obtained by Kjeldahl nitrogen assay. However, the protein concentrations calculated from the total mass of the recovered residues appear to give relatively low estimates in almost all cases. Thus, it is concluded that amino acid analysis is an appropriate reference method only when stable residues are employed for quantitation, particularly for highly purified proteins of rDNA origin.

Amino Acids↗

PubMedDate not supplied

Protein assays for monoclonal antibodies.

Several techniques were evaluated for the quantitation of the total protein content of an IgG2a monoclonal antibody, KS1/4, and its deacetylvinblastine (DAVLB) conjugate. The UV assay is rapid, but it requires an extensive calibration of the response factor, and impurities may cause a high bias. Amino acid analysis (AAA) is an absolute method that has few interferences, but it requires evaluation of hydrolysis recovery factors. Kjeldahl nitrogen is very sensitive to minor impurities, and it requires a conversion factor to calculate percent protein. The Kjeldahl assay also is less precise (observed RSD of 3-4%) than the UV and AAA assays (observed RSD of 2-3% for both). The bicinchoninic acid assay, a representative of colorimetric assays, inherently requires comparison to a calibrated standard of the same material and tends to be less precise than the other assays. Thus, the UV and AAA assays are the techniques of choice for measurement of the total protein content of KS1/4 and its DAVLB conjugate.

Amino Acids↗

PubMedDate not supplied

Recommendations for establishment of reference standards for recombinant-DNA-derived proteins and polypeptides.

An integrated approach is presented for the establishment of purified protein and peptide reference standard materials suitable for both biological and chemical assays. Preliminary considerations, handling and storage conditions, and a variety of chemical methods for defining the reference standards are examined in detail. Of the chemical methods, liquid chromatography (LC), amino acid analysis, Kjeldahl protein assay, electrophoresis, and ion chromatography are key assays in determining the potency, purity, and identity of the reference standard preparation. Finally, the role of liquid chromatography in assessing reference standards for identity and chemical purity is examined and correlated with other methods.

Amino Acids↗