Immunoglobulins and virus antibody titers: of past needs, current requirements, and future options.

BACKGROUND: Immunoglobulins (Igs) have been in clinical use for almost 70 years, and early on were also used in conjunction with exposure to the measles virus or polio virus. The US regulations that describe functional Ig lot release thus require the demonstration of minimum antibody titers against these two viruses, although the use of vaccines has now dramatically reduced their incidence. The lower clinical importance of these viruses raises the question of whether other virus antibodies might be more informative for patients with immunodeficiency. STUDY DESIGN AND METHODS: A literature survey was conducted to identify viruses of potential clinical concern for people with immunodeficiency. The viruses selected have stable seroepidemiology and associated functional antibody assays. As a result, neutralizing antibody titers to human adenovirus 5 (HAdV5), respiratory syncytial virus (RSV) serotypes A and B, and human parainfluenza virus 3 (hPIV3) were determined in Ig lots produced from plasma collected in either the United States or the European Union. RESULTS: The virus antibody titers measured were high and consistent among the Ig lots tested. Use of either US- or EU-derived plasma as starting material resulted in equivalent virus antibody titers, with the exception of RSV serotype B, for which a lower titer was seen in EU plasma-derived Ig lots. CONCLUSION: With the significant decline in measles virus and polio virus circulation, and even their potential eradication, measurement of antibody titers against other viruses in Ig products may be more informative for functional lot release testing.

Applications of isothermal titration calorimetry in protein science.

During the past decade, isothermal titration calorimetry (ITC) has developed from a specialist method for understanding molecular interactions and other biological processes within cells to a more robust, widely used method. Nowadays, ITC is used to investigate all types of protein interactions, including protein-protein interactions, protein-DNA/RNA interactions, protein-small molecule interactions and enzyme kinetics; it provides a direct route to the complete thermodynamic characterization of protein interactions. This review concentrates on the new applications of ITC in protein folding and misfolding, its traditional application in protein interactions, and an overview of what can be achieved in the field of protein science using this method and what developments are likely to occur in the near future. Also, this review discusses some new developments of ITC method in protein science, such as the reverse titration of ITC and the displacement method of ITC.

Advances in the analysis of isothermal titration calorimetry data for ligand-DNA interactions.

Isothermal titration calorimetry (ITC) is a well established technique for the study of biological interactions. The strength of ITC is that it directly measures enthalpy changes associated with interactions. Experiments can also yield binding isotherms allowing quantification of equilibrium binding constants, hence an almost complete thermodynamic profile can be established. Principles and application of ITC have been well documented over recent years, experimentally the technique is simple to use and in ideal scenarios data analysis is trivial. However, ITC experiments can be designed such that previously inaccessible parameters can be evaluated. We outline some of these advances, including (1) exploiting different experimental conditions; (2) low affinity systems; (3) high affinity systems and displacement assays. In addition we ask the question: What if data cannot be fit using the fitting functions incorporated in the data-analysis software that came with your ITC? Examples where such data might be generated include systems following non 1:n binding patterns and systems where binding is coupled to other events such as ligand dissociation. Models dealing with such data are now appearing in literature and we summarise examples relevant for the study of ligand-DNA interactions.

Improving method capability of a drug substance HPLC assay.

The assay of a drug substance (DS) is one of the tests required to confirm the active pharmaceutical ingredient (API) quality at release. In the past, usually volumetric titration methods were performed, that were precise, but often non-specific. Nowadays specific chromatographic assay procedures are preferred. However, high performance liquid chromatographic (HPLC) methods, the way they are usually executed, tend to be less precise and have a larger total method variation compared to titration methods. The capabilities of fully validated titration and HPLC assay methods were determined and compared. It was studied which factors had the largest effects on the capability of chromatographic HPLC methods in order to improve their precision and precision-to-tolerance ratio. This was done using multiple Gage R&R (repeatability & reproducibility) studies and an experimental design approach. The investigations showed that it was feasible to define an HPLC method with a similar capability as the titration method. The most important factor determining the precision was demonstrated to be higher sample and reference material weights. When low weights are to be used, increasing the number of sample preparations and the number of reference solutions may enhance the method capability.

Determination of promethazine hydrochloride and its preparations by highly accurate nephelometric titration.

A highly accurate nephelometric titration for the determination of promethazine hydrochloride and its preparations was presented. The titration operating conditions were studied and the solubility product constant of promethazine tetraphenylboron precipitation was determined. The result of the titration is comparable to those of control experiments. The proposed method has been found to be accurate, precise, specific and linear.

Processive action of cellobiohydrolase Cel7A from Trichoderma reesei is revealed as 'burst' kinetics on fluorescent polymeric model substrates.

Reaction conditions for the reducing-end-specific derivatization of cellulose substrates with the fluorogenic compound, anthranilic acid, have been established. Hydrolysis of fluorescence-labelled celluloses by cellobiohydrolase Cel7A from Trichoderma reesei was consistent with the active-site titration kinetics (burst kinetics), which allowed the quantification of the processivity of the enzyme. The processivity values of 88+/-10, 42+/-10 and 34+/-2.0 cellobiose units were found for Cel7A acting on labelled bacterial cellulose, bacterial microcrystalline cellulose and endoglucanase-pretreated bacterial cellulose respectively. The anthranilic acid derivatization also provides an alternative means for estimating the average degree of polymerization of cellulose and, furthermore, allows the quantitative monitoring of the production of reducing end groups on solid cellulose on hydrolysis by cellulases. Hydrolysis of bacterial cellulose by cellulases from T. reesei revealed that, by contrast with endoglucanase Cel5A, neither cellobiohydrolases Cel7A nor Cel6A produced detectable amounts of new reducing end groups on residual cellulose.