That T. Ngo

Kosmotropes enhance the yield of antibody purified by affinity chromatography using immobilized bacterial immunoglobulin binding proteins.

Abstract The yield of antibody purified using affinity chromatography on immobilized Protein A or Protein G was increased up to 5‐fold (500%) by including kosmotropic salts in the binding buffer. The binding buffer is used to equilibrate the affinity column before applying a sample to the column and also to dilute the sample prior to loading onto the affinity column to optimize conditions for a maximal binding of antibodies to affinity gels. In this study, the kosmotropic salts that were effective in greatly increasing antibody binding to Protein A included both inorganic and organic salts of ammonium; sodium; or potassium sulfate, phosphate, polycarboxylates; for example, succinate, citrate, isocitrate, N‐(2‐hydroxyethylene diamine triacetate (HEDTA), ethylene diamine tetraacetate (EDTA), and ethylene glycol‐O,O′‐bis(2‐aminoethyl)‐N,N,N′N′‐tetra acetate(EGTA). On an equal‐molar basis, the greater the number of carboxylic groups within the polycarboxylate molecule, the greater the increase in the yield of the purified antibody that was observed. The data show that kosmotropes can be used as effective additives to enhance the binding of immunoglobulins to Protein A or Protein G gels with a resultant increase in the yield of the purified antibodies. Thus, it appears that strongly hydrated anions (citrate, sulfate, and phosphate) and weakly hydrated cations (ammonium, potassium) increase the yield of antibody purified on either Protein A or Protein G affinity gels.

The use of a sulfide ion selective electrode to study O-acetylserine sulfhydrylase from germinating rapeseed

Abstract O-Acetylserine sulfhydrylase was partially purified from germinating seeds of Rape (Brassica napus L. var Target), and a sulfide ion specific electrode used to study the properties of this enzyme. It was demonstrated that the sensitivity of the electrode to sulfide ion concentration was not affected by pH, or by various concentrations of cysteine, sulfate, sulfite, and acetate. Kinetic studies showed that the Km for O-acetyl- l -serine was 1.74 × 10−6 m , while the Km for sulfide was 4.3 × 10−4 m . The implications of the data, together with the methods used to obtain them, are discussed.

Peroxidase in Chemical and Biochemical Analysis

The structure, reaction kinetics and mechanism, substrate specificity, stability and chromogenic, and fluorogenic and chemiluminogenic substrates of peroxidase are reviewed. The applications of peroxidase in the quantitative analysis of hydrogen peroxide, clinically important metabolites via oxidase-coupled reactions, as a label in quantitative immunochemical and DNA hybridization reactions and in immuno-blot and immuno-histochemical investigations will also be reviewed.

Flow injection analysis of lactose using covalently immobilized β-galactosidase, mutarotase, and glucose oxidase/peroxidase on a 2-fluoro-1-methylpyridinium salt-activated fractogel support

Milk samples were analyzed for their lactose content using flow injection analysis and incorporating immobilized beta-galactosidase or beta-galactosidase/mutarotase and glucose oxidase/peroxidase bioreactors. These enzymes were immobilized, under mild conditions, on to a 2-fluoro-1-methylpyridinium salt-activated Fractogel support. The use of a phosphate buffer (0.15 M) was found to facilitate the rapid mutarotation of alpha-D-glucose and hence could obviate the need for the more expensive mutarotase. The chromogenic agents of choice for monitoring the reaction were 3-methyl-2-benzothiazolinone hydrazone and 3-dimethylaminobenzoic acid. Linearity was observed over the concentration range 16-160 micrograms/ml using lactose standards (r = 0.996). Between 30 and 40 milk samples/h can be analyzed. Comparisons are made with existing HPLC and alkaline methylamine methods for a range of milk matrices. The FIA method consistently gives the lowest standard deviations and coefficient of variation for the various milk matrices analyzed.

Affinity Chromatographic Purification of Antibodies

Abstract Affinity chromatographic methods for purifying antibodies are reviewed. Topics reviewed include (a) the matrices used in the preparation of the affinity supports; (b) the chemistries commonly used to attach affinity ligands directly on hydroxyl‐carrying supports and indirectly through the use of bifunctional agents to crosslink the affinity ligands to the supports; (c) methods for detecting ligand leakage; (d) macromolecular affinity ligands; (e) low molecular weight peptidyl affinity ligands; (f) low molecular weight non‐peptidyl affinity ligands; (g) optimal conditions for achieving improved product yield; and (h) optimal elution systems that minimize the denaturation of the purified antibodies.

Ligand Displacement Immunoassay

Abstract Ligand displacement immunoassay (LDI) is an immunoassay that uses an immunocomplex consisting of immobilized antibodies that have been presaturated with labeled analyte ligand, or conversely, it can use a complex of immobilized analyte ligands that are prebound with corresponding labeled antibodies. The complex of antibody–antigens dissociates when it is exposed to analytes. Thus, the labeled reagents are displaced by the analyte in the test sample. This review focuses on three aspects of immunochemistry that are germane to the development of an LDI. The interaction of antibody with antigen, the solid phase and methods used for the immobilization of immuno‐reagents and the labels and labeling methods used in LDI are discussed in details.

Improved spectrophotometric determination of lactose in milk using flow-injection analysis

Abstract Milk samples were analysed spectrophotometrically for their lactose content using flow-injection analysis. The methodology is based on the carmine colour that develops (λMax = 540 nm) when lactose reacts with alkaline methylamine (pH 12.7). Lactose concentrations as low as 0.6 mg ml−1 can be readily detected and up to 40 samples can be analysed per hour. Excellent correlations (r > 0.999) are obtained when the results are compared with the batch and liquid chromatographic methods for lactose determination. Interference from maltose, if present, should be eliminated by pretreatment with maltase.

Ligand Displacement Fluorescence Immunoassay for Gentamicin and Human IgG

Abstract A ligand displacement fluorescence immunoassays (LD‐FIA) for serum gentamicin and for human IgG have been developed. The LD‐FIA for gentamicin used affinity purified anti‐gentamicin antibodies immobilized onto ¼ inch polystyrene balls by adsorption. The immobilized anti‐gentamicin antibodies were presaturated with fluorescein labeled gentamicin to form an immuno‐complex consisting of fluorescein labeled gentamicin bound to anti‐gentamicin antibodies coated balls (F‐GAB). F‐GAB released the fluorescein labeled gentamicin upon contact with the gentamicin containing sample. The amount of released fluorescein labeled gentamicin is directly proportional to the amount of gentamicin in the test serum. LD‐FIA for human IgG was constructed in a similar way using immobilized antihuman IgG and fluorescein labeled human IgG. LD‐FIA is a rapid and convenient immuno‐analytical technique that requires minimal manipulation, (i.e., it only requires the addition of diluted sample to one reagent—the F‐GAB). The technique can be used for determining either small molecules or macromolecules as demonstrated here. The authors acknowledge the excellent technical work of R. C. Wong.

Multivariate statistical {using factor and discriminant} analyses of some selected chemical and physical freshness indicators of fish [Scomberomorus brasiliensis (carite)] muscle tissue samples

Abstract Seventy-eight samples of [ Scomberomorus brasiliensis (carite)] muscle tissue were analyzed for % fat, hypoxanthine content (determined by a flow injection method), total volatile acids and bases, total bacterial count, and textural parameters (firmness, hardness, fracturability, cohesiveness, chewiness, and elasticity) were subjected to discriminant and factor analyses. The apparent error rate obtained by discriminant analysis with all the parameters in the analysis was about 5.6%. However, classification was not improved (apparent error rate 6.9%) when cohesiveness and chewiness were excluded from the analysis. The stepwise methods of selection (Wilks and Minresid) H×FI ranked second, and fourth when the Mahal and Maxminf methods were used. In the factor analysis, using the principal component analysis, five factors were extracted. Factor loadings relate factor 1 (tastiness) with hypoxanthine concentration, total volatile acids and bases: factors 2, 3, 5 (texture) with the textural attributes; and factor 4 (rancidity) with the % fat content. Rotation of the reference axes showed that factors 1, 2 and 5 remained unchanged, while factors 3 and 4 were interchanged.

In‐Tandem Column Arrangement for Rapid Purification of IgG using a Blue Avid Gel P and an Aza‐Arenophilic Affinity Gel

Abstract An in‐tandem study comprising two affinity chromatographic columns, Blue Avidgel P (for the selective removal of albumin) and an aza‐arenophilic gel (Gel A) containing a nitrogen capping nucleophile (for the selective removal of IgG), was conducted to obtain high purity IgG from serum. The results demonstrated that highly pure IgG (>97%) can be obtained from the phenethylamine capped aza‐arenophilic gel column after the serum was passed through the Blue Avidgel P column in contrast to the 38% IgG purity obtained when the serum was passed through a Gel A column alone under similar conditions. Further studies show that IgG selectivity is not dependent on the nature of the support material used in the synthesis of these aza‐arenophilic gels since the synthesis of aza‐arenophilic gels using either Sepharose or Fractogel yield similar results.