Membrane filtration affinity purification (MFAP) of dehydrogenases using cibacron blue.

The method for purification of biomolecules by a combination of affinity interactions and membrane filtration for separation of unwanted material has been found to be of interest for large-scale work. This study examines the suitability of silica nanoparticles as carriers in the process. Alcohol dehydrogenase and lactate dehydrogenases were chosen as target molecules to be purified. The binding capacity was found to be comparative to what is obtained for high-performance liquid chromatography (HPLC) packing material. Both binding and desorption of the enzymes were found to be effective. The limiting factor of the process was the filtration flow rate.

Flow-injection binding assays: a way to increase the speed in binding analyses.

A flow-injection analysis system was equipped with a small column containing immobilized concanavalin A. Pulses containing glucosides or glycoproteins were passed over the column, the lectin bound the carbohydrates. By using horseradish peroxidase as a labeled carbohydrate and letting it compete with other glucosides or mannosides a competitive binding assay for the latter was set up. When the enzyme activity had been evaluated, the column was rinsed and reconditioned, allowing a new assay to be run. To speed up the assay, substrates for the enzyme marker, peroxidase, were present in the perfusing buffer. A computerized evaluation of the absorbance peak allowed the time of the assay cycle to be reduced to 70 s. The sensitivity of this binding assay was fully comparable with those reported for other systems using the same reactants.

Immunoassays in aqueous two-phase systems.

Aqueous two-phase systems provide a novel and convenient method for separating bound from free fractions in a binding assay. The ease of automation of this type of procedure makes it particularly attractive for separations based on immobilized ligands or binders or on adsorbents.

A general study of the binding and separation in partition affinity ligand assay. Immunoassay of beta 2-microglobulin.

Partitioning in aqueous 2-phase systems was used to separate free and bound ligand in an immunoassay for beta 2-microglobulin. In order to get efficient separation in the phase system, the antibodies were modified to favour their partition in a different phase from that of antigen. However such modification of antibodies significantly decreased their binding capacity. This was overcome by using antibodies bound to previously modified staphylococci, which had proper partitioning behaviour. Alternatively, antibodies conjugated with biotin could be used in combination with modified avidin. This paper presents a method for the evaluation of data from immunoassays whereby 2-phase systems have been used to separate free and bound antigen.

Comparison between binding analyses performed by equilibrium dialysis and partitioning in aqueous two-phase systems exemplified by the binding of Cibacron Blue to serum albumin.

Partitioning in two-phase systems was used as a separation method in binding analyses and compared with equilibrium dialysis. A phase system was chosen to give partitioning conditions with the reactants in different phases. The interaction between serum albumin and Cibacron Blue F3G-A was used as a model system. The protein partitioned to the bottom phase and the ligand to the top phase in a phase system consisting of poly(ethylene glycol) and dextran. When both albumin and Cibacron Blue were present in the phase system, the degree of binding was observed as a translocation of ligand from the top to the bottom phase. An evaluation method that compensates for the amount of free reactant in the phase with the binding protein is presented.

Partition Affinity Ligand Assay (PALA) : A simple binding assay procedure.

In a competitive binding assay, the ligand to be quantified competes with a fixed amount of labeled ligand for the sites on a limiting amount of binding protein. The amount of label bound is therefore dependent on the ratio between native and labeled ligand. In a binding assay, one must separate the free ligands from bound. The better the separation, the higher the sensitivity of the assay. But effective methods are often laborious and time-consuming and thus we have developed a novel approach, the Partition Affinity Ligand Assay (PALA).

Application of partition affinity ligand assay (PALA) in quick test for quantitation of Staphylococcus aureus bacterial cells.

A novel immunochemical method for the quantitation of bacterial cells is described. The method, which is based on separation of bound from free reactants in aqueous two-phase systems has been studied both as direct and competitive binding assays, with a system consisting of intact cells of Staphylococcus aureus and human immunoglobulin G molecules. To achieve high resolution, one of the reactants was modified so that the two reactants, the bacterial cells and the immunoglobulin molecules, occurred in different phases of the phase system. When binding takes place, the partition of one of the reactants is changed. The degree of change is then correlated to the amount of reactant present. Using this method, cell numbers in the region 10(5)-10(7) can be quantified. An assay takes 40-90 min.

Partition affinity ligand assay (PALA). A new approach to binding assays.

A two-phase partitioning technique was used to separate free from bound labelled ligand in a binding assay. Efficient separation was accomplished by modifying one of the reactants in such a way that it partitions preferentially to one of the two phases. The biospecific interactions between the lectin concanavalin A and various carbohydrates and glycoproteins were investigated in this model study.