Adriaan Bantjes

Colloidal carbon particles as a new label for rapid immunochemical test methods: quantitative computer image analysis of results.

Colloidal carbon particles can serve as label in sol particle immunoassays. The universal applicability of these particles in qualitative and (semi)quantitative immunoassays has been demonstrated. Sol particle and/or dipstick immunoassays, not yet optimized in terms of sensitivity, are discussed. The colloidal label has been used successfully in a mouse immunoglobulin isotyping kit. Human serum albumin spotted onto nitrocellulose in a concentration range of 7.8 to 1000 ng could be detected using anti-albumin antibody absorbed onto colloidal carbon particles. It was also possible to perform a competitive assay with this conjugate for a concentration range of free human serum albumin varying from 0.25 to 6.75 micrograms. The Kunitz-type trypsin inhibitor from soybean was determined by a colloidal carbon based immunoassay in a range of 2.5 to 160 ng. In this assay, free and colloidal carbon-bound inhibitor competed for binding specific antibodies spotted onto a nitrocellulose membrane. An image- and data-processing procedure has been developed that enables a rapid and simple quantification of colloidal carbon sol particle immunoassays. The average grey level of a spot is taken as a measure for quantitative purposes. This so-called Sol-particle Image Processed ImmunoAssay (SIPIA) procedure is equally well applicable to assays using other colloidal particles.

Effects of Tween 20 on the desorption of proteins from polymer surfaces

The effects of Tween 20 on the desorption of proteins from polyethylene and polyurethane were studied, using single protein solutions of the human proteins fibrinogen (Fb), immunoglobulin G (IgG), serum albumin (HSA), high density lipoproteins (HDL), and plasma. The surfactant may partly or even completely desorb the proteins, depending on the type of polymer and protein. About 40% of adsorbed HSA and 80% of adsorbed HDL from the corresponding single protein solutions were desorbed by Tween 20 from polyethylene, whereas Tween 20 had a small effect on the desorption of adsorbed Fb and IgG under the same conditions. However, the desorption of Fb and IgG by Tween 20 was much higher in the case of a diluted plasma solution compared to a pure protein solution. These findings may be explained by the differences of the interaction strengths between polymers and the adsorbed proteins. The displacement of HSA from polyethylene by Tween 20 occurred in the first few minutes and did not increase in time. It was also observed that preadsorbed Tween 20 was able to prevent in a large extent the adsorption of HSA onto polyethylene. Thus, the effect of Tween 20 on the desorption of protein is due to either the displacement of protein or prevention of protein adsorption onto the surfaces.

Iron(III) chelating resins-I. Preparation and properties of Sepharose-desferrioxamine gels

For the removal of iron(III), Sepharose-desferrioxamine gels were prepared by the coupling of CNBr-activated Sepharose with desferrioxamine (DFO) at pH 7.8-8.3. DFO densities of the gels were 12-23 mumol/ml gel with iron(III) chelating capacities of 8.5-18 mumol/ml gel. The Sepharose-DFO gels with a high affinity for iron(III) were used for the removal of iron(III) from aqueous iron(III) solutions, wine, milk and whey.

Iron(III) chelating resins II. 3-Hydroxy-4(1H)-pyridinones-sepharose gels.

3-Hydroxy-4(1H)-pyridinones(HP)-Sepharose gels were prepared to study their iron(III) chelating properties. As ligands, derivatives of 3-hydroxy-4(1H)-pyridinone were coupled to CNBr-activated Sepharose gels. HP-Sepharose gels were obtained with HP densities of 23-28 mumol/ml gel and iron(III) chelating capacities of 19-23 mumol/ml gel at pH 6.8. From preliminary experiments, it was found that with the gels 19-27% iron could be removed from milk. In addition, 74% of iron(III) was removed from 100% iron(III) saturated lactoferrin within 24 h at pH 6.8 in the presence of citrate and a Sepharose gel, onto which 1-(2-aminoethyl)-3-hydroxy-2-methyl-4(1H)-pyridinone had been immobilized as a ligand. The properties of the gels make them potentially useful as water-insoluble iron(III) chelating agents.

Large-scale purification of factor VIII by affinity chromatography: optimization of process parameters

The optimization of a new process for the extraction of human coagulation factor VIII (FVIII) from plasma with the tailor-made affinity matrix dimethylamino-propylcarbamylpentyl-Sepharose CL-4B (C3-C5 matrix) is described. First, plasma is applied to DEAE-Sephadex A-50 anion exchanger in order to separate a number of proteins, including coagulation factors II, IX and X (prothrombin complex), from FVIII. Subsequently, the unbound fraction of the ion exchanger, containing FVIII, is contacted with the C3-C5 affinity matrix. Optimization of the FVIII affinity chromatographic procedure is accomplished in terms of the ligand density of the matrix, adsorption mode (batch-wise versus column-wise adsorption and matrix to plasma ratio), and conditions of pH and conductivity to be applied on washing and desorption. In scale-up experiments, by processing 20 l of plasma, the recovery (340 U VIII:C/kg plasma) and the specific activity (s.a.) (1.2 U VIII:C/mg protein) are better than those obtained by cryoprecipitation (recovery 300 U VIII:C/kg plasma, s.a. 0.3 U VIII:C/mg protein). The newly developed process using the specially designed C3-C5 affinity matrix has potential application in the process-scale purification of FVIII.

Structure of a complex of iron(III) with a crosslinked copolymer of 1-(β-acrylamidoethyl)-3-hydroxy-2-methyl-4(1H)-pyridinone and N,N-dimethylacrylamide

Chelation is of great significance in many chemical and biomedical systems such as the isolation of Fe3+ or A13+, Ga3+ and actinides,-3 the treatment of iron ~ v e r l o a d , ~ ~ and the inhibition of bacterial Recently, some of us reported the synthesis and properties of several iron(II1) chelating resins with immobilized natural or synthetic iron(II1) chelators. One of these resins (AHMP-DMAA) was prepared by the copolymerization of l-(/3-acrylamidoethyl)-3-hydroxy-2-methyl-4(lH)pyridinone (AHMP) and N,N-dimethylacrylamide (DMAA) in the presence of a crosslinking agent. The structures of the monomer AHMP and of the AHMPDMAA resin are represented in Figure 1. This resin exhibits a high affinity and selectivity for iron(II1) and was found to be chemically stable and reusable. The resin has been used in the removal of iron from iron-binding proteins, milk, wine, or beer, for the inhibition of bacterial growth, and for iron detoxification of poisoned human p1a~ma.I~ Furthermore, the stability constant of its iron(II1) complex was determined recently, and was found to be even higher than that of the corresponding monomer c0mp1ex.I~ Although the AHMP-DMAA resin can be effectively applied for iron(II1) chelation, the structure of the resin-iron(II1) complex so far has been unknown. Iron binding studies with soluble iron(II1) chelating ligands containing the same groups as present in AHMP, showed tha t a ligand/iron(III) ratio of 3 is observed at a p H range of 5-11, with a n octahedral coordination around the iron(II1) i ~ n . * ~ ~ Ho wever, i t is possible tha t differences occur in the coordination mode for the soluble iron(II1) chelate and the resiniron(II1) complex. This article describes the results of a study about the coordination structure using infrared spectroscopy (IR), electron paramagnetic resonance (EPR) , and diffuse reflectance spectroscopy (electron spectroscopy).

Colloidal carbon particles as a new label for rapid immunochemical test methods: Quantitative computer image analysis of results

Colloidal carbon particles can serve as label in sol particle immunoassays. The universal applicability of these particles in qualitative and (semi)quantitative immunoassays has been demonstrated. Sol particle and/or dipstick immunoassays, not yet optimized in terms of sensitivity, are discussed. n nThe colloidal label has been used successfully in a mouse immunoglobulin isotyping kit. Human serum albumin spotted onto nitrocellulose in a concentration range of 7.8 to 1000 ng could be detected using anti-albumin antibody adsorbed onto colloidal carbon particles. It was also possible to perform a competitive assay with this conjugate for a concentration range of free human serum albumin varying from 0.25 to 6.75 ?g. The Kunitz-type trypsin inhibitor from soybean was determined by a colloidal carbon based immunoassay in a range of 2.5 to 160 ng. In this assay, free and colloidal carbon-bound inhibitor competed for binding specific antibodies spotted onto a nitrocellulose membrane. n nAn image- and data-processing procedure has been developed that enables a rapid and simple quantification of colloidal carbon sol particle immunoassays. The average grey level of a spot is taken as a measure for quantitative purposes. This so-called Sol-particle Image Processed ImmunoAssay (SIPIA) procedure is equally well applicable to assays using other colloidal particles.

Coating and Blood Compatibility of Amberlite XAD-4

Two different coatings for XAD-4 were investigated. XAD-4 was coated either with cellulose acetate (CA) by the Wurster technique, or with a synthetic polyelectrolyte (PLE) which had anticoagulant activity by adsorption and cross-linking induced by cobalt-60 gamma radiation. With regard to amino acids, short-chain fatty acids, octopamine and bile acids, CA-coated XAD-4 showed in vitro the same adsorption properties as the uncoated resin. In PLE-coated XAD-4, the adsorption of vitamin B12 and phenylalanine was about 95% of that of uncoated resin. In vitro blood compatibility tests revealed a marked reduction of thrombocyte losses after 3 h by PLE-coated XAD-4 (loss of 20%) and CA-coated XAD-4 (loss of 50%) compared to uncoated XAD-4 (loss of 90%).

Conversion of Creatinine in the Presence of Activated Carbon

Activated carbon (a.c.) coated with different types of polymer has been used as a sorbent in haemoperfusion techniques for the removal of toxic products (drug overdose, metabolites, etc). The adsorption capacity of different types of a.c. for creatinine, uric acid, etc, has to be determined to select the types of a.c. best suited for specific purposes. Dedrick et al. (1967) have studied the adsorption of uric acid onto the a.c. Columbia NXC (Union Carbide Corporation). They reported that uric acid in contact with a.c. was converted at least partially into yet unknown products. We report on the conversion of creatinine in the presence of a.c. (Norit RBX1) and oxygen.

Synthesis, Structure, and Properties of Polymers Based on Pivalolactone

Abstract Since the introduction of poly(ethy1ene terephthalate) (PET) and polyamide 6, many attempts have been made to synthesize other polymers with a large variety of properties. One of the polymers which has been studied extensively is polypivalolactone (PPVL), formed by ring-opening polymerization of pivalolactone (PVL). PPVL is a highly crystalline aliphatic polyester (≥75% crystallinity) with mechanical properties comparable with those of PET and polyamide 6. Although the technology and expertise to prepare PPVL on a commercial scale was already available in the early 1970s, most of the projects concerning PPVL, especially in fiber applications, were discontinued on the basis of economic considerations.