Chromatographic study of the adsorption kinetics of albumin on monoclonal and polyclonal immunoadsorbents

Summary High-performance liquid chromatography (HPLC) has been used to study the adsorption kinetics of proteins on immunoadsorbents. The adsorption rate constant of human serum albumin (HSA) on monoclonal and polyclonal anti-HSA antibodies immobilized on a silica HPLC support was determined by saturating the column with repeated pulse injections. Studies on polyclonal immunodsorbents of different capacities enable evaluation of the contribution of transport to the binding sites. The adsorption properties of two different monoclonal anti-HSA antibodies immobilized on a chromatographic support were characterized by different approaches. The location of the epitope on the HSA molecule was determined by enzyme-linked immunoassay (ELISA) with albumin fragments. The chromatographic method was used to determine the column capacity and the adsorption rate constant of HSA on the immunoadsorbent. To compare the affinity of the antibodies for the antigen, an indirect ELISA method was used to determine the equilibrium constant of antigen-antibody association in solution Presented at the 21^st ISC held in Stuttgart, Germany, 15^th–20^th september, 1996.     

Immunoenzymatic determination of antibody-bound soluble antigens ofTrypanosoma cruzi

Anti-Major Cathodic Antigen (MCA) monospecific immunoglobulins ofT. cruzi have been used for the preparation of polyamide-linked immunoadsorbents. A high proportion of serologically positive patients with Chagas disease show the presence of a soluble antigen complexed with human immunoglobulins, which complex binds to those immunoadsorbents as determined by a double sandwich reaction and a peroxidase final determination.     

吸附型“人工肝”辅助材料的制备及其性能研究Ⅱ.免疫吸附剂的制备及其吸附性能

本文介绍以醋酸乙烯酯为单体,二乙烯苯为交联剂制得大孔共聚物;经皂化、活化后,偶联IgG制得免疫吸附剂。讨论了不同活化剂对偶联配体的影响,观察其对人血清中的HBsAg的吸附性能。     

Immunoaffinity purification of subcellular particles and organelles

The application of immunoaffinity techniques to subcellular fractionation is reviewed and the basic principles underlying the various methods that have been successfully employed, identified. The requirement for organelle-specific antigens and high-avidity antibodies is discussed, as is the widespread use of indirect immunoadsorbents. Approaches for the optimization of immunoaffinity-based subcellular fractionation are suggested.     

Compared stability of Sepharose-based immunoadsorbents prepared by various activation methods.

During the use of chromatographic supports for the purification of proteins or the selective removal of substances by immunoaffinity, leakage of the antibodies immobilized on the matrix is systematically observed. When the cleansing of blood plasma by extracorporeal circulation is concerned, it is of prime importance that the immunoadsorbents exhibit an extensive chemical stability over the whole range of experimental conditions. To study and minimize this leakage, a matrix, Sepharose CL-4B, was activated by various chemical reagents and coupled to goat anti-apolipoprotein B polyclonal antibodies. Immunoadsorbents thus prepared were compared with those obtained earlier by cyanogen bromide activation. It turns out that divinyl sulphone- and tresyl chloride-activated supports lead to similar results in terms of coupling yield and adsorption capacity, but to a significant reduction in released antibodies.     

Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome

Guillain-Barré syndrome is a postinfectious, autoimmune neuropathy resulting in neuromuscular paralysis. Auto-antibodies, often induced by bacterial infection, bind to human gangliosides possessing monosialoside and diasialoside epitopes and impair the function of nerve junctions, where these ganglioside structures are highly enriched. Truncated gangliosides representive of GD3, GQ1b and GM2 epitopes have been synthesized as methyl glycosides and as a glycosides of an eleven carbon tether. The synthetic oligosaccharide ligands are structural mimics of these highly complex ganglioside epitopes and via their ability to neutralize or remove auto-antibodies have the potential for therapy, either as soluble blocking ligands administered systemically, or as immuno-affinity ligands for use as extracorporeal immunoadsorbents.     

Leakage of immobilized IgG from therapeutic immunoadsorbents

In developing therapeutic immunoadsorbents (IAs), antibodies (IgG molecules) covalently immobilized on porous carriers, a leak of IgG was determined both in the storage test with buffers at 25 and 4 degrees C and in contact with plasma at room temperature (RT). The amount of antibody released from therapeutic IAs must be minimized to avoid side effects during treatment. The amount of IgG released was a. Dependent on the amount of IgG immobilized b. Much greater with CNBr-activated Sepharose 4B, or Formyl-Cellulofine as a support material than with aminopropyl CPG (controlled pore glass) c. Found to yield again during another storage in buffers after the IAs were washed and their buffers replaced with fresh ones and d. Decreased after the IAs were treated with glutaraldehyde (GA) solutions. Whereas treating the IAs with GA solutions significantly reduced the amount of IgG released, it caused some deterioration of the adsorption characteristics of the IAs. An irradiation dose of 2.5 Mrad as a crosslinking procedure also reduced the amount of IgG released; its effect was comparable to that of 0.025% GA, the lowest concentration used.     

Ligand location and biochemical productivity of silica-based immunoaffinity adsorbents.

Ligand location within particles, detected by immunogold labelling, was shown to influence the biochemical productivity of a silica-based solid phase, Sorbsil C-500, using a model ligand-biomolecule system (immobilised human immunoglobulin G-anti-human immunoglobulin G monoclonal antibody). The distribution of the ligand was in turn affected by the initial ligand challenge used to prepare the immunoadsorbents. Maximal productivity was achieved with adsorbents prepared with an initial challenge of about 3 mg human immunoglobulin G per ml: the ligand in these cases was shown to be more uniformally distributed within the adsorbent particles than adsorbents, exhibiting low productivity, prepared with either low (1 mg/ml) or high (9 mg/ml) concentrations of human immunoglobulin G. The ligand in the latter was restricted to the periphery of the particles.     

Affinity extraction of emerging contaminants from water based on bovine serum albumin as a binding agent

Affinity sorbents using bovine serum albumin as a binding agent were developed and tested for the extraction of environmental contaminants from water. Computer simulations based on a countercurrent distribution model were also used to study the behavior of these sorbents. Several model drugs, pesticides, and hormones of interest as emerging contaminants were considered in this work, with carbamazepine being used as a representative analyte when coupling the albumin column on‐line with liquid chromatography and tandem mass spectrometry. The albumin column was found to be capable of extracting carbamazepine from aqueous solutions that contained trace levels of this analyte. Further studies of the bovine serum albumin sorbent indicated that it had higher retention under aqueous conditions than a traditional C₁₈ support for most of the tested emerging contaminants. Potential advantages of using these protein‐based sorbents included the low cost of bovine serum albumin and its ability to bind to a relatively wide range of drugs and related compounds. It was also shown how simulations could be used to describe the elution behavior of the model compounds on the bovine serum albumin sorbents as an aid in optimizing the retention and selectivity of these supports for use with liquid chromatography or methods such as liquid chromatography with tandem mass spectrometry.     

EARLY CHANGES IN LIGHT-IRRADIATED SOLUTIONS OF BILIRUBIN: A SPECTROPHOTOMETRIC ANALYSIS

Abstract— Early changes in irradiated solutions of bilirubin were evaluated by spectrophotometric techniques. By absorbance difference (AD) spectroscopy, the photoproducts formed in solutions of bilirubin combined with human serum albumin were found to differ from those formed in naturally or artificially jaundiced rat serum, bovine serum albumin solutions, or chloroform or carbon tetrachloride solutions.
With a technique based on variable path-length compensation for bilirubin loss (compensated absorbance difference or CAD spectroscopy) the approximate shapes of photoproduct absorption spectra could be evaluated. Chloroform and bovine serum albumin solutions produced single-peaked products absorbing at slightly shorter wavelengths than bilirubin. Human serum albumin solutions produced a twin-peaked chromophore absorbing most strongly near 490 nm.     

Glass electrode measurements of sodium in albumin solutions

Measurements of the sodium level of albumin solutions and their ultrafiltrates were made with commercially available sodium-sensitive glass electrodes. The potential of the electrode was found to vary considerably with the depth of immersion. The potentials of albumin solutions which were allowed to remain at room temperature or higher for any length of time were found to increase, and in time gave unrealistic levels of sodium activity. An albumin solution stored in a refrigerator for a month did not show this effect. The sodium levels of albumin solutions as determined by the electrode were lower than the flame photometric values for the same solutions.     

Albumin‐bound low molecular weight thiols analysis in plasma and carotid plaques by CE

We describe a new method for the quantification of low molecular weight thiols, as homocysteine, cysteine, cysteinylglycine, glutamylcysteine and glutathione bound to human plasma albumin. After albumin isolation and purification by SDS‐PAGE, thiols were freed from protein with tri‐n‐butylphosphine and successively derivatized with 5‐iodoacetamidofluorescein. Samples were then injected and quantified in about 18 min by CE with laser induced fluorescence detection. Precision tests indicate a good repeatability of the method both for migration times (RSD<0.63%) and areas (RSD<2.98%). The method allows to measure all five low molecular weight thiols released from just 3 μg of albumin thus improving the other described methods in which only three or four thiols were detected. Due to the elevated sensitivity (LOD of 0.3 pM for all thiols), also low molecular weight thiols bound to albumin filtered in tissues could be quantified.     

Isolation and purification of cat albumin from cat serum by copper ion affinity chromatography: further analysis of its primary structure

Proteins, regardless of their origin, have to be highly purified, particularly from the immunochemical point of view, if they are to be used to study their allergenicity. It is shown that cat albumin, a highly potent allergen for cat-sensitive humans, can be isolated and purified from cat serum using immobilized metal ion affinity chromatography (copper ions) instead of a salting-out process or precipitation with alcohol, techniques generally used for the preparation of serum proteins. During the process described, immunoglobulins are concomitantly isolated in a relatively pure form. Cat albumin amino acid composition and sequence were analysed after an ultimate purification by ion-exchange chromatography. The highest homology (greater than 80%) was found with the rat serum albumin.     

A study of embolizing materials for chemo-embolization therapy of hepatocellular carcinoma: embolic effect of cisplatin albumin microspheres using chitin and chitosan in dogs, and changes of cisplatin content in blood and tissue.

Hepatic artery of dogs was embolized with cisplatin (CDDP) albumin microspheres containing chitin and chitosan to investigate the in vivo CDDP release kinetics from CDDP albumin microspheres, the CDDP cumulative characteristics in the liver, and the influence of microsphere administration on hepatic tissue. Results showed that changes in blood CDDP content were dependent on CDDP albumin microsphere type and that release kinetics were better sustained when chitin was added to the microspheres or when the microspheres were treated with chitosan. In particular, the administration of CDDP in the chitin-containing CDDP chitosan albumin microspheres showed a blood CDDP content of approximately 0.26 micrograms Pt/ml 14 d after administration. The administration of chitin-containing or chitosan treated CDDP microspheres showed a CDDP content in the hepatic tissue of 0.14 to 0.23 micrograms Pt/g 28 d after administration. They also showed better control of CDDP release than those without chitin or chitosan treatment. No CDDP influence on hepatic tissue was observed. We conclude that, even in vivo, chitin and chitosan are effective embolic materials.     

Binding affinities of commonly employed sensitizers of viral inactivation

Methylene blue (MB), riboflavin (RB) and psoralen sensitizers (4' aminomethyl-4,5',8-trimethylpsoralen [AMT] and derivatives) are under study as sensitizers of viral inactivation of blood products such as plasma proteins, platelets and red cells, all of which lack genomic nucleic acid. To predict where these sensitizers accumulate in viruses and in cells, their relative affinities for calf thymus DNA, neutral and negatively charged phospholipids and albumin were determined by dialysis. MB has a strong affinity for nucleic acid and negatively charged phospholipid, but little affinity for albumin or neutral phospholipid. RB has modest affinity for nucleic acid and little affinity for albumin or either phospholipid. AMT has substantial affinity for nucleic acid, neutral and negatively charged phospholipids and albumin. Neither AMT nor RB binds to poly G, although MB has some affinity for this polymer. Evidence of association of RB with guanosine monophosphate, adenosine monophosphate and tryptophan methylester hydrochloride in PBS buffer in the presence and absence of formamide was obtained from nonlinear Stern-Volmer plots and shifts in the ground state absorption spectrum of RB.     

Development of a negligible depletion-solid phase microextraction method to determine the free concentration of chlorpromazine in aqueous samples containing albumin

The addition of proteins to in vitro systems influences the free concentration of the test compound in the medium. The objective of this study was to set up a negligible depletion-solid phase microextraction method, coupled to high-performance liquid chromatography (nd-SPME-HPLC) to measure the free concentration of chlorpromazine (CPZ) in medium containing albumin. The nd-SPME method was optimized for coating thickness (polyacrylate coating) and exposure time, and potential effects from the addition of bovine serum albumin (BSA) were studied. It was shown that the addition of albumin did not cause fouling or influenced the uptake kinetics of CPZ into the fiber. At a realistic in vivo albumin concentration of 40 g/L of albumin, 94% of CPZ was protein bound. This is in line with findings in vivo, reporting a protein binding for CPZ of 92-99%. The nd-SPME-HPLC method described in this study can be used to measure the free concentration of chlorpromazine in medium containing proteins. These findings can be used to correct in vitro data for protein binding of chlorpromazine and this information is essential for the extrapolation to in vivo data.     

Preparation and chromatographic behavior of acetate-fiber filter rods with dye affinity ligands

Summary The dyes cibacron blue F3GA and reactive red 120 have been immobilized on acetate fiber filter rods to produce potential affinity matrixes. The isothermal adsorption of bovine serum albumin or human serum albumin on these matrixes was investigated and proved to conform to the Freundlich equation. In the static adsorption of human plasma the adsorption capacity for human serum albumin was 12.5 mg g⁻¹ fiber filter and one band was observed in SDS-PAGE electrophoresis of human serum albumin isolated by the immobilized cibacron blue F3GA filter rod. Ligand utilization efficiencies and breakthrough volumes were obtained for adsorption of HSA on the cibacron blue F3GA filter rod when the feed-rates were from 0.5 to 6 mL min⁻¹. In the affinity chromatography of human plasma the yield of human serum albumin was 1.27 g per 100 mL plasma.     

Flocculation of diatomite by methylated egg albumin

A common and inexpensive protein, egg albumin, was applied to the solid-liquid separation or flocculation of diatomite. Egg albumin was methylated in a 0.05 M HCl methyl alcohol solution at room temperature. About 90% of the carboxylic groups of egg albumin could be methylated within 24 h. The adsorption of egg albumin onto diatomite at pH 6.8 was remarkably enhanced by methylation. The adsorption constant of methylated egg albumin to diatomite at 30 degrees C was about 100-fold larger than that of native egg albumin; however, the adsorption constant of methylated egg albumin decreased to about 1/100 with temperature decreasing from 30 to 6 degrees C. The saturated adsorption amount of egg albumin was also increased by the methylation. The flocculating ability of methylated egg albumin was examined with a diatomite suspension at 6 and 30 degrees C in the pH range from pH 2 to 11. The diatomite suspension was effectively flocculated by the addition of small amounts of methylated egg albumin (only 0.5-1 wt% against diatomite) over a wide pH range from pH 3 to 10.     

Adsorption of egg albumin onto methylated yeast biomass

A new biosorbent, methylated yeast (MeYE), was prepared for the adsorptive separation of proteins from aqueous solutions. Yeast was methylated in a 0.1 M HCl methyl alcohol solution at room temperature. About 80% of the carboxylic groups of yeast could be methylated within 9 h. The adsorption of egg albumin onto MeYE was studied to evaluate the protein adsorption ability of MeYE. At near neutral pH, egg albumin was scarcely adsorbed onto unmethylated yeast and the adsorbed amount of egg albumin increased with increasing methylation degree. The amount of egg albumin adsorbed onto MeYE increased with increasing pH from 4 to 7 and steeply decreased above pH 7. The Langmuir isotherm was applied to determine the apparent adsorption constant and the saturated adsorbed amount of egg albumin on MeYE. Both the apparent adsorption constant and the saturated adsorbed amount increased with the degree of methylation. The saturated adsorbed amount of egg albumin onto MeYE having methylation degree 77% was 8.41 x 10(-6) mol g(-1) or 0.378 gg(-1) at near neutral pH.     

Properties of an albumin inhibiting lysosomal acid cholesteryl ester hydrolase in rat liver.

Lysosomal acid cholesteryl ester hydrolase (acid CEH, EC. 1.1.1.13) activity was inhibited by addition of an increasing amount of d = 1.21 bottom fraction from rat serum (Lipids in press). To clarify the mechanism of this inhibition, rat native and modified albumin were added to the assay mixture and their effects on acid CEH activity were examined. The inhibitory effect on acid CEH activity was dependent on the concentration of rat albumin. Albumin of various mammalian species, including human, bovine and rabbit, also inhibited acid CEH activity. This inhibitory activity was markedly increased by heat treatment, the effect increasing in parallel with the prolongation of the treatment. Moreover, this albumin-dependent inhibition of acid CEH activity was also markedly increased by methylation of albumin. In contrast, the inhibition of acid CEH activity by modified albumins, such as acetyl albumin, succinyl albumin and glycine methyl ester albumin, was much lower than that of albumin, and no stimulatory effect of heat treatment on the albumins was observed. The heat-treated albumin-dependent inhibition of acid CEH activity was not abolished in the presence of sodium deoxycholate. The values of Vmax obtained were similar with or without heat-treated albumin. These results suggest that the inhibitory effects of heat-treated albumin may be due to an intrinsic and characteristic property of the lipid/water interface, and that the stimulatory effects of heat treatment on albumin-dependent inhibition may be due to heat-induced changes in the affinity and conformation of albumin.