Taizo Watanabe

Preparation of adsorbents for pyrogen adsorption

Abstract Preparation of adsorbents having a higher affinity for “pyrogen” was investigated. Adenine, cytosine, histamine and histidine showed high affinities as ligands for pyrogen. Cellulose and agarose were the most suitable as matrices for the preparation of adorbents having a high affinity for pyrogen. When the chain length of the spacer was 19.7–29.0 », the adsorbents showed the highest affinity for pyrogen. The adsorbent immobilized histamine to aminohexyl-Sepharose CL-4B with glutaraldehyde had the highest affinity for pyrogen originating from Escherichia , Klebsiella and Salmonella cells.

Immobilized tannin- a novel adsorbent for protein and metal ion

Abstract In order to prepare an adsorbent suitable for protein adsorption, we chose tannin as a ligand and attempted to bind it covalently to a water-insoluble matrix. The most favourable adsorbent was obtained by binding tannin to aminohexylcellulose. This adsorbent is called immobilized tannin, and can adsorb proteins well. Further, since it is known that tannin binds with metal ions as well as proteins, the adsorption specificity of immobilized tannin for metal ions was tested and immobilized tannin was found also to adsorb metal ions. This review summarizes the preparation method, characteristics and applications of immobilized tannin.

Specific assay for endotoxin using immobilized histidine and Limulus amebocyte lysate.

The LAL test is inhibited or enhanced by many substances. To overcome these problems, we have developed a specific endotoxin assay method using an ultrafiltration unit, a fluorometric LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This method is composed of two steps. The first step is the adsorption of endotoxins. Using immobilized histidine, endotoxins are quantitatively adsorbed on the adsorbent, and the adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the ultrafiltration unit. The second step is the reaction of adsorbed endotoxins with the LAL reagent. The endotoxins adsorbed on immobilized histidine are directly reacted with the LAL reagent in a filter cup and show enough activity for assay. The reproducibility and the accuracy of this method are high, and the recovery of endotoxins from a sample solution is more than 95%. The new endotoxin assay method using immobilized histidine can be utilized for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics instead of requiring employment of the more common gel-clot technique.

Characteristics of immobilized tannin for protein adsorption

Abstract The adsorption of proteins on immobilized tannin has been found to fall into one of three classes: in the first the proteins are well adsorbed at neutral to weak acidic pH values, and the adsorption is not affected by salt concentration; in the second the proteins are well adsorbed at acidic or alkaline pH values, but the adsorption is affected by salt concentration; in the third class the proteins are only slightly adsorbed at all pH regions. On the basis of these results, a mixture of three different proteins has been separated using an immobilized tannin column.

Specific assay for endotoxin using immobilized histidine, Limulus amoebocyte lysate and a chromogenic substrate

The Limulus amoebocyte lysate (LAL) test is inhibited or enhanced by many substances. In order to overcome this problem, a specific endotoxin assay method using a membrane filter unit, a chromogenic LAL reagent, and immobilized histidine (which is a specific adsorbent for endotoxins) was developed. Endotoxins are quantitatively adsorbed on immobilized histidine. The adsorbed endotoxins are separated from LAL-inhibiting or -enhancing substances by the membrane filter unit, and their activities are directly assayed with the LAL reagent in a filter cup without any inhibition or enhancement. The reproducibility and the accuracy of this method are high. This new endotoxin assay method using immobilized histidine can be used for the determination of endotoxins in a solution containing LAL-inhibiting or -enhancing substances such as amino acids and antibiotics, as an alternative to the more common gel-clot technique.

Assay of Endotoxin in Human Plasma Using Immobilized Histidine, Limulus Amoebocyte Lysate and Chromogenic Substrate

The Limulus amoebocyte lysate test for endotoxin is inhibited or enhanced by many substances. It is particularly difficult to determine endotoxin in plasma. In order to overcome this problem, we have modified the specific endotoxin assay method by using a membrane filter unit, a chromogenic Limulus amoebocyte lysate reagent, and immobilized histidine (which is a specific adsorbent for endotoxins). This immobilized histidine method consists of the endotoxin adsorption step on immobilized histidine, the separation step, in which Limulus amoebocyte lysate-interfering substances are removed, and the Limulus amoebocyte lysate test. Preheating of plasma samples (40-fold dilution with distilled water, at 100 degrees C for 7.5 min) was necessary, and it was necessary to dilute the sample more than 100-fold for the adsorption step. Under these conditions, the fraction of endotoxin recovered from plasma by the immobilized histidine method was almost 1. Moreover, by increasing the sample volume and extending the Limulus amoebocyte lysate reaction time, the sensitivity could be increased. By using the immobilized histidine method, 50-200 units/l of endotoxin in plasma samples can be accurately assayed. The method was used for the determination of plasma endotoxin in rabbits.

PREPARATION OF IMMOBILIZED TANNINS FOR PROTEIN ADSORPTION

Preparation and adsorption specificity of tannins immobilized by covalent binding on water-insoluble matrices were investigated. Immobilized tannins were prepared by condensing cyanogen bromide activated tannins with aminohexyl derivatives of several kinds of matrices. The most suitable matrix for the immobilization of tannin was alkali-treated cellulose powder. The concentration of sodium hydroxide solution for alkali treatment influenced the adsorption capacity of immobilized tannin for a protein, but temperature and time for alkali treatment did not. Immobilized tannins having spacers of long chain length exhibited high adsorption capacity for a protein. Chinese gallotannin was the most favorable ligand for protein adsorption. The immobilization of tannin on aminohexyl matrices was also possible by using epichlorohydrin instead of cyanogen bromide. The maximum adsorption capacity of the immobilized tannin for a protein was about 50 mg/ml of the absorbent. Immobilized tannin adsorbed proteins specifically but did not absorb low molecular weight compounds.

Use of immobilized histidine in assay for endotoxin in patients with liver disease

TheLimulus amebocyte lysate (LAL) test has the disadvantage of being influenced by various inhibitors and activators. We have developed a method for the LAL reaction that involves the specific adsorption and isolation of endotoxin using a membrane filter unit and immobilized histidine; in this present study we used the method to measure endotoxin in the plasma of patients with acute or chronic liver disease. The adsorbed endotoxins are separated from LAL-inhibitors or-activators by the membrane filter unit, and their activity is directly assayed with the LAL reagent in a filter cup without any inhibition or activation. The study population consisted of 23 subjects, 3 with fulminant hepatitis and 20 with cirrhosis (9 with esophageal varices and 11 without). All 3 (100%) of the samples of plasma from patients with fulminant hepatitis were positive for endotoxin, as were the samples of 7 (78%) of the 9 patients with cirrhosis and esophageal varices, and 2 (18%) of the 11 patients with cirrhosis but without such varices. The results suggested that this method appears to be useful for assaying the concentration of endotoxin in patients with fulminant hepatitis or cirrhosis of the liver.

Affinity Chromatographic Removal of Pyrogens

Pyrogens are classified into two groups, exogenous and endogenous. Exogenous pyrogens originating in the cell wall of gram-negative bacteria have the strongest pyrogenicity (Nowotny, 1969), and for them synonyms such as endotoxins, lipopolysaccharides (LPS), and O-antigens are used. If a pharmacologically active substance is contaminated with pyrogens, its intravenous administration produces transient fever in homoiothermic animals. Therefore, it is necessary to remove any pyrogen present in medicines or drugs for intravenous administration.