Detection Of Sialic Acid Research Articles

The Variable Domain Glycosylation in a Monoclonal Antibody Specific to GnRH Modulates Antigen Binding

Functionally important glycosylation has been identified in the antigen binding domain of an anti-GnRH monoclonal antibody. Presence of mannose and sialic acid residues is revealed from con A immunoblots and positive staining with a sialic acid detection kit, respectively. Desialylation of the antibody reduces GnRH binding, suggesting the role of terminal sialic acid residues in modulating antigen binding. The crystal structure of the Fab fragment shows electron density adjacent to the antigen binding site which may be attributed to the covalently attached carbohydrate moiety. Thus, the presence of sialic acid containing mannose-rich carbohydrate moiety near the antigen binding site of a monoclonal antibody Fab fragment is relevant for defining antibody specificity.

A Biomimetic Receptor System for Sialic Acid Based on Molecular Imprinting

Abstract A new type of a fluorescent receptor system specific for sialic acid has been prepared. For the preparation of this artificial receptor a polymerization reaction using allylamine, vinylphenylboronic acid and ethylene glycol dimethacrylate in the presence of sialic acid as template was carried out. After splitting off the template molecules, a suspension of the polymer was used in reaction with OPA reagents, both in the presence and absence of sugars. It was found, that sialic acid promoted the fluorescence development in this system, probably as a result of the so-called “gate effect”, resembling natural occurring receptors. Rapid detection of sialic acid in the concentration range of 0.5–10 μM was shown possible within 40 minutes. Our method seems applicable for the development of optical sensors; combining the high sensitivity and selectivity of biological receptors and high stability of synthetic polymeric materials.

Carbohydrate depletion of immunoglobulin A1 by oral species of gram‐positive rods

Bacterial deglycosylation of immunoglobulin Al (IgA1), the dominant isotype of antibody in the oral cavity, probably provides both nutrition as well as protection to the oral bacterial community. Representative strains of oral gram-positive rods were tested for their ability to remove carbohydrates from IgA1. Detection of sialic acids was performed by means of high-performance liquid chromatography (HPLC) separation (Aminex HPX-87H) and ultraviolet light absorption at 190 nm, and neutral carbohydrates were measured by HPLC separation (Capcell Pak C-18 SG 120) and ultraviolet light absorption at 245 nm after derivatization. Four strains of Actinomyces naeslundii, two strains of Corynebacterium matruchotii and one of two strains of Actinomyces odontolyticus partially or totally removed sialic acid, while two strains of Propionibacterium propionicus and the other strain of A. odontolyticus did not. Complete correlation was observed between sialic acid removal, neuraminidase activity measured with fluorogenic substrate and with one exception, altered immunoelectrophoretic mobility of IgA1. Only limited removal of other carbohydrates was observed with poor correlation to exoglycosidase activities measured with chromogenic substrates. Desialylation increases the susceptibility of glycoproteins, including IgA1, to proteolysis. Therefore, the desialylation of IgA1 by oral gram-positive rods may facilitate the proteolytic activities of other oral bacteria, and the concerted action may positively influence the survival of the bacteria in the oral community.

Quantitative Detection of Whey Cream in Sweet Cream and Butter

A simple and sensitive assay is described that can detect trace amounts of whey cream in sweet cream and in butter made from that cream. The method is based on the detection of sialic acid and involves a color reaction between Ehrlich's reagent and sialic acid. The procedure is faster than other methods for detection of whey. The method could be termed semiquantitative because of the seasonal variability of sialic acid in cow's milk. However, with a calibration curve, the method is quantitative and can easily detect as little as 1% whey cream in sweet cream and in the resulting butter. With 1% whey cream in sweet cream, the purple color indicating the presence of sialic acid is visible to the naked eye.

Human IgG preparations isolated by ion-exchange or protein G affinity chromatography differ in their glycosylation profiles

IgG from patients with rheumatoid arthritis (RA) is abnormally glycosylated in the Fc region, with sialic acid and galactose levels lower than normal. Protein G and DEAE purify populations which are differentially glycosylated. Significantly increased exposure of sialic acid was detected in normal IgG compared with that of RA IgG when ion exchange was used to prepare samples. However, when the same samples were prepared using protein G, no difference in the detection in the detection of sialic acid was seen between the two groups. When examining the heavy chain of IgG, more sialic acid, galactose and N-acetylglucosamine were detected in DEAE purified IgG compared with that prepared by protein G Detection of sialic acid and N-acetylglucosamine was also increased on light chains from IgG prepared by ion exchange chromatography Since this occurs notably on rheumatoid light chains it would appear that this arrangement would contribute to the overall glycosylation changes in IgG. In the case of molecules lacking galactose the discrimination between the RA and normal IgG is significantly improved when ion exchange chromatography is used. Since differentiation between disease and normal groups relies on the purification technique used, we recommend that more than one method is employed before undertaking an analysis of glycosylation changes.

Detection of sialic acid on cultured cells by binding of a lectin from Limax flavus.

(J. Am. Soc. Nephrol. 1992; 3:113-1 15) washed gently in PBS and solubibized in 1 mL/wehl of 0. 1 % sodium dodecyb sulfate (BioRad, Richmond, CA), and the radioactivity was estimated with a gamma counter (LKB-Bnomma, Sweden). To study the effects of fixation, the lectin binding was measured In cells fixed in 95% ethanol. Because the fixed cells could not be solubihizcd completely in sodium dodecyl sulfate, the cell layers were scraped by cotton tips and the tips were counted.

Detection of glomerular sialic acids in patients with diabetic nephropathy.

A study on immunofluorescence of sialic acids in glomeruli from patients with diabetic nephropathy is described. Measurement of sialic acid in sera from 25 patients with diabetes mellitus was also performed. Renal biopsy specimens from 12 patients with diabetic nephropathy were stained with FITC-labeled antihuman IgG antiserum and rhodamine-labeled Triticum vulgaris (WGA) or Limulus polyphemus (LPA). These specimens were also stained with such reagents after treatment with neuraminidase, trypsin or citrate buffer. Both deposition of IgG and binding of WGA in the glomerular capillary walls were observed in all patients with diabetic nephropathy. The binding of WGA in the glomerular capillary walls in diabetic nephropathy was significantly increased compared with that in four normal renal tissues. However, the binding of LPA was hardly observed in the glomerular capillary walls of patients with diabetic nephropathy. The binding of WGA in the glomeruli was markedly decreased after treatment with neuraminidase although it was hardly decreased after treatment with trypsin or citrate buffer. The levels of sialic acid in sera from patients with diabetic nephropathy were markedly increased. It is suggested that accumulated substances in the glomerular capillary walls with an affinity for WGA are mainly composed of N-acetyl glucosamine and/or N-acetyl neuraminic acid in patients with diabetic nephropathy.

Serum sialic acid in normals and in cancer patients.

A simple procedure is described for the detection of sialic acid in serum. After a direct addition of Ehrlich reagent to serum and an incubation at 56 degrees C for eight hours, the resulting mixture is diluted with saline. After centrifugation, the color in the supernatant is determined at 525 nm in a spectrophotometer. Serum sialic acid was significantly greater in cancer patients than in normals. Cancer patients with metastases had significantly greater sialic acid than cancer patients without metastases. In two cancer patients, sialic acid levels returned to normal after surgery. The diagnostic usefulness of 95.6% was similar to that reported with lipid-soluble sialic acid and seemed to be superior to CEA and other tumor antigens associated with a limited spectrum of tumors. However, patients with inflammatory diseases such as arthritis, Crohn's disease and psoriasis also showed elevated sialic acid levels. Ultrafiltration showed that almost all of the sialic acid was retained on an Amicon filter, which suggests that sialic acid was bound to a macromolecule. A quality control serum run 25 times had a coefficient of variation (CV) of 8.4% and the same serum ran on 42 days had a CV of 11.6%.

Gas-liquid chromatographic analysis of the monosaccharide composition of acid glycosaminoglycans (mucopolysaccharides) derived from animal tissues

Quantitative, gas-liquid chromatography was investigated for analysis of the monosaccharide composition of acid mucopolysaccharides from animal tissues. The method entailed the analysis of the trimethylsilyl (Me 3Si) derivatives of methyl glycosides on two liquid phases. Good resolution of monosaccharides was achieved by use of columns of SE-30 and Apiezon-M. The procedure was tested with chondroitin 4-sulfate, and the results were slightly different from those of Mathews et al. When the analysis is performed according to this method, important points are:(1) absolutely anhydrous, methanolic hydrogen chloride is necessary, to ensure detection of hexosamines and sialic acid; and (2) high moisture in the air obstructs high recovery of methyl glycosides and their Me 3Si derivatives, except in the case of neutral sugars.

Detection of sialic acid containing compounds and the behaviour of gangliosides in polyacrylamide disc electrophoresis

A modified method is described for staining of sialic acid in glycoproteins and gangliosides in polyacrylamide gels using resorcinol reagent. The electrophoretic properties of ganglioside are discussed in view of the stability of their micelles in aqueous solutions.

EXAMINATION OF EIAL'S REACTION, WHICH IS A HISTOCHEMICAL DEMONSTRATION METHOD OF SIALIC ACID

Various methods have been introduced for chemical detection of sialic acid. Among those methods we applied resorcinol reaction, orcinol reaction (Bial) and thiobarbituric acid reaction to histochemical study as they appeared to be useful. As the result, it was found that only orcinol reaction (Bial) shows reaction on specimen. When orcinol reaction was conducted by using pure reagents, fructose, sorbose, tryptophan and sialic acid (N-acetylneuraminic acid) indicated positive reaction. Fructose and sorbose were discriminated from sialic acid because of their color and its quick fading. Discrimination of tryptophan from sialic acid was possible by tryptophan staining by Serra's method and tetrazonium method as its reaction part is different. It was also possible to discriminate these substances by spectrophotomter and thin-layer chromatography. It was therefore concluded that the substance which develops color in Bial's reaction on specimen is most probably sialic acid.

Effect of the removal of cell surface sialic acids on cell aggregation in vitro.

THE extensive investigations of Gottschalk1 and Warren2 have established that sialic acids (9-carbon sugars) occur widely in all orders of the Vertebrata. In many species histochemical studies using both the light and the electron microscope have shown that these acids constitute part of a carbohydrate-rich substance found at the surfaces of both normal3,4 and tumour cells5,6. The utilization of the specific enzyme N-acetylneuraminate glycohydrolase15 has enabled investigators to demonstrate by microchemical tests that sialic acids occur at the surfaces of Ehrlich ascites tumour cells7,8, of red blood cells9,10 and of normal and malignant rat liver cells11. Studies employing cell electrophoresis have resulted in the detection of sialic acids in many other cell types12, and have demonstrated that the increased negative mobilities of some transformed cell lines13 and of RPMI No. 41 cells (derived from human osteogenic sarcoma) in mitotic peak phase14 are caused by an increased amount of cell surface sialic acids.

Sensitive location reagent for the simultaneous detection of sugars, amino sugars and sialic acids.

MOST methods for the detection of sugars on paper and thin-layer chromatograms are tedious and frequently involve the use of toxic reagents. No single location reagent so far described is capable of detecting reducing sugars, non-reducing sugars, amino sugars, and sialic acids simultaneously. In the case of thin-layer chromatography there is an additional disadvantage in that, unless a cellulose layer is used, the sensitivity of many of the reagents is reduced, probably because of the nature of the adsorbants used. Schwartz has described a very sensitive method for the detection of serine on paper chromatograms1. The chemical basis for the method is the liberation of formaldehyde from serine after periodate oxidation. The liberated formaldehyde, after condensation with acetylacetone in the presence of ammonium salt yields 3,5-diacetyl-1,4-dihydrolutidine, which is a yellow coloured, highly fluorescent compound. In his original paper, Schwartz pointed out that the reaction was not necessarily limited to serine or hydroxyamino-acids and suggested that a variety of compounds would be expected to give a positive reaction on chromatograms containing biological materials other than protein hydrolysates.

Detection of sialic acid in various Escherichia coli strains and in other species of bacteria.

IT was recently reported1 that an acidic aminopolysaccharide termed colominic acid was elaborated into the culture medium by the colocinogenic strain of Escherichia coli known as K235. This polysaccharide was afterwards shown to be composed of repeating units of N-acetylneuraminic acid or ovine sialic acid2. Since this was the first time that sialic acid substances had been found in other than mucopolysaccharides derived from mammalian sources a survey was made to ascertain whether these substances occurred in other Escherichia coli strains and in different species of bacteria. In addition, an attempt was made to correlate the pressure of sialic acid with other known properties of these bacteria.