Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1987-08-20
1992-06-16
Kepplinger, Esther L.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
424450, 427 2, 435 7, 435177, 435181, 435810, 435814, 436501, 436518, 436527, 436529, 436531, 436530, 436543, 436547, 436808, 436809, 436823, 436824, 530359, 530395, 530403, 530409, 530412, 530811, 530812, 530816, 536 187, G01N 3353, G01N 33543, C01K 1514, C07H 504
Patent
active
051224500
DESCRIPTION:
BRIEF SUMMARY
This invention relates to a biochemical reagent, its preparation and its uses. More particularly, the reagent of this invention is an immobilised oligosaccharide and has its principal application in the biochemical investigation of oligosaccharides which occur in combination with proteins, as glycoproteins and proteoglycans, and which may be immunologically active.
Production of antibodies by the body is triggered by an encounter between a foreign molecule (an "antigen") and cells of the adaptive immune system. The biochemical mechanisms of antibody production are complex and have not been fully elucidated. What is known is that antigens trigger the immune system to produce antigen-specific antibodies which on subsequent encounters will bind to the antigen resulting in its inactivation. Regions on the antigen to which antibodies bind are known as "antigenic determinants" or, synonymously, "epitopes". In the interests of simplicity, the term "antigenicity" is used herein and in the claims to indicate the ability of a molecule to bind to an antibody.
It is known that when antibodies bind to proteinaceous antigens such as glycoproteins and proteoglycans, the epitope to which the antibody is directed may be a sequence of amino acids on the protein or a sequence of sugar units on one of the glyco group oligosaccharide chains. Investigation of the antigenicity of the oligosaccharides is of significant biochemical and medical interest.
Oligosaccharides, when isolated as such from immunogenic glycoproteins and proteoglycans, do not bind to their known antibodies in vitro; for example, their simple solutions do not bind to antibodies. The probable reason for the absence of antibody-binding ability is that epitopes are only recognised by antibodies when they are presented to the antibodies in specific orientation and concentration.
In Analytical Biochemistry, 139, 168-177, Raja, R. H. et. al. describe a method of bonding oligosaccharides, obtained by enzyme degradation of hyaluronic acid, to immobilising supports to give, for example, affinity chromatography media and polyacrylamide cell culture surfaces. Hyaluronic acid is a polysaccharide, occurring in connective tissue, with a molecular weight of several million and composed of repeating units of N-acetylglucosamine and glucuronic acid. Partial digestion of hyaluronic acid with the enzyme hyaluronidase, under the conditions descibed by Raja, produces a mixture of fragments of which about fifty percent have an average size of from six to eight disaccharide units (i.e. from twelve to sixteen monosaccharide units). This oligosaccharide mixture is bonded to the immobilising support by a method which involves the following sequential steps: (a) reduction of the oligosaccharide with sodium borohydride, which results in cleavage of the terminal sugar ring and formation of a methylol group at the site of the ring opening, (b) selective oxidation of the methylol group with sodium periodate to form an aldehyde group, (c) coupling of the aldehydo-oligosaccharide to an alpha, omega alkyldiamine such as hexane diamine in the presence of sodium cyanoborohydride resulting in a derivative which has the oligosaccharide residue at one end of a polymethylene chain and an active amino group at the remote terminus (omega postion); and, (d) bonding of the derivatized oligosaccharide, via the omega amino group, to a chromatography support, such as Sepharose (Trade Mark), under the action of cyanogen bromide or, alternatively, to a polyacrylamide cell culture surface under the action of sodium cyanoborohydride.
It is known that reducing disaccharides (i.e. those having active aldehyde groups) may be coupled to proteins or to aminoethyl polyacrylamide gels by selective reduction of a Schiff base, formed between the reducing (aldehyde) group of the disaccharide and the amino group of the protein or the aminoethyl gel using sodium cyanoborohydride [Gray, G. R. (1974), Arch. Biochem. Biophys.; 163, 426-428]. It is the reductive animation method of Gray which is utilised by Wood and Kabat to
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Feizi Ten
Tang Ping W.
Hoffer Florina B.
Kepplinger Esther L.
Research Corporation Limited
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