Patent ID: 6824997
Filing Date: 2004-11-30
Classification: C07K,G01N

Abstract:
A method of detecting the presence of the cell wall C-polysaccharide antigen of Streptococcus pneumoniae, in a liquid sample, which method comprises the following steps:a) culturing Streptococcus pneunoniae bacteria, to obtain a desired size of culture and harvesting therefrom cells therof as a wet cell pellet; b) separating from the wet cell pellet the cell wall C-polysaccharide antigen containing not more than 10% protein by performing a series of steps which comprises; (i) suspending the wet cell pellet in an alkaline solution and nixing; (ii) adjusting the pH to an acid pH with a strong acid; (iii) separating the mixture from step (ii) into two layers; (iv) removing the upper layer and adjusting its pH to approximate neutrality; (v) adding to the product from step (iv) a broad spectrum protease enzyme and digesting to destroy residual proteins; (vi) adjusting the pH of the product from step (v) to alkaline pH with a weakly alkaline aqueous solution: and (vii) separating out the cell wall C-polysaccharide antigen containing not more than 10% proteing; c) coupling to a chromatographic column through a spacer molecule the cell wall C-polysaccharide antigen containing not more than 10% protein obtained in step (b); d) passing polyvalent antibodies to Streptococcus pneumoniae over the chromatographic affinity column of step (c) to produce purified antigen-specific antibodies; and e) conducting an immunoassay upon a liquid sample suspected of containing Streptococcus pneumoniae and/or its C-polysaccharide cell wall antigen which immunoassay comprises the steps of (i) contacting the liquid sample with conjugates of purified antigen specific antibodies from step (d) hereof and a labelling agent capable of manifesting a color or a detectable signal upon completion of the immunoassay, whereupon C-polysaccharide cell wall antigen of Streptococcus pneumoniae in the sample, whether or not in free form, will react with said conjugates to form labelled antibody-antigen conjugates, (ii) further contacting the liquid and all of the conjugates it contains with a solid surface upon which a mass of unlabeled antigen-specific antibodies from step (d) hereof have been immobilized, whereupon any labelled antibody-antigen conjugates present will react with the immobilized antibodies on the surface to form labelled antibody-antigen-immobilized antibody sandwiches, and (iii) detecting any label thereby accumulated on the solid surface by a detection means appropriate to the nature of the label so as to confirm the presence of the Streptococcus pneumoniae C-polysaccharide cell wall antigen in the sample.