US 6,824,997 B1Granted

Process and materials for the rapid detection of streptococcus pneumoniae employing purified antigen-specific antibodies

AssigneeBinax, Inc.

InventorsNorman James Moore, Mary Kathleen Fent, Vladimir Andrei Koulchin, Elena Valentin Molokova

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Abstract

A process is disclosed for obtaining a C-polysaccharide cell wall antigen containing not more than about 10% protein from Streptococcus pneumoniae bacteria. The antigen thus obtained is conjugated to a spacer molecule, and the free end of the latter is then conjugated to a chromatographic affinity column. The column is then utilized to purify raw antibodies to S. pneumonia bacteria, thereby producing antigen-specific antibodies. A portion of such antibodies is conjugated to a labeling agent which displays a visible color change upon reaction of the antibodies with their antigenic binding partner and embedded in a first zone of an immunochromatographic assay device. Another portion of such antibodies is bound to the reaction zone of the device which has a view window. When a liquid sample, such as patient urine, cerebrospinal fluid or blood is applied to the first zone, the conjugate of antibodies and labeling agent and the sample move along a flow strip of bibulous material to the reaction zone wherein, if the sample contains S. pneumoniae or its cell wall antigen, a sandwich is formed among the labeled conjugate, the antigen and the bound antibodies and a color change is observed. The immunochromatographic assay thus performed is completed within about 15 minutes. This assay affords a basis for rapid and reliable diagnosis of various pathogenic states caused by S. pneumoniae including pneumonia, bronchitis, otitis media, sinusitis, meningitis, and secondary disease states that commonly occur when primary pneumonic infection caused by this bacterium persists undiminished over a time period of 3-5 days.

Claims18

Dependent claims

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.

13 more elements

An immunochromotographic (“ICT”) device for the detection of the C-polysaccharide cell wall antigen of Streptococcus pneumoniae in a liquid sample, which device comprises a housing equipped with a window and containing a strip of bibulous material, at least having at least a first zone and a second zone, said strip being so positioned within said housing that its second zone appears directly beneath said window, said strip being further characterized in that

a) said first zone has movably embedded therein a conjugate of a labeling agent and purified antibodies specific to the cell wall C-polysaccharide antigen of
b) said second zone is located downstream of said first zone, to which second zone is immovably bound a stripe of unlabeled purified antibodies specific to the cell wall C-polysaccharide antigen of Streptococcus pneumoniae,
wherein all of said antibodies specific to the cell wall C-polysaccharide antigen of Streptococcus pneumoniae in both zones have been obtained by passing polyvalent antibodies to Streptococcus pneumoniae over a chromatographic affinity column to which is coupled to a purified cell wall C-polysaccharide antigen of Streptococcus pneumoniae obtained from a culture of Streptococcus pneumoniae bacteria according to a method comprising the steps of:
(i) harvesting cells from the said culture in the form of a wet cell pellet;
(ii) suspending the wet cell pellet in an alkaline solution and mixing;
(iii) adjusting the pH of the resultant mixture to an acid pH with a strong acid;
(iv) separating the acidified product from step (iii) into two layers;
(v) removing the upper layer and adjusting its pH to approximate neutrality;
(vi) adding to the product from step (v) a broad spectrum protease enzyme and digesting to destroy residual proteins;
(vii) adjust the pH of the product from step (vi) to an alkaline pH with a weakly alkaline aqueous solution;
and
(viii) separating out the cell wall C-polysaccharide antigen of Streptococcus pneumoniae having no more than 10% protein.

9 more elements

Dependencies are inferred from the claim text.

Facts

Application: 09/397,110Filed 16 Sep 1999 · continuation in part
Priority: 18 Sep 1998
Granted: 30 Nov 2004
Term ends: Not estimatedThe source records don’t support an estimate.

Classification11 codes

G01N 33/56944
Bacteria: streptococcus
C07K 16/1275
Gram-positive bacteria: streptococcus
G01N 21/78
By observing the effect on a chemical indicator: producing a change of colour
G01N 2021/752
Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated: devices comprising…