Use of blocking protein with high pH extraction in method to determine a microorganism associated with periodontal disease and kit useful therefor

A method for the determination of at least one microorganism associated with a periodontal disease comprising the steps of:

• A. extracting an antigen from said microorganism which is present in a specimen, using an extraction composition which is buffered to a pH of from about 8.5 to about 11.5,
• B. simultaneously with or immediately after the extraction in step A and without lowering the pH of the extraction composition below about 8, mixing said extraction composition with a blocking composition to reduce nonspecific interactions, said blocking composition consisting essentially of a non-immunological blocking protein in an amount sufficient to provide a mixture having at least about 0.2 weight percent of said protein, said protein being casein or a protease, the pH of said mixture being at from about 8.5 to about 11.5,
• C. contacting said mixture formed in step B with a microporous membrane having thereon, in a discrete zone of a surface of said membrane, a water-insoluble reagent comprising water-insoluble particles having affixed thereto antibodies specific to said extracted antigen, to form in said zone, a water-insoluble complex between said antibody and the extracted antigen on said membrane,
• D. prior to, simultaneously with or immediately subsequent to the contact in step C, but after step B, contacting said extracted antigen with a detectably labeled, water-soluble second antibody specific to said extracted antigen so as to form a detectably labeled, water-insoluble sandwich complex specific for said microorganism, the complex being formed from both said water-soluble labeled and water insoluble antibodies with said extracted antigen in said zone on said membrane,
• E. simultaneously with or subsequently to step D, washing uncomplexed materials through said membrane, and
• F. detecting said labeled, water-insoluble sandwich complex in said zone on said membrane as a determination of said microorganism in said specimen.