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Method of detecting mycoplasma infection in poultry and compositions therefor
| Details |
Inventors: Avakian, Alan P.; Kleven, Stanley H.;
Assignee: The University of Georgia Research Foundation, Inc. (Athens, GA)
Primary Examiner: Nucker; Christine M.
Assistant Examiner: Preston; D. R.
Attorney, Agent or Firm: Needle & Rosenberg
The present invention relates to the identification and purification of antigens from M. gallisepticum and M. synoviae and their use in serological assays. Cell proteins from these organisms that possess substantial immunoreactive responses with poultry antisera to the mycoplasma from which they are isolated in both the acute and convalescent stages of infection are identified. Of these immunogenic proteins, those that also lack immunogenic cross-reactivity with antisera to other poultry mycoplasmas and pathogens are identified. These species specific proteins are purified and incorporated into enzyme-linked immunosorbent assays (ELISAs). The resulting assays are able to detect sensitively and selectively both field or experimentally-induced M. gallisepticum or M. synoviae infection in poultry. A combination of these assays are incorporated into a diagnostic kit to allow the rapid, sensitive, and selective testing for and differentiation between M. gallisepticum and M. synoviae. This invention also provides anti-serum to these mycoplasma antigens and an immunoassay that employs this anti-serum to detect infection in poultry. Furthermore, a method to produce genetic clones that express the antigenic portions of the mycoplasma antigens is provided. |
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DETAILED DESCRIPTION OF THE INVENTION As used herein, the term "antigen" refers to a protein, polypeptide, or Coomassie blue staining band purified by the method described below and proteins or polypeptides which are substantially similar thereto, whether synthetic or naturally occurring.
An antigen is said to be "substantially similar" to another antigen if it is capable of performing substantially the same function, and has substantially the same chemical structure.
Thus, the term is intended to include synthesized proteins or polypeptides, proteins or polypetides purified from any stain or isolate within the species M.
gallisepticum or M.
synoviae, and proteins or polypeptides produced using recombinant technology.
The term further includes proteins or polypeptides which are substantially the same as the naturally occurring protein or polypeptide but which differ therefrom by one or more amino acids while still having substantially the same antigenic activity.
The term "immunoassay" is intended to refer to a system for detecting the presence of particular immunoglobulins.
Examples of immunoassays include, but are not limited to, enzyme-linked immunosorbant assays (ELISA), Western immunoblotting, agglutination, radioimmunoassays and immunodiffusion.
1.
Identification of Species-Specific Immunoqenic Polypeptides Western immunoblots of cell proteins of M.
gallisepticum and M.
synoviae with antisera to M.
synoviae and M.
gallisepticum, respectively, were examined to determine the extent of immunogenic cross-reactivity between these two mycoplasmas.
A comparison between these immunoblots and those of the cell proteins of both mycoplasmas developed with their own antiserum revealed which protein bands were immunospecific to either M.
gallisepticum or M.
synoviae.
The R strain of M.
gallisepticum and the F10-2AS isolate of M.
synoviae were filter-cloned 3 times through a 450 nm filter and checked for purity by immunofluorescence.
Cloned mycoplasmas were grown in Frey's medium with 12% swine serum (FMS) to log phase (until culture medium turned orange) at 37
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