IMMUNOELECTROPHORETIC ANALYSES OF AN AQUEOUS FRACTION OF SOME MYCOPLASMATA AND BACTERIAL L‐FORMS*

Abstract

The primary purpose of this report is to offer for consideration a method whereby certain strains of the Mycoplasmata and bacterial L-forms might be differentiated and hopefully identified. The urgency for such work arises out of the belief that any study of the group of microorganisms must be based on some fundamental identifying property, and further, that a possible pathological relationship may depend considerably on even subtile differences in characteristics of closely related strains.Protein characterization was considered as probably one of their most distinctive properties. The propensity of these organisms for assuming certain characteristics of the medium, particularly lipids, is generally acknowledged. Because of their small size and presumably limited potential for synthesis, and the probability that larger protein molecules would not pass the limiting membrane, it was further reasoned that their protein make-up would be quite characteristic (unless such material were to be phagocytized) .Further justification for such an approach arises out of the importance of protein components in relation to the immunologic activity of the organisms. In combination with immunoprecipitation techniques then, it was felt that it might be possible to obtain the best species resolution with a minimum of manipulations. MethodsDemonstration of the appearance of the organisms before and after disruption was accomplished by adding 3 % (final concentration) of purified gelatin to the culture which permitted gelling and trapping of the organisms within 10 minutes when placed in the refrigerator at 4°C. Fixation at the same temperature in the resultant gel is believed to cause a minimum of physical distortion. Each sample was treated with osmium tetroxide in a final concentration of 1 % and embedded in methacrylate prior to sectioning.All organisms were grown in a pancreatic digest of beef heart enriched with 20% bovine serum for 48 hours prior to harvest for immunizations and electrophoretic studies. Details have been published.' The concentration of the soluble portion after centrifugation was adjusted for the polyacrylamide experiments, so that 600-700 micrograms of protein could be applied in five drops.Six-pound albino rabbits were inoculated intraperitoneally each week with 0.5 ml of antigen mixed with an equal volume of Freund's incomplete adjuvant (Difco) representing two to three mgm protein nitrogen. Reactive titers were generally achieved within a six-week period.

Immunoelectrophoretic Analysis of Mycoplasma mycoides var. mycoides

“…There are few reports describing IEP using mycoplasma (4,12,15,26). This may be because, due to electrophoresis, a good precipitating antiserum is required and a much higher concentration of mycoplasma antigen is needed as it must be spread over a larger area than in the direct double-diffusion procedure.…”