Grass pollen allergens. IV. The isolation of some of the principal allergens of Phleum pratense and dactylis glomerata and their sensitivity spectra in patients.

Cocksfoot and Timothy pollen extracts are each found to contain at least fifteen components antigenic in rabbits. Most of these can also be allergens for man, but only a few are regularly so. These principal' allergens have now been isolated in highly purified form. Procedures are given for a simple method of preparing extracts for clinical purposes and for the partial separation, concentration and purification of the allergens by means of differential extractions of the pollens and by means of ultrafiltration, isoelectric precipitation and salt fractionations (at acid and neutral pH) of the extracts. Isoelectric precipitations gave highly pigmented acid complexes, two of which moved as single sharp peaks at pH 7.4 in free electrophoresis, but proved to be hardly active by skin tests. Acid NaCl fractionation of the remainder resulted for Cocksfoot and Timothy in the isolation of a nearly white powder (T21.111121112 = T21B) which was weight for weight 1000–10,000 times as active as the pollen from which it had been derived. The powders have retained their activity for 7 years. By gel diffusion tests, they were found to contain two antigens (one in each preparation) which were immunologically partially related, but the Timothy preparation contained in addition the innermost' twin' antigens specific for Timothy that we had discovered previously in the crude extracts by gel diffusion methods. Skin reactions could be elicited in hay-fever subjects by prick tests with concentrations of 10–10 g./ml., which is equivalent to intradermal injections of 10–10 mg. and represents a 300-fold purification with respect to the concentrates of crude pollen extracts prepared by ultrafiltration and dialysis. Fractionation on DEAE-cellulose of one of the highly purified Timothy preparations (T21.11112112 = T21A) and other, crude Timothy and Cocksfoot extracts resulted in considerable and reproducible separation of the various antigens, with no indication of the continuous dissociation into smaller fragments reported in recent publications. The immunologically related antigens of Cocksfoot and Timothy were among a number not retained by the DEAE-cellulose at pH 7.4–8.5. These proved to be skin reactive in all the patients tested. When this fraction was prepared from T21.11112112(= T21A) only one main line (plus a faint subsidiary line) was found in the gel diffusion tests. At pH 6.4 the important dissimilar Cocksfoot and Timothy allergens were eluted; the fraction derived from T21A contained only the special innermost' twin antigens in roughly a further ten-fold purification over and above that achieved in the acid NaCl fractionation.