Biologic potency and immunoblotting studies of extracts of three cockroach species.

BACKGROUND: Although cockroaches are responsible for severe allergic disease, cockroach extracts are not yet standardized by the ID50EAL method, nor have the allergens been completely characterized. OBJECTIVE: To determine biologic potency and immunoblotting patterns of whole-body extracts of three cockroach species: Periplaneta americana, Blattella germanica, and Blatta orientalis. METHOD: Twenty-four subjects puncture test positive to mixed cockroach extract were puncture tested with the three extracts. Fourteen qualified for endpoint titration testing by the ID50EAL method to P. americana with a sum of erythema (sigma E) > or = 20 mm by puncture testing, while 13 qualified for B. germanica and 12 qualified for B. orientalis. RESULTS: The mean D50s were 9.67 +/- 1.33 (mean +/- SD) for P. americana, 10.24 +/- 1.16 for B. germanica, and 10.36 +/- 1.40 for B. orientalis, resulting in a potency of 1000 BAU/mL for each extract. Omitting data from subjects with puncture sigma E < 30 mm did not change the calculated potency. Sixteen serum specimens revealed up to 17 bands with molecular weight 14 to 263 kD by SDS-PAGE immunoblotting. Four specimens showed no bands; of these, one had a positive RAST only to P. americana and one only to B. germanica. There was no correlation between number of bands or RAST counts with D50. CONCLUSIONS: IgE immunoblots of cockroach-allergic patients vary considerably as to the number and intensity of bands. Most patients react similarly to all three species. Current nonstandardized products (1:10 wt/vol) in glycero-Coca's solution appear to contain approximately 1000 BAU/mL. Ideally these extracts should be more potent, eg, 10,000 BAU/mL.

Standardization of allergenic extracts by basophil histamine release.

BACKGROUND: Allergenic extracts are standardized by using the ID50EAL (Intradermal Dilution for 50 mm sum of Erythema Determines the Allergy Unit) skin test technique to assign allergy units to reference preparations. When new batches of extracts are manufactured, they are compared with the reference by RAST inhibition or other in vitro techniques. This study was designed to determine whether basophil histamine release might be an additional useful in vitro test for the standardization of allergenic extracts. METHODS: Basophil histamine release, skin tests, and RAST were compared using several different allergenic extracts at many different strengths. Allergy units were calculated using skin test results and basophil histamine release. RESULTS: Basophil histamine release, skin tests, and RAST correlated well (P < .01). Basophil histamine release-derived allergy units and skin test-derived allergy units were highly correlated (P < .01). CONCLUSION: Basophil histamine release appears to be a useful tool for the standardization of allergenic extracts.

In vivo and in vitro characterization of Allpyral grass pollen extracts.

The Food and Drug Administration, Center for Biologics Evaluation and Research (CBER) has developed methodology to standardize both aqueous and freeze-dried (lyophilized) extracts. Thus far, it has not been determined whether or not this methodology can be used to standardize alum-adsorbed extracts. This study was designed to examine the in vivo and in vitro potency of selected Allpyral grass pollen extracts, including timothy, orchard grass, perennial ryegrass, sweet vernalgrass, and meadow fescue. Puncture testing was performed on highly grass-sensitive subjects with the concentrate of each of the five Allpyral grass extracts. Additionally, puncture testing was done on 22 subjects to compare Allpyral timothy grass with a lyophilized, standardized timothy grass extract. The ID50EAL (Intradermal Dilution for 50 mm sum of Erythema determines the Allergy Unit) skin test method was used to determine allergy units of the Allpyral extracts. Relative potency of the Allpyral timothy extracts to a timothy laboratory standard was determined using an ELISA-inhibition assay. Intradermal tests were also performed to examine the potency of the supernatant obtained after centrifugation of the whole Allpyral timothy extract. The puncture test responses to the Allpyral timothy extracts were less than those to the lyophilized extract. Those 10,000 PNU/mL Allpyral grass pollen extracts tested were determined to contain a calculated 10,000 BAU/mL. By ELISA inhibition, the Allpyral timothy extracts were determined to be approximately 1,000-fold less potent than the laboratory standard. The estimated concentration of the supernatant preparation to elicit a target response was notably (mean = 1,175 times) greater than that of the whole Allpyral timothy extract needed to elicit the same erythema response.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of mixing allergenic extracts containing Helminthosporium, D. farinae, and cockroach with perennial ryegrass.

Allergenic extracts used for immunotherapy often contain mixes of different allergens. Studies have shown that certain allergenic extracts contain enzymes that can cause a decrease in grass pollen allergenicity when mixed with grass extracts. Glycerin and buffered saline with phenol (BSP) extracts containing Helminthosporium interseminatum, D. farinae, and cockroach were mixed with perennial ryegrass extracts and analyzed 7, 30, 60, and 90 days after mixing to determine the mixing effect of these extracts on the ryegrass pollen potency. Analysis was performed using RAST inhibition, SDS-PAGE and, to determine biologic potency, a quantitative intradermal skin test technique. All tests showed significant decreases in ryegrass potency when mixed with Helminthosporium and cockroach. This decrease was not seen with the D. farinae mix. Glycerin seemed to have some protective effect. Even in the situation showing the most decrease in ryegrass potency (Helminthosporium mixed with ryegrass in a BSP extract), the resulting extract still contained an estimated biologic potency of 10,000 AU/mL. This may explain why such mixes, which have been used clinically for many years, appear to give adequate therapeutic results. The study also suggest that clinicians may be well advised not to mix grass pollen extracts with extracts that may contain proteolytic enzymes.