Martha C. Anderson

A standardized quantitative skin-test assay of allergen potency and stability: studies on the allergen dose-response curve and effect of wheal, erythema, and patient selection on assay results.

A quantitative skin-test assay of allergenic potency was developed and tested on 28 allergic patients. The best-fit linear regression line was calculated from the sum of erythema or wheal diameters obtained from four intradermal threefold serial dilutions near the endpoint. Each line must have a correlation coefficient greater than 0.85 and the slopes of reference and test extract lines must not be significantly different. The relative potency can be calculated from the horizontal distance between the reference and test lines. The between-assay reproducibility was determined by comparing references against themselves. The 95% confidence limits were 54% to 186% when erythema was used and 27% to 367% when wheal was used. The narrower limits for the assay using erythema maybe due to the 5.5 times steeper slope of erythema lines than wheal lines. Potency results on five commercial extracts were highly correlated with potency results by RAST, AgE assay, or rye I assay. Extracts with isolectric focusing patterns dissimilar from the references were of significantly lower potency by in vivo and in vitro assays. The measured potency of two low-potency short ragweed extracts varied significantly with patient sensitivity to heat-stable and heat-labile ragweed allergens, indicating that patient selection may significantly affect assay results. Dose-response lines using either histamine or short ragweed by puncture (P) and intradermal (ID) techniques in the same patient showed that P/ID dose for equal erythema response was 909 for histamine and 31,250 for ragweed. This quantitative skin-test assay is highly re[rpdicob;e, yields potency data comparable to those of in vitro tests, can be applied to studies of extract stability, and is suitable as a primary bioassay of allergenic activity.

A comparative study of the allergens of cat urine, serum, saliva, and pelt

In direct RAST analyses of sera from 43 individuals with a history of cat allergy, 39.5% were positive to cat pelt, 37.5% to cat saliva, and 12% each to cat urine and serum. The cat pelt and saliva extracts contained allergen 1, but cat serum and cat urine collected by bladder puncture had no detectable levels of this allergen. A crossed immunoelectrophoresis/crossed radioimmunoelectrophoresis analysis failed to reveal any allergen in urine or serum that was not also present in the saliva or pelt preparations, although urine had two allergens not present in serum. When serum from a patient who was direct RAST positive to cat pelt, serum, saliva, and urine was tested by crossed radioimmunoelectrophoresis, it was determined that a total of six allergens were detectable in cat pelt, three in cat urine, and six in cat serum. Since cat serum contains no detectable cat allergen 1, it may be concluded that at least seven allergens derived from the cat are capable of binding to IgE antibody in humans.

Protein components of fire ant venom (Solenopsis invicta)

Abstract H. Baer , T.-Y. Liu , M. C. Anderson , M. Blum , W. H. Schmid and F. J. James . Protein components of fire ant venom ( Solenopsis invicta ), Toxicon 17, 397–405, 1979.—Venom of the fire ant ( Solenopsis invicta ), long thought to contain only alkaloids, is shown to contain proteins, which undoubtedly accounts for the induction of anaphylactic reactions from stings. Failure of previous investigators to identify proteins in this venom is probably due to the low protein content of approximately 0·1% of the venom weight. In common with other hymenoptera venoms, it contains phospholipase and hyaluronidase activity. Sephadex chromatography showed the presence of at least 3 proteins which were allergenically active, as determined by RAST using sera of individuals allergic to fire ant sting. The RAST also indicated that S. invicta venom was different from the venoms of S. richteri, S. xyloni and S. geminata , but probably shows extensive cross-reactivity. Commercially prepared extracts were shown to contain venom by RAST.

Antigenic and allergenic changes during storage of a pollen extract

The changes that occur in the antigens and allergens of a timothy grass pollen extract during storage were measured by radioallergosorbent test (RAST) inhibition, isoelectric focusing, (IEF), crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) and a dye-binding protein assay. Storage intervals varied from 7 to 92 days at temperatures of 4 degrees, 22 degrees, and 35 degrees C and the freeze-dried extract was rehydrated and stored in either phosphate-buffered saline with 50% glycerol (PBSG) or Cocas solution (CS). For extracts stored in CS at 35 degrees C, allergenic activity as measured by RAST inhibition dropped by almost one half at 7 days and certain of the IEF, CIE, ad CRIE bands were no longer visible, showing that these properties correlated with allergenic activity. After 65 dys, no IEF bands were visible but three CIE bands could be visualized. At 22 degrees the changes were slower and at 4 degrees C no changes were observed for the 92-day storage time. Extracts stored in PBSG showed some of the same changes but were much more stable at all temperatures. During storage, the protein content as measured by dye binding was also reduced, showing tha proteins were fragmenting. Because some of the allergenic proteins disappeared more rapidly than others, there was not only a loss in total allergenic activity but also a change in specificity. This raises serious questions concerning the use of such extracts, especially for diagnosis.

The role of specific IgE and beta-propiolactone in reactions resulting from booster doses of human diploid cell rabies vaccine

Reactions after booster injections of human diploid cell rabies vaccine (HDCV) were investigated to determine the possibility of IgE-type antibody involvement. Although normal manufacture of HDCV involves the inactivation of the virus with beta-propiolactone (BPL), the effect of BPL on nonviral vaccine components, such as host cell components or stabilizing proteins, may be typical of the haptenic action of small molecular weight chemicals. Specific IgE to commercial HDCV preparations, BPL-treated preparations of noninfected host MRC5 cell sonicate (BPL-MRC5), and a human albumin (HA) (BPL-HA) used as a stabilizing agent were detected in sera from five individuals who reported reactions after booster doses of HDCV. However, these patients had no detectable IgE to normal HA. Sera from nonvaccinated individuals, vaccinated individuals who reported no reaction after HDCV booster, and pollen-allergic individuals had no detectable IgE to HDCV, BPL-MRC5, or BPL-HA. Changes in the ratios of pre- to postbooster serum levels of specific IgE to HDCV and BPL-HA were significantly different in a group of 19 individuals who reported reactions to HDCV boosters; these changes in pre- to postbooster IgE levels in nonreactive vaccinees were not significant. Prebooster serum IgE RAST ratios to HDCV or BPL-HA were not predictive of potential reactions to HDCV. A number of experimental BPL-HA reaction mixtures were assayed to examine the effect of variable concentrations of BPL to HA. Increasing relative molar concentrations of BPL to HA resulted in increased electrophoretic mobility, whereas the highest relative specific IgE binding was detected in BPL-HA molar reaction mixtures of approximately 12.5:1.(ABSTRACT TRUNCATED AT 250 WORDS)

The preparation and testing of the proposed International Reference (IRP) Bermuda grass (Cynodon dactylon)-pollen extract.

A lyophilized candidate extract of Bermuda-grass (Cynodon dactylon) pollen, intended for use as an International Reference Standard, was prepared by pooling four individual candidate extracts. In preliminary investigations, the four candidate extracts encompassed a variety of extraction methods. The collaborative testing program simultaneously analyzed the proposed reference and the individual extracts and included 11 laboratories performing RAST inhibition, histamine release, crossed immunoelectrophoresis, crossed radioimmunoelectrophoresis, isoelectric focusing, sodium dodecyl sulfate gel electrophoresis, and protein determinations with a variety of reagents and methods. The four candidate extracts and the pooled reference were found to be equivalent. The stability of this extract has also been studied. This International Reference Preparation of Bermuda grass-pollen extract should be useful for research and industry.

Chapter 9.16 Allergens

Publisher Summary This chapter discusses allergenic extracts used to diagnose and treat allergies in man, and consist of water- or buffer-soluble proteins derived from plants, animals, insects, fungi, and foods. These proteins induce allergic response within minutes of exposure, and are found on the surface, or readily extractable, or secreted from the source material. Common allergens include pollen grains, dusts from animal skin surfaces, animal bedding materials, animal saliva and urine, and venoms of stinging insects, such as bees. The procedures found to be useful are polyacrylamide gel electrophoresis (PAGE), PAGE with the addition of sodium dodecyl sulphate (SDS-PAGE), isoelectric focusing (IEF), crossed immunoelectrophoresis (CIE), crossed radioimmunoelectrophoresis (CRIE), and to a limited extent, agar, or agarose gel immunoelectrophoresis. Immunoelectrophoresis is not a widely used technique and has limited sensitivity, but is used to examine the antigens produced by Aspergillus under different growth conditions.