Studies on Alternaria allergens: II. Measurement of the relative potency of commercial Alternaria extracts by the direct RAST and by RAST inhibition

The relative potency of 12 commercial Alternaria extracts was analyzed by end-point skin test titrations and compared to in vitro measurements of potency, including (1) the radioallergosorbent test (RAST) inhibition procedure; (2) the direct RAST procedure; and (3) the protein nitrogen unit (PNU) content. Potencies determined by skin testing 10 sensitive patients were strongly correlated among the various patients. Measurements of potency by both RAST inhibition and direct RAST assay were strongly correlated to potency as measured by skin testing. In contrast, neither the weight:volume nor the PNU content bore any relationship to allergenic potency as measured by skin testing or by either of the RAST procedures. Extracts differed by as much as 3,000-fold in allergen content by skin testing. Moreover, the extracts appeared to contain different allergenic determinants when tested by RAST inhibition. RAST inhibition offered several technical advantages over the direct RAST procedure, in that only one solid-phase RAST reagent was required, slopes of dose-response curves could be more easily compared, and a greater discrimination in allergenic potencies among extracts could be made. The RAST appears to offer an excellent method for measuring the potency of allergy extracts, pending the isolation and characterization of actual allergens.     

Studies on Alternaria allergens: III. Isolation of a major allergenic fraction (ALT-I)

The allergens present in crude Alternaria alternata extract were fractionated by sequential ammonium sulfate precipitation, DEAE cellulose ion-exchange chromatography, preparative flat-bed electrofocusing, and Sephadex G-100 gel filtration. The carbohydrate-rich fraction isolated (Alt-I) was heterogeneous by polyacrylamide gel disc electrophoresis and by thin-layer electrofocusing. By preparative polyacrylamide gel electrophoresis the Alt-I fraction could be further separated into at least five subfractions, all of which contained similar allergenic activity. Significant elevations in IgE antibody to Alt-I were found in $\text{21}\text{25}$ patients with asthma having positive skin tests to crude Alternaria extract but were not found in 15 patients with non-IgE-mediated asthma. Alt-I appears to contain glycoproteins (7% N) and is a major allergenic fraction of Alternaria.     

Measurement of the potency of allergy extracts by their inhibitory capacities in the radioallergosorbent test

The potencies of 11 commercial extracts of short ragweed pollen were analyzed by skin test end point titration and compared to potency as measured in vitro: (1) by the radioallergosorbent test (RAST), (2) by AgE concentrations, and (3) by protein nitrogen determinations (PNU). RAST potency was determined by the capacity of the extract to inhibit the binding of IgE antibody to solid-phase allergen in the first step of the RAST, and it was expressed as the quantity of extract needed for 50 per cent inhibition of binding. Potencies determined by skin testing in 7 patients were strongly related among the various patients in spite of a 1,000-fold difference in the patients' sensitivity to the extracts. Similarly, potency as measured by RAST inhibition showed about a 1,000-fold difference among the extracts, and the RAST potencies were strongly related to potency as measured by skin testing. Purified antigens derived from short ragweed inhibited binding of IgE antibodies, but the slopes of the inhibition curves were significantly less than those produced by crude or partially purified short ragweed. AgE concentration and PNU concentrations also were correlated with skin test potency, but the range of potencies was less than that found by RAST inhibition. Measurement of the potency of extracts by RAST inhibition should prove useful as a general procedure for the standardization of allergy extracts.     

Measurement of IgE antibodies by the radioallergosorbent test. I. Technical considerations in the performance of the test.

Since its introduction by Wide, Bennich, and Johansson in 1967, the radioallergosorbent test (RAST) has been increasingly used for measurement of specific IgE antibodies. In this study we have varied the conditions during the performance of the RAST and have investigated the effect of altering: (1) the speed of rotation of tubes, (2) the number of washes in both steps of the test, (3) the length of rotation of tubes, (4) the temperature, (5) the conditions during centrifugation, (6) the quantity of antibody to IgE in the second step of the test, (7) the composition of buffers in the reactions, (8) the type of tube employed in the test, (9) the quantity of antigen employed during the coupling procedure, and, finally, (10) the iodine isotope, 125I or 131I, used to radiolabel anti-IgE. Of these variables the most significant were the quantity of anti-IgE used in the second step of the test and the quantity of antigen used in the coupling procedure. Other variables of importance were the choice of radioiodine, whether the tubes were rotated or not, the type of tube used, and finally the diluent employed.     

Measurement of IgE antibodies by the radioallergosorbent test: II. Analyses of quantitative relationships in the test

IgE antibodies have been measured by the radioallergosorbent test (RAST) and the relative amounts present in serum determined by comparison with reference standards. In this study we analyzed the quantitative aspects of the binding of IgE antibody to solid-phase ragweed antigens. With the volumes of allergic serum usually tested, IgE antibodies are in excess in the first step of the RAST. The inability of solid-phase antigens to remove IgE antibody appeared to be due to an insufficient quantity of antigen on the particles rather than steric interference with binding of IgE antibody. Dose-response curves with serums from several ragweed-sensitive subjects were not parallel when plotted on a semilog scale. In contrast, log-log plots of dose-response curves with serums from ragweed-sensitive subjects were nearly parallel. Log-log plots of dose-response curves with serums from subjects with ragweed and grass sensitivity and tested with the appropriate solid-phase antigen also were nearly parallel. Because of the latter finding, RAST could be standardized using a reference serum in every assay and plotting the results on a log-log scale. Finally, because IgE antibody is in excess in the RAST as it is usually performed, the final result reflects both the quantity and the affinity of the IgE antibody.     

Preseasonal IgE ragweed antibody level as a predictor of response to therapy of ragweed hay fever with intranasal cromolyn sodium solution.

Intranasal administration of a 4% solution of cromolyn sodium for the treatment of ragweed hay fever was tested in an 8-week double-blind matched-pair study involving 66 patients. Patients on active drug received 5.2 mg into each nostril 6 times daily; control patients received a placebo spray. The treated group showed a significant reduction in mouth breathing (p less than 0.001), stuffy nose (p less than 0.002), runny nose (p less than 0.003), and postnasal drip (p less than 0.035). Patients receiving the active drug also reported fewer sneezing episodes (p less than 0.003) and nose blowing episodes (p less than 0.015). One patient using cromolyn solution developed nasal ulceration, tongue swelling, coughing, and wheezing. Other side effects were minimal and occurred with equal frequency in both groups. In the treated group relief of symptoms was most marked in patients with high preseasonal levels of IgE ragweed antibody. Intranasal 4% cromolyn solution appears to be an effective drug for the treatment of ragweed hay fever; measurement of the preseasonal level of IgE ragweed antibody is a useful screening test to identify patients most likely to achieve a maximal beneficial response to treatment.     

Reactivity of ragweed allergens with IgE antibodies. Analyses by leukocyte histamine release and the radioallergosorbent test and determination of cross-reactivity.

Five distinct proteins with allergenic activity have been isolated from short ragweed pollen. We initially tested three of these, AgE, AgK, and Ra3, for reactivity with IgE antibodies by leukocyte histamine release and by the radioallergosorbent test (RAST). We found highly significant correlations between the reactivities of these allergens by leukocyte histamine release and by the RAST, consistent with the view that both procedures detected comparable allergenic activity. We next tested the allergenic cross-reactivity of all five ragweed allergens. AgE, AgK, Ra3, Ra4, and Ra5, by RAST inhibition. With solid-phase AgE the only nonhomologous inhibitor was AgK, which cross-reacted weakly and required a 140-fold mass excess of AgK compared to AgE. With solid-phase AgK both AgK and AgE produced significant inhibition; AgE was slightly more potent than the homologous AgK, Ra3 and Ra5 were allergenically unique, because only the homologous allergen produced 50% inhibition. Ra4 was weakly inhibited by AgE, Ra3, and Ra5 when these allergens were added in 300- to 5---fold mass excesses; this weak inhibition may represent either cross-reaction or cross-contamination. We found that RAST inhibition could be used as an assay for the individual ragweed allergens and we demonstrated the presence of all of the allergens in a whole ragweed extract. The sensitivity of the RAST inhibition assay ranged from 10 ng to 100 ng for 50% inhibition. Finally, the solid-phase ragweed allergens were used to determine the frequency of elevated IgE antibody levels in 65 patients with ragweed hay fever. Virtually all of the patients reacted with AgE (97%), while 88% reacted with AgK, 51% reacted with Ra3, 28% reacted with Ra4, and 17% reacted with Ra5. These results highlight the usefulness of the RAST as a specific and sensitive tool for immunochemical studies of allergens.     

Immunological and biochemical studies in beekeepers and their family members

To determine the immunological and biochemical consequences of chronic honeybee venom administration to humans, we studied beekeepers and their family members in two separate investigations. In the first of these studies serums were analyzed for their content of antibodies to honeybee constituents. IgE antibody levels to honeybee venom (HBV), venom sac (HBVS), and phospholipase-A (PLA) were significantly elevated in beekeepers and their family members as compared to non-beekeeper controls. However, IgE antibody levels to HBVS, HBV, and PLA did not distinguish sting-sensitive from non-sensitive beekeepers. IgE antibody levels to honeybee whole body (HBWB) were highest in beekeepers with histories suggestive of inhalant sensitivity to bee body dust. IgG antibody levels to PLA were highest in non-sensitive beekeepers stung daily or weekly; there was a significant positive correlation between IgE and IgG antibody levels to PLA in these beekeepers. In the second study the potentially harmful effects of chronic venom administration were sought, especially evidence of chronic renal disease. While a number of beekeepers and family members showed minor abnormalities in their urinalysis or blood chemistry determinations, in most instances the incidence of abnormalities was not greater among persons receiving a larger number of stings. Commercial beekeepers had a significantly greater incidence of urinary free lipids and hyaline casts, but in no case were large amounts of these substances found. The results discourage the belief that chronic honeybee venom administration is associated with any clinical disease in humans.     

Interference in ragweed pollen and honeybee venom radioallergosorbent tests

We studied sera from patients sensitive to short ragweed (SRW) and honeybee venom (HBV) to investigate serum factors able to interfere with the measurement of IgE antibody levels by the radioallergosorbent test (RAST). We heated sera to destroy IgE antibodies and tested them for interference in the RAST. Heating sera for 4 hr at 56 °C destroyed up to 98% of the IgE antibody activity. After immunotherapy sera from patients sensitive to SRW and HBV produced striking interference in the RAST. The interference was most marked in the RAST employing 50-μg quantities of microcrystalline cellulose—linked allergens, but it was also evident in the RAST employing 500-μg quantities of such allergens and in the commercial RAST in which allergen is linked to paper disks. The interfering substance eluted from diethylaminoethyl (DEAE) cellulose in the IgG fraction. The interference could be eliminated by increasing the relative quantity of solid-phase allergen; RAST interference was not detected when SRW extract was linked to Sepharose 4B. The results indicate that serum factors, presumably IgG antibodies, produce interference in the RAST. Thus immunotherapy studies that measure IgE antibody levels by the RAST must consider the possibility that IgG antibodies can appear to depress IgE antibody levels. Furthermore, because commercially available RAST disks are susceptible to RAST interference, they must be used with caution for the diagnosis of allergy in patients whose sera may contain significant quantities of IgG antibodies.     

Anaphylaxis after ingestion of millet seeds

We report the case of a patient with anaphylaxis after ingestion of millet seeds, a previously unreported allergen. Skin testing with freshly prepared millet seed extract produced positive intradermal responses at a concentration of 100 pg/ml. The reaction was mediated by IgE antibodies to millet, as demonstrated by elevated specific IgE antibody levels by radioallergosorbent test (RAST) and by significant in vitro histamine release from leukocytes incubated with millet seed extract.     

Detection of allergy to nuts by the radioallergosorbent test.

The diagnosis of food allergy is often difficult to make by conventional means. Histories are frequently ambiguous, and skin testing is of dubious reliability because of the number of false-positive and false-negative reactions. We have evaluated the radioallergosorbent test (RAST) for the in vitro measurement of the specific IgE antibodies to nuts, including Brazil nut, almond, walnut, pecan, cashew, and the legume, peanut. Serums were obtained from 18 patients with a history of nut allergy and IgE level and specific IgE antibodies were measured. Thirteen of the 18 patients had significantly elevated IgE antibody (greater than twice control) to one or more of the allergens. Prausnitz-Küstner tests on selected serums in general corroborated the results of the in vitro studies. Five patients had RAST elevations to 2 or more nuts. As a group RAST-positive patients had elevated mean serum IgE levels and more severe clinical symptoms (p less than 0.01). The specificity and cross-reactivity of IgE antibodies to different nut antigens was investigated by RAST inhibition with serums from 5 patients having high levels of IgE antibody. In 4 patients no cross-reactivity between Brazil nut and peanut was found. In contrast, several nut extracts inhibited the reaction of pecan allergen with IgE antibodies. These results indicate that specific IgE antibodies can be measured by RAST in patients with nut allergy and the cross-reactivity of nut antigens can be investigated. RAST would appear to be most useful in confirming the diagnosis of nut hypersensitivity in children or in highly allergic patients in whom skin testing poses a risk of anaphylaxis.     

The effect of cromolyn sodium powder as a treatment for ragweed pollinosis

The effect of treating ragweed pollinosis with intranasal cromolyn sodium powder was evaluated in 26 patients in a double-blind clinical study. Thirteen matched patient pairs were treated by either nasal insufflation of cromolyn sodium powder, 20 mg three times daily, or by a placebo powder. Patients were monitored for severity of symptoms by means of daily symptom diaries collected weekly throughout the pollen season. Serum samples were obtained in July prior to the ragweed season and following the ragweed season during the first and fourth weeks of October for the determination of ragweed-specific IgE antibody levels by the radioallergosorbent test. A significant reduction in symptom severity was observed in the treated patients with high preseasonal levels of IgE antibody to ragweed. Initially the levels of IgE antibody to ragweed did not differ between the two groups. IgE antibody levels increased in both groups after pollen exposure, but the treated group showed a significantly greater rise in IgE antibody levels. The results suggest that treatment with cromolyn sodium enhanced IgE antibody response to pollen exposure and significantly reduced the severity of hay fever symptoms in patients with high preseasonal levels of IgE antibody to ragweed.     

Comparison of procedures for measurement of IgE protein in serum and secretions.

Because of conflicting reports in the literature concerning the value of various procedures for measurement of IgE in serum and secretions, we compared four different methods, the radioimmunosorbent test (RIST), the double-antibody radioimmunoassay (RIA), the paper disc immunosorbent test (PRIST), and radial immunodiffusion (RID). The standards used in the assays were tested initially in the double-antibody RIA with the use of the reference from the World Health Organization. The results showed that RID as expected was relatively insensitive and IgE was reliably measured only above approximately 1,000 international units (IU). Moreover, certain sera containing low levels of IgE by the other procedures gave distinct precipitin zones and presumably falsely high levels of IgE protein. Thus RID may yield apparently erroneous results when used as a screening procedure for measurement of IgE levels. Among the other procedures PRIST and the double-antibody RIA showed the best agreement. With serum samples RIST yielded values for IgE in the low level range higher than those given by the PRIST and double-antibody RIA. With breast milk and colostrum, values of IgE between 120 and 690 ng/ml were found by RIST, whereas IgE was not detected by double-antibody RIA and PRIST. No evidence of an inhibitor of IgE was found in breast milk, so that the apparent elevation of IgE in breast milk by the RIST is likely false. These findings confirm prior reports of spurious elevations of IgE with the RIST and indicate the usefulness of the PRIST and double-antibody RIA for the measurement of IgE in sera and secretions.     

Measurement of the absolute levels of IgE antibodies in patients with ragweed hay fever: Effect of immunotherapy on seasonal changes and relationship to IgG antibodies

The effect of immunotherapy with aqueous ragweed pollen extract on changes in IgE antibody was analyzed in 40 untreated patients with ragweed hay fever and compared to changes in 63 treated patients. Treated patients received cumulative doses prior to and during treatment ranging from 1.4 × 10⁵ to 4.8 × 10⁶ protein nitrogen units (PNU). IgG antibody to ragweed antigen E (AgE) was measured by radioimmunoprecipitation, while IgE antibody to allergens in crude ragweed extract was measured by the radioallergosorbent test (RAST). The RAST procedure was calibrated using a serum whose content of IgE antibody in nanograms per milliliter had been determined by immunoabsorption, and with this method the absolute quantities of IgE antibodies to ragweed allergens could be measured. Control experiments indicated that the IgG antibodies in the sera of the treated patients did not interfere with the measurements of IgE antibodies. The levels of IgE antibody in serum varied from less than 5 ng/ml to approximately 3,000 ng/ml. IgE antibodies decreased from October, 1973, to July, 1974, and rose sharply from July to October, 1974, following the pollination season. In both untreated and treated patient groups the magnitudes of the decreases were related (p < 0.001) to the levels of IgE antibody in October, and the magnitudes of the rises were related to the levels of IgE antibody in July. In the treated patients with IgE antibody less than 71 ng/ml in October, the rate of decrease (October to July) was less than in the untreated patients. Comparison of the rises in IgE antibody from July to October revealed that these were partially suppressed in the treated group (p < 0.001), and this effect was most marked in patients with July levels less than 36 ng/ml. Levels of IgG antibody to AgE in the treated patients were approximately 70-fold greater than in untreated patients and showed little change over the study period. The levels of IgG and IgE antibody in the treated patients were positively correlated (p < 0.001) at all time periods, and patients with high levels of IgG antibody also had greater declines and rises in IgE antibodies. The results indicate that immunotherapy is associated with (1) a reduction in the seasonal decrease in IgE antibody from October to July in patients with the low levels of IgE antibody, (2) a partial suppression of the rises in IgE antibody from July to October, and (3) that both these effects are related to the level of IgE antibody before the change. In many treated patients both IgE and IgG antibodies were low in spite of parenteral administration of ragweed extract. This result is consistent with the view that overall humoral immunologic reactivity to ragweed antigens is reduced in these patients.     

Rush venom immunotherapy program for honeybee sting sensitivity.

The clinical and immunological effects of honeybee venom (HBV) immunotherapy were studied in 20 HBV-sensitive patients. Patients were treated once monthly in a hospital setting by “rush” immunotherapy; we aimed for a maximum single dose of 200 μg. One patient withdrew shortly after beginning therapy. Eleven patients required epinephrine during treatment for reversal of anaphylaxis. Patients who tolerated maximum doses were readmitted one month later for deliberate honeybee sting challenges. Stings were tolerated by 11 of 19 patients; 5 of the remaining 8 required epinephrine therapy. Twelve of 16 patients achieved long-term protection as determined by deliberate stings; 4 of 16 achieved partial protection. Three additional patients, who were treatment failures, were switched to weekly immunotherapy. IgE antibodies to HBV and phospholipase A (PLA) rose shortly after immunotherapy was begun and in many patients remained elevated above baseline values. Serum IgG antibodies to PLA rose steadily during therapy; there was no absolute level of IgG antibody which was protective for all patients. Although satisfactory long-term clinical results were achieved for most patients, the frequency of systemic reactions during the rush immunization caused us to abandon this form of treatment in favor of a once-weekly outpatient injection program.     

Changes in the antigenic composition of the short ragweed plant during maturation.

Whole ragweed plants were collected before and during the ragweed pollination season and extracts of various portions of the plants were analyzed for antigen E (AgE) and for their inhibitory potency in the radioallergosorbent test (RAST). Ragweed stems and roots contained very low, if any, AgE. AgE was present in very small amounts in anthers prior to pollination season, increased with anther and pollen grain maturation, peaked with the appearance of significant airborne pollen grains in mid-August, and fell slowly through late September. Leaf AgE content also rose during mid- and late August, but brief rinsing reduced antigen E content by almost 50%. Significant RAST inhibitory activity was found only in anthers and the activity paralleled the content of AgE.     

Melittin: An allergen of honeybee venom

The presence of serum IgE antibodies to melittin was tested by the radioallergosorbent test (RAST). Melittin, the principal protein of honeybee venom, was isolated by gel filtration on Sephadex G-75 and covalently bound to cyanogen bromide-activated microcrystalline cellulose. The melittin preparation was homogenous by immunoelectrophoresis with the use of rabbit antiserum to whole honeybee venom and by polyacrylamide electrophoresis in gels containing 1% sodium dodecyl sulfate. Elevated serum IgE antibodies to melittin (three times greater than binding by normal sera) were found in 7 of 24 honeybee venom-sensitive persons and in 5 of 20 nonsensitive beekeepers. In one venom-sensitive patient a particularly high titer of IgE antibody was found. The reaction between solid-phase melittin and IgE antibody could be inhibited by fluid-phase melittin but not by phospholipase A (PLA). Similarly, the reaction of IgE antibody with solid-phase PLA was inhibited by PLA but not by melittin. In passive transfer skin tests with the sensitive patient's serum, positive wheal-and-flare reactions were obtained in 3 nonallergic recipients following melittin challenge; appropriate controls were negative. These results indicate that melittin is an allergen in some honeybee venom-sensitive patients and in an occasional patient melittin may be a major allergen.     

Topical ocular administration of cromolyn sodium for treatment in seasonal ragweed conjunctivitis.

We studied topical ocular administration of a 4% solution of cromolyn sodium (CS)for treatment of 112 patients with ragweed conjunctivitis in two 8-wk, double-blind, matched-pair studies. During the 1977 and 1978 ragweed seasons, patients instilled a daily total of 12.8 or 19.2 mg CS into each eye, control patients received placebo drops. Treatment efficacy was assessed by daily symptom score diaries, ophthalmic examinations, and a subjective report by the patient at the end of the trials. In the 1977 trial, CS pair members whose preseasonal immunoglobulin E antibody level was <99 ng/ml showed a significant reduction in eye itching (p < 0.01), eye irritation (p < 0.02), visual blurring (p < 0.025), and nasal congestion (p < 0.025) and required less supplemental medication (p < 0.02) during the peak pollen period. In the 1978 trial all patients had IgE antibody levels >100 ng/ml. CS pair members showed a significant reduction in rhinorrhea (p < 0.004) and nasal congestion (p < 0.007) only; no significant reduction in eye symptoms was noted. Transient eye burning was noted by a total of 38 CS and 39 placebo patients from both studies; ophthalmic examinations were unremarkable; and no other side effects were noted. The 4% CS ophthalmic solution provided significant relief of nasal symptoms in both the 1977 and 1978 trials, but significant relief of ocular symptoms was noted only in the 1977 trial and only in those patients with lower preseasonal IgE ragweed antibody levels.     

Measurement of the potency of pollen extract by radioimmunoprecipitation.

We describe a novel procedure for standardization of allergen extracts using radioiodinated crude extracts. Radiolabeled extracts were reacted with immunoglogulin E (IgE) antibody, and the resulting complexes precipitated by anti-IgE. Experiments with ragweed pollen, grass pollen, honeybee venom, and Alternaria extracts revealed that 1% to 14% of the radioactivity was precipitated, these percentages ranged from 2 to 20 times blank values. Binding of labeled ragweed extracts to IgE antibody could be inhibited by unlabeled extracts and linear inhibition responses could be derived. The potencies of 11 short ragweed extracts were ranked by their inhibitory capacities in such an IgE radioimmunoprecipitation (RIP) test and compared to potencies determined by radioallergosorbent test (RAST) inhibition; these were related, rs = +0.97 (p less than 0.001). Labeled extracts were also reacted with IgG antibodies, and the resulting complexes were precipitated by anti-IgG. From 1% to 34% of the radioactivity associated with the various extracts was precipitated by anti-IgG, and these percentages were from 2 to 20 times blank values. The reaction of IgG antibody with radiolabeled pollen extracts could be inhibited by unlabeled extract, and in the case of short ragweed the measurements of potencies using IgG and IgE antibodies were correlated, rs = +0.96 (p less than 0.001), suggesting that similar determinants were recognized by both IgG and IgE classes. The RIP procedure provides an alternative to the RAST for the standardization of allergy extracts and eliminates the need for affinity chromatography-purified antibody to IgE.