- Email: [email protected]
Allergy testing
Allergy testing = R.G. Hamilton Johns Hopkins
University Current
School of Medicine, Baltimore,
Opinion
in Immunology
Introduction The field of allergy testing encompasses clinical and laboratory methods that are used in the diagnosis of individuals suspected of having &E-mediated hypersensitivities, in the identification and quantification of offending allergens in the environment, and in the monitoring of immunologic indicators of therapy efficacy. This subject deals with reagents and assays that are constantly moving towards improved standardization, sensitivity and specilicity. An issue of Clinic& Reviews in Allergy was recently devoted to a series of papers on the role of the laboratory in the evaluation of allergic disease [l-7]. These papers examine new developments in skin testing, in vitro assays for IgE, bronchial challenges, nasal challenges, methods for assessing the role of basophils, eosinophils and platelets in allergy, and diagnostic methods and tests for food allergy.
Maryland,
USA
1990, 2:558-564
and house dust mite (Ford et al, JAllergy Clin Immunol 1985, 75:676). Wahl et al. (JAllergy Clin Immunoll989, 84:44&456) applied the Phleum protense IS to the characterization of both in-house and commercially available timothy grass pollen extracts. Using their antiserum, they detected an allergen in their extract that was not described in the initial IS collaborative study. They attribute this failure to differences in the source of the timothy grass pollen. The IS was also useful in validating their in-house grass pollen human serum pool in comparison with that prepared by the World Health Organization.
Standardization
An additional live international allergen standards are being prepared, to extracts of dog dander/hair, birch, bermuda-grass pollen, cat dander/hair and Alternuriu. Iarsen et al. [9] report the characterization of the standard dog (Can& domesticus) hair/dander extract. The total allergenic activity of test dog hair and dander extracts were compared by radioallergosorbent test (RAST) inhibition, skin testing and leukocyte histamine release. Allergen complexity was assessed by electrophoretic separation of the individual extract components and by using IgF antibodies to detect the allergens in crossed immunoelectrophoresis (CIE), rocket immunoelectrophoresis, isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE) with immunoblotting. Twenty-two precipitin lines were detected in the CIE of the proposed IS, 11 that were identified as being of serum origin by precipitation with dog serumspeci6c antiserum. The CIE pattern detected by Ford et al. (Clin Exp Allergy 1989, 19:183-190) in a more recent study with a different dog extract showed 28 distinct antigens, 11 of which were common to dog serum proteins.
Reed et UC [8] examine the major problems in allergy tests that may be associated with the variable qualities of an allergen. Internationally documented allergen stardards are being prepared as ‘yard sticks to compare and validate the allergen content and biological activity of in house or national reference extracts. International standards (IS) have been finalized for timothy grass (Gjesing et al, J Allergy Clin Immunol 1985, 75:258), short ragweed (Helm et al, JAllergy Clin Immunol1984, 73:79(I)
Arntzen et al. [lo] describe the collaborative testing of birch (Bet& verrucasu) pollen extracts for identilication of a suitable IS. Of the six extracts tested, all but one ~fullilledthe desired specifications for an IS: > 0.5mg of protein per ml, > 20 bands by IEF, > 3 detectable al lergens (Ag 34, Ag 19 and Ag 251, > 4Oug of Ag 23 per mg of protein and > 15% of total added radioactivity retained in direct RAST. The chosen standard was shown to be stable for 36 months when stored at 5°C.
Allergens The allergen has been the single most scrutinized component of the allergy test. Allergens are extracted using different solvents and protocols from crude weed, grass and tree pollens, venoms, foods, molds, epidermal scrapings, parasites and insects. Extracts, by their very nature, vary widely in allergen content, biological potency and IgE antibody-binding capacity.
Abbreviations AU--allergy IEF-isoelectric
558
unit; Cl&crossed
focusing
IS--International
immunoelectrophoresis; Standards;
@
FAV-fire
PAC&polyacrylamide
ant venom;
HEP-histamine
gel electrophoresis;
Current Science Ltd ISSN 0952-7915
equivalent
prick;
RAST-radioallergosorbent
test.
Allergy
The most definitive statement this year on allergen and methodOlOgy standardization was presented in the American Academy of Allergy task force report on standardization of old and new technologies used in the diagnosis and treatment of allergic diseases [ 111. These proceedings are divided into four sections relating to percutaneous and intracutaneous skin tests, late-phase reactions, in vitro diagnostic tests, and an overview of diagnostic tests for cell-mediated immune reactions (delayed hypersensitivity). The section on standardization of skin tests examines performance factors, interpretation of test results and biological equivalency tests using 1 mg/ml of histamine dihydrochloride as a reference (e.g. histamine equivalent prick; HEP). Alternatively, biological activity has been expressed in activity units for the allergen extracts without reference to the HEP. The allergy unit (AU) of potency is equivalent to the mean three-fold intradermal dilution of an allergen extract that produces a 50 mm sum of perpendicular eiythema diameters in a patient known to be puncture-test positive. The in vitro diagnostic test section examines technological approaches, reporting schemes, threshold criteria, and quality control. The authors recommend the use of assays with well documented solid-phase allergens and anti-IgE reagents and the use of quantitative reporting methods in which units are proportional to antibody content. Allergen
cross-reactivity
Cross-reactivity among allergens also complicates allergy testing. Hoffman et al. (I Allergy Clin Immunol 1988, 82:81@327) report on the properties of four allergens (Sol i I-N) isolated from fire ant (S0lenopsi.s invictu) venom (FAV). Sol i II (28 kD) and III (26 k~) were shown to be major proteins in FAV, while Sol i I (37kD) and N (20 kD) were only detected in small amounts. All four components were shown to be signilicant allergens that are recognized by human IgE antibodies from FAV-allergic individuals. Hoffman et al. [ 121 used these purified Sol i I-N allergens to study the cross-reactivity of IgE antibodies from patients sensitive to cross-reactive bee and wasp venom. Immunoassays using solid-phase FAV, bee and wasp venoms were used to screen sera from individuals who were: (1) not allergic to venoms; (2) exposed but not allergic to FAV; (3) allergic to FAV; (4) allergic to bee and wasp venoms but not FAV; and (5) allergic to bee and wasp venoms and exposed to FAV. Reactivity with FAVwas 0% (group l), 24% (2),100% (3), 54% (4) and 87% (5). Sol i I was shown by immunoblot analysis to be the major cross-reactive allergen of imported FAV. Commercial sources of FAV vary widely in how much of these components they contain (Butcher and Reed, J Alleqy Clin Zmmunoll!X38, 82:770-777), indicating that more rigorous testing and standardization are needed. Penicillin is one of many antibiotics with a p-lactam structure. Bianca et al. (J Allergy Clin Zmmunol 1989, 83:381-385) challenged a group of patients allergic to penicillin with a new cephalosporin, cephamadole, which contains side-chain structures similar to peni clllin. Two out of 19 penicillin-allergic patients reacted to a cephamadole challenge, supporting previous ob-
testing
Hamilton
servations about the cross-reactivity between penicillin and cephalosporins (Batchelor et al, Immunology 1966, 10:21). Another study examined the possible cross-reactivity between penicillin and imipenem, the first member of new class of jG~tam antibiotics called the carbapenems. Saxon et al. (J Allera Clin Zmmunoll988, 82:213217) found 20 patients who were skin-test positive to one or more penicillin determinants and also reacted to imipenem. These studies indicate the importance of controlled challenge tests in the study of penicillinallergic patients for possible sensitivities to cephalosporins and carbapenems. Allergen
identification
One of the first steps in the preparation of a well documented allergen reference preparation is the identlfication of the major and minor allergens in particular extracts. This year, several studies have enhanced our knowledge of specific allergenic and antigenic components in extracts. Aqueous white oak pollen extracts prepared by Loria et al. (I Allergy Clin Zmmunoll989, 84:!+18) were shown to contain 45 distinct protein bands using sodium dodecyl sulphate polyacrylamide gel electrophoresis. IgE antibodies from 30 out of 38 oak-sensitive patients bound 23 bands from 13.2-108kD. There was no single band that could be recognized by IgE antibody from every patient. Two proteins (74-77.9 k~ and 16.2-17.7 kD) appeared to be important allergens because they were bound by IgE from 71% of patients’ sera. Iranitalab et al. (Allergy 1989, 44:314-321) studied four extracts of Micropolyqora faeni by western blot analysis using serum from farmers with (n = 25) and without ( n = 14) extrinsic allergic alveolitis, all of whom were exposed to hay, and 27 healthy controls. Major antigens of 11,12,25,35 and 60 kD were identiIied with serum from this group. Although farmers with extrinsic allergic alveolitis had higher levels of IgG, IgM and IgA antibodies to these M. faeni antigens than farmers without the disease, there was no correlation between the antibody response and the onset of disease. Variation in antigen content between commercial M. faeni extracts led the authors to conclude that the extracts must have at least the 11, 25 and 60kD components before they can be used in in vitro diagnosis of M. faeni-induced extrinsic allergic alveoli&. Baur et al. (JAllergy Clin Zmmunol1989,83:839+44) examined whether different stages of allergic bronchopulmonary aspergillosis may correlate with variations in the level and specificity of the antibody response. Western blots were performed using @ergillusfumigatus allergen extracts with serum from 28 patients with allergic bronchopulmonary aspergillosis in stages II to V. The pattern of IgG and IgE antibody binding to R jkmigatus did not discrimina te between the stages of disease, as both reacted with the same antigens. Only the level of IgE antibody appeared to be stronger in stages III to V. To identify new allergens, human serum pools from well characterized patients and polyclonal and monoclonal
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Atopic allergy
and other
hypersensitivities
antibodies made to the total allergen extract, as well as individual components, are often required. Van Ree et al. (j Allergy Clin Immunoll989, 83:144151) demonstrated the value of both monoclonal and polyclonal antibodies in the identification of new allergens including an as yet unidentified major allergen from grass pollen in the group I molecular weight range. De Grout et al. (I Allergy Clin Immunol1988, 82:778-786) have prepared monoclonal antibodies to Fe1 d I (the major allergen in cat dander) which have been useful in immunoassays that detect the allergen,
-While accurate immunoassays are available for measuring the level of major cat allergens in house dust, the measurement of dog allergens awaits the preparation of specific antibodies. Breed-specific dog dandruff allergens have been identified by Lindgren et al. (I Al/era Clin Immunoll988, 82:196-204) using CIE and serum from patients who were clinically allergic to dogs. Dog breed extracts contained common allergens and also allergenic components that only occur in one or two dog breed extracts. Although the study identified skin-test reactivity to breed-specific allergens in about 15% of individuals allergic to dogs, the degree of reactivity did not correlate with allergic symptoms to that specific dog breed.
Environmental
1988, Schumacher et al. (r Allergy Clin Immunol 82:6Oti16) used immunoblot microscopy to differentiate grass pollen aeroantigens collected in air sam plers. Antigen particle counts were performed by apply ing pollen to adhesive tape, blotting it onto nitrocellulose and detecting the antigen using specific antibodies and a fluorescein-conjugated second antibody. Fluorescence microscopy-derived ‘antigen particle counts’ were compared with light microscopy ‘pollen counts’. This method has potential use in the quality control of source pollen; it may be able to detect contamination of commercially available grass pollen by pollen of other grass types. Moreover, the study showed that the number of airborne particles associated with specific antigens was greater than the number of grass pollen grains, indi eating that morphologically unrecognizable grass pollen antigens were present in such samples.
allergen
testing
One of the most direct methods of allergy treatment is to remove the allergen from the patient’s environment or vice versa. The availability of new monoclonal antibocty reagents and assays has allowed the diagnostic al lergy laboratory to quantify the level of allergen in the home environment. Several recent reviews examine the rationale and practical approaches to the evaluation of allergen levels in the home. Dust containing dust mite, cockroach and animal (cat and dog) and mold allergens is most often found in old houses. Klein and Ziering [ 131 provide a home evaluation questionnaire which is useful in identifying major offending household allergens. Pollart et al. [ 141 focus on house dust mite allergen as a risk factor in allergies and on methods for dust control. The level of dust mite allergen in a specimen of house dust can be used as a bench mark upon which to judge effective removal of allergen by rigorous cleaning techniques (Green et al, Clin Exp Allergv 1989, 19:203_207). Moreover, the level of allergen can be used to assess the relative importance of a particular allergen in the environment to a patient who is skin-test-positive and/or RASTpositive. Assays are available for measuring group I allergens from Dermutopbagoidespteronyssinus (Der p I) and D. farinae (Der f I). Patients exposed to levels of Der p I or Der f I of more than 10 ug per gram of dust often experience allergic symptoms, while those in envi ronments containing < 1 ug/g seem to be symptom-free. While these levels cannot be directly translated into mite counts, 10 gg/g of Der p I is approximately equivalent to 200 mites per gram of dust (Platts-Mills et al, J Allergy RAST inhibition with house Clin Immunoll986,78:398). dust extracts has been used as an indirect approach to estimate the level of D. farinue allergen in house dust (Twiggs et al, Clin Rev Allergy 1988, 6:3542). Wood et al. [ 151 studied the rate of cat allergen reduction in dust specimens collected from 15 homes over a 943.week period after removing the cat. Baseline levels of Fe1 d I in houses with at least one cat were 7-436.7 Food and Drug Administration units per g of dust (median 61.2 units/g). After removal of cats, Fe1 d I levels in most homes declined gradually to < 1 unit/g, which is similar to homes without cats, in 2&24 weeks. This study shows that even when the allergen source (the cat) is removed, elimination of allergen from the environment can be a slow and variable process and monitoring of major allergen levels is useful in documenting allergen removal.
Jensen et al. (Allera 1989, 44:52) studied the use of a high-volume air sampler that was equipped with 0.3 l,trn glass fiber lilters to collect both amorphous air-borne allergens of respirable size and pollen grains. Birch and timothy allergens eluted from lilters were analyzed by an IgG RAST inhibition assay. The amount of airborne allergen collected by the high-volume air sampler and measured immunochemically correlated signiIicantly with pollen counts obtained using a pollen trap located in the same place. 0
Humoral immune responses Allergen-specific
IgE
Diagnostic allergy testing is based on the detection of allergen-specific IgE antibodies by in vivo challenge methods in which the skin, nasal passages and bronchi are exposed to allergen, or by in vitro methods in which .speciIic IgE from serum and nasal secretions is bound to solid-phase allergens in the RAST. Discrepancies have been observed between the levels of allergen-specific IgE detected by skin test and those found in serum by RAST, the most common being a positive skin test with a negative RAST. Van der Zee et al. [ 161 performed paired intracutaneous skin tests and RASTs on 660 adult patients to detect specific IgE to grass pollen, house dust mite and cat dander. Out of 1980 skin test and RAST combinations, there were 140 discrepancies, more than 80%
Allergy
of which were positive skin reactions without detectable allergen-specific IgE antibodies in serum (negative RAST). A second RAST analysis using commercial and in-house reagents and skin tests reproduced > 80% of these discordant results. Thus, discordant skin-test and RAST results are not a consequence of random experimental errors. Histamine was released from the washed leukocytes of 82% of 28 patients with positive skin tests and negative RASTs upon in vitro challenge with the relevant allergen extract. Moreover, low levels of specific IgE in the serum of skin-test-posit-negative individuals were confirmed when a positive RAST was obtained using atlinity-purified IgE from serum of these patients. IgG* antibodies were not detected in most of these preparations. Thus, it was concluded that the majority of positive skin reactions that were accompanied by a negative RAST are caused by IgE antibodies whose levels are too low or whose allergenic components are present on the solid phase in quantities too small to be detected in vitro. Serologists have questioned whether more quantitative estimates of spectic IgE would be useful in monitoring allergy patients. Seasonal exposure to aeroallergens is known to increase serum IgE antibody levels towards the end of the pollination period but the relationship between IgE antibody levels and the patients’ symptoms has remained unclear. Somville et al. [ 171 assessed variations in symptoms and serum IgE antibody levels in 78 grasspollen-allergic patients during the pollen season. They showed that specific serum IgE antibodies are of little use in predicting the clinical symptoms of grass pollen-hypersensitive patients since IgE levels strongly depend on individual immune responses. This conclusion was analogous to that of Reisman and De Masi (Znt Arch Allergy Appl Zmmunol1989, 8967-70) who showed that absolute levels of serum venom-specific IgE in Hymenoptera venom-allergic patients were unrelated to any clinical feature of the disease. These studies suggest that the absolute measurement, in weight-per-volume units, of specific IgE in preseason sera is of minimal value in predicting the extent of clinical symptoms during the season. Technical
Hamilton
82:812) demonstrated the usefulness of solid-phase antiIgE in binding IgE from serum. When it was used with a labeled allergen, isotypic interference of the binding of IgE antibody to limited allergen binding sites by IgG antibody could be minimized. This works best, however, when the ratio of specific IgE to total IgE is high. The value of these modifications is difficult to assess from these reports because many assay and specimen variables alfect the accuracy of an IgE antibody measurement. However, these reports do provide a perspective on trends in in vitro IgE detection methods that relate to: (1) improved quality of the allergen; (2) enhanced binding capacity of the solid-phase allergosorbent; (3) methods for documentation and quality control of the labeled anti-&E; and (4) methods for Improvement of IgE antibody detection in the presence of high levels of IgG antibody.
less common
allergen
specificities
A number of studies examining IgE antibodies to al lergens that are not discussed frequently have been reported this year.
(1)
(2)
(3)
issues
There have been few advances in specific IKE assays during the past year. Several reviews [2,3] and the task force report [ II] continue to recommend that maximum sensitivity is obtained by the modified RAST format with 0.1 ml of serum and an overnight incubation. Some alterations in the solid phase have been proposed to enhance sensitivity of the RAST for detecting IgE antibodies secreted by lymphocytes in culture fluid (Poulsen et al, Allergy 1989, 44:174) and in nasal secretions (Marcucci et al, Clin Exp Allergy 1989, 19:157). A new aluminium hydroxide-adsorbed allergosorbent did not perform as well as the paper disk-insolubilized allergen in the Phadebas RAST (Heinlg et al, Allergy 1989, 44:260). A dipstick enzyme immunoassay for IgE antibodies (Twiggs et al., Ann Al lergy 1989, 63:225) and the multi-allergosorbent chemiluminescent assay (Finnerty et al, Clin Exp Allergy 1989, 19:51) were shown to detect IgE to a number of allergen specificities. Lee et al. (IAllergy Clin Zmmunol1988,
testing
(4)
(5)
(6)
Spaner et al. (I Allergy Clin Zmmunol 1989, 83:1135-1137) presented the case study of an in dividual with hypersensitivity to natural latex who has specific IgE antibodies detectable by skin tests and RASTs. Boxer et al. (r Allergy Clin Zmmunol 1988, 82:622426) presented evidence from experiments in which IgE antibodies to metabisullites from patients with a clinical history and positive skin test were able to sensitize monkeys passively. However, IgE anti-metabisuliite antibod ies could not be detected by immunoassays using metabisulliteprotein conjugates as the solidphase antigen. Clin Zmmunol 1988, Enberg et al. (I Allera 82:795-800) studied the serum of patients with a history of sensitivity to water melon. IKE and IKE antibody responses to extracted and immunoblotted water melon antigens were shown to have extensive heterogeneity. Neither IgE nor IgG antibodies specific for water melon allergens were predictive of clinical symptoms. Kramps et al. (Clin Exp Allergy 1989, 19:509-514) studied individuals who had been exposed to formaldehyde as a result of their occupation. They concluded that formaldehyde may provoke the production of specific IgE antibodies, but rarely does. Although the cross-reactivities of beef, pork and human insulin are well known, Grammer et al. (Clin Exp Allergy 1989, 19:551-553) described a patient with anaphylaxis to beef and pork insulin who did not cross-react with recombinant human insulin. Chloramine T is an oxidative agent used in antiseptics and disinfectants. Wass et al. (Clin Exp AC lergy 1989, 19:463471) examined the specificity
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Atopic allergy and other hypersensitivities
of IgE antibodies to chloramine T using different ratios of human serum albumin carrier to antigen. No matter what the carrier protein form, IgE antibodies from a chloramine T-sensitive patient remained specific only to the hapten. Interestingly, no IgG antibodies to chloramine T could be detected despite the presence of high, inhibitable levels of IgE antibody.
Allergen-specific
IgC
Allergen-specilic IgG antibodies have been used: (1) as indicators of exposure to a particular antigen; (2) in identification of the offending allergen among a group of related allergenic substances such as the Hymenoptera venoms; and (3) in selected cases, to study the efficacy of an immunotherapy regimen. The levels and specificity of IgG antibodies are highly dependent on the allergen, the patient, and the mode and duration of exposure. The level of IgG antibodies generally correlates with the extent of exposure to the allergen either by accidental or intentional exposure (van Metre et al, J Allergy Clin Immunoll988, 82:10551068; Nordvall et al, Allergy 1989, 44:380-384; Wass et al, J Allergy Clin Immunol 1988, 82679686; Price et al., J Alleqy Clin Immunol 1989, 84:520-528). Allergen immunotherapy is known to induce the formation of IgG antibodies that are highly restricted to IgG subclasses 1 and 4. Because of its compact hinge, inability to activate complement and reported functional monovalency, IgG4 has good properties as a ‘blocking’ antibody. Devey et al. [ 181 used a competitive inhibition enzyme immunoassay to show that the functional aifinities of IgGl anti-phospholipase A2 appear to decrease over the course of honey bee venom immunotherapy, while an increase in the quantity and relative affinity of IgG* antibodies can be observed. Golden et al. [ 193 investigated how long venom immunotherapy should be continued to ensure protection from a systemic allergic skin reaction to a subsequent sting. Thirty patients chose to stop immunotherapy after 5 years of treatment. Skin-test reactivity decreased during the im munotherapy of 18 out of 30 patients, but it remained positive in 23 out of 30 patients. Serum venom-specific IgE was at the lower limit of detection (1 ng/ml) in 11 out of 30 patients. Mean serum IgG anti-venom declined from 5.5 f 0.6 clg/ml to 2.4 f 0.3 cLg/mlby 9 months af ter stopping treatment. One year after stopping, no sys temic reactions were obsetved in 29 patients given a live sting challenge. One month after the sting, the IgG anti-venom increased to 4.1 f 0.5 ug/ml with no signif icant increase in venom-specific IgE. Prolonged venom immunotherapy, therefore, appears to Induce IKE antivenom suppression and may provide persistent protection by mechanisms other than specific IgG antibody. Protection from sting reactions persists for at least 1 year following venom immunotherapy despite a decrease in venom-specific 1gG antibody levels back to baseline and
a persisting venom sensitivity as measured by skin test or RAST. The debate about the ideal method for measuring serum IgG ‘blocking’ antibodies specific for clinically relevant allergens is continuing. One approach has been to mimic the RAST and use a solid-phase allergen to bind antibodies of all isotypes and a labeled anti-human IgG or Protein A to detect bound IgG. Lee and Adkinson [20] describe such a microtiter plate-based enzyme immunoassay for ragweed-specific IgG which uses a fluorescent substrate and performs in a manner comparable to the established Protein A solid-phase immunoassay (Hamilton and Ad122:1073-1079). This assay dekinson, J Immunoll979, sign is constrained by its fixed antigen-binding capacity, which means that some minor isotype antibodies are underdetected. Moreover, some of the antigens on the solid phase are not allergens (they do not recognize specific IgE antibodies). Thus, because this assay detects all IgG antibodies, only some of which are allergen-specific, it may overestimate IgG ‘blocking’ antibody levels which, by definition, must be able to compete with IgE antibody in vitro for the binding of allergen. A second assay design involves the use of a capture antibody to bind all antibody of a given isotype. Labeled allergen is then used to detect bound antibody which is specilic. Ice et al. (IAllergy Clin Immunoll988,82:812-817) report such an assay for ragweed-specific IgG antibodies. While this assay works well for IKE, it has limitations when IgG antibodies constitute only a small percentage of the total serum IgG. Total serum IgE The total serum 1gE has been used as a serological
marker of a predisposition of individuals to develop atopic disease. From a technical perspective, there have been no major advances in total serum IgE quantication methods during the past year. Saryan et al (Ann Allera 1989, 63:323) systematically studied the previously reported problem of the high IgE dose hook effect, which in several immunometric assays leads to falsely low estimates of IgE. The hook-effect-related bias can be idealied and minimized by analysing each test serum at two or more dilutions to conlirm parallelism between the test and reference serum. Cord blood total serum IgE has been used to assess the predisposition of newborn infants to the development of atopic disease. Haus et al. (IAllergy Clin Immunoll988, 82:179) measured cord blood IgE in a multiethnic crosssection of black, white and mixed race newborn infants. After exclusion of infants with an atopic family histoty and maternal ascariasis, mean cord blood IgE levels were signiiicantly higher in the black than in the mixed race or white newborns (0.2 kU/litre, black; 0.06 kU/litre, mixed race; and 0.05 kU/litre, white). They concluded that while cord blood serum IgE in white newborns may reflect a hereditary predisposition for atopy in whites, it appears to have no value in identifying ‘high allergic risk newborn infants in black populations which appear to represent genetically predisposed high I@ responder phenotypes.
Allergy testing Hamilton
The thirtieth reported IgE myeloma patient was identified by McIachlan et al. (Clin Cbem 1988,34:2168-2171). They used zone electrophoresis and IEF in ultra-thin agarose gels in combination with immuno!ixation by antihuman IgE to detect the paraprotein. The IEF spectrotype of this IgE myeloma ranged from p1 5.ti.5. Although this and other IgE myeloma proteins are useful as specificity proteins and invaluable in the study of the IgE structure, Neuberger et al. (Nature 1985, 314:268) reported the most novel human IgE antibody. Their human-mouse chimeric antibody has been engineered by recombinant DNA techniques to contain a mouse V-region specificity for the hapten 4-hydroxy-3-nitro-phenylacetyl, and human IgE constant regions. This reagent has been applied to the quality control of anti-human IgE immunoreagents (Hamilton, Ann Biol Clin 1990, in press).
/n vivo indicators of mast cell degranulation
Improved methods have been reported for monitoring cutaneous mast cell degranulation, especially in patients on drugs that are known to inhibit mast cell and basophil degranulation in viva. Weiss et al. (I Allergy Clin Zmmunol1989, 83:1040) have demonstrated the utility of F(ab’)2 fragments of rabbit anti-human IgE as a universal reagent to evaluate I@-dependent mast cell function in the skin. McBride et al. (JAllergy Clin Zmmunoll988,82:63E&46) reported a monoclonal antibody-based immunoassay for histamine that displayed a linear response from 0.1 to 5ng/ml. Using this assay, they detected 0.193 f 0.08 r&ml histamine in the plasma of 40 healthy individuals. They then examined plasma histamine levels in allergic patients exposed to allergen and observed a detectable increase 5-15 min after antigen challenge; it returned to baseline over a period of 60 min (r Allergy Clin Zmmunoll989, 83:374-380). The suggestion is that plasma histamine may be useful as a direct monitor of mast cell degranulation in allergic individuals.
Annotated
references
and recommended
reading ?? ??.
Of interest Of outstanding interest
1.
GUEIUNB, WATSONRD: Skin tests. Clin Rev Alkrgy 1988, 6:211-227. Review of intradermal and prick skin testing methods with an emphasis on the pathophysiology of the skin response, allergen extract prepara tion and storage and precautions. ??*
2. 00
JOHAN~~ON SGO, YMANL: In vitro assays for immunoglobu-
lin E: Methodology, indications and interpretation. Clin Rev
Allergy 1988, 6:9%139. Comprehensive overview of reagents and in vitro techniques for the quantitation of total IgE and allergen-specilic IgE in human serum. The authors highlight the use of these measurements in the differential diagnosis of atopy and procedures for standardization and quality control of RASTs. HAUJERNGM: Evaluation of in vitro testing to diagnose 3. 0 atopic diseases. Clin Rev A&r&y 1989, 7~23-47. Overview of in vitro methods for the assessment of the allergic patient.
4. CHOI H: Bronchial challenges. Clin Rev Auergv 1988, 6:231-257. 0 Examination of the goals, methods and problems associated with bronchial challenge of the allergic patient. Exercise and direct contract induced bronchial contraction is contrasted to challenge methods using allergen, methacholine and histamine inhalation.
Clin Rev Allergy 1988, PIFKORN U: Nasal provocation. 5. 0 6:285301. Overview of delivery systems, challenge agents and response measurements in diagnostic nasal basophils. HAXXK IB, MA WS: Basophil histamine release: assays and interpretation. Clin Rev Alkrgy 1988, 6:141-165. Summary of the rationale and methods in the study of histamine release from human basophils.
6.
??
BAHNASL: Diagnostic test for food allergy. Clin Rev Alkrgy 1988, 6:25!+283. Review of diagnostic elimination diets, the food/symptom diary, skin testing and in vitro tests used in the assesment of patients with sw petted food allergies.
7.
??
REED CE, YUNGINGER JW, EVANSR Quality assurance and standardization of allergy extracts in allergy practice. JAIkrgy Clin Immunol1989, 84:4-s. Succinct examination of the issues related to allergen standardization and quality control.
8. 0
Conclusions This year has seen advances in our ability to assess the patient’s home environment for the levels of dust mite, animal dander and mold allergens. Furthermore, advances have been made in the standardization of allergens through careful characterization of extracts for specific allergens and the preparation of several nw IS for allergens. The utility of specifk IgE measurements continues to be in diagnosis; semiquantitative (positive/negative) measurements are satisfactory for most applications. Quantitative specific IgG measurements continue to be used to identify specific allergen exposure and for monitoring patients on immunotherapy.
9. 0
LARSEN JN, FORDA, GJEXNGB, LEVYD, PETRUNOV B, SILVESTFU L, LOWENSTEIN H: The collaborative study of the intemation~
standard of dog, Cunis domesticus, hair/dander extract. J AIkrgy Clin Immunol 1988, 82:31%330. Collaborative study of an IS for dog hair/dander. The major analytical methods used to characterize allergens are presented. 10. 0
FC, WIIIIELMSEN TW, ENG A, IIXVENSTEIN H, GJESING B, MA.GCH HJ, STROMBERGR, EINARSSONR, BACHMAN4 MAKINEN-~JNNENS, FORD A The internationalcokbora-
ARNTZEN
study on the first international standard of birch (Betula verrucosa) pollen extract. JAlkrgy Clin Immunoll989, 83:6&82. tive
study used the same methods as in [9]. It pre%ZntspatternS of reactivity for a pollen extract.
This 11. ??*
BERNSTEIN L; Proceedings of the task force on guidelines for standardizing old and new technologies used for the diagnosis and treatment of allergic diseases. J Allergy Clin
563
564
Atopic allergy and other hypersensitivities This report recommends procedures for the standardization of reagents and methods in the diagnosis and treatment of IgEmediated diseases. Four areas were examined: percutaneous and intracutaneous skin tests, late-phase cutaneous reaction methods, in vi&-odiagnostic tests, and tests of cell-mediated immune reactions. 12.
HOFFMANDR, DOVE DE, MOFFITTJE, STAFFORD CT: Allergens in Hymenopteru venom. XXI. Crossreactivity and multiple reactivity between tire ant venom and bee and wasp venoms. J Aller~ Clin hnmunol 1988, 82:82=34. Study of the structural similarity of fire ant and other common Hymenoptera venoms using human serum and immunoblotting methods. ??
KLEINGL, ZIEMNG RW: Environmental control of the home. 13. 0 Clin Rev Allergy 1988, 6~3-22. Principles and rationale for environmental sampling and analysis of house dust for environmental allergens. 14.
POW S, CHAPMAN MD, PIA~-Mms TAE: House dust mite and dust control. Clin Rev Allergy 1988, 6:2%33. Detailed review of methods for monitoring and eliminating dust mite allergen from a house, ??
PA The effect of cat removal on allergen content in household-dust samples. J Alhgy Clin Immunol 1989, 83:73&734. Study of the effect of removing the cat on the level of cat allergen content in the home. Cat allergen elimination from an indoor environment can take 2@24 weeks after removal of the cat. 15. 00
WCOD
16. me
VAN DER ZEE JS, DE GROOT AABERSE RC: Discrepancies
RA, CHAPMAN MD,ADKINSONNFJR,EGGLESTON
H, VANSWIETENP, JANSENHM, between the skin test and IgE antibody assays: studies of histamine release, complement activation in vitro and occurrence of allergen-specific IgG. J Allergy Clin Immunol 1988, 82:27&281. Systematic examination of the causes of discrepancies between skintesting and in vitro testing in aeroallergen disease. Most discordant positive skin test/negative RAST cases studied resulted from the inability of the RAST to detect low levels of IgE antibody or to bind IgE of minor
@ergen specificities onto the solid phase, not because of an absence of speciiic IgE in the patient, 17.
SoMvrLLEMA, ivLic~1~1.5 J, G~u!zs JGG, SAINT~REME JMR: Seasonal variation in specitic IgE antibodies of grass-pollen hypersensitive patients depends on the steady state IgE concentration and is not related to clinical symptoms. J Alkqy Clin Immunol 1989, 83:486494. Study of the relationship between specific IgE antibody levels and symptoms before and after the pollen season, ??
18. ??e
DEVF( ME, LEE SR, RKHARDS D, KEMENYDM: Serial studies on the functional a@Inity and heterogeneity of antibodies of different subclasses to phospholipase A2 (PLA) produced in response to bee-venom immunotherapy. J Allergy Clin Immunol 1989, 84:326330. Functional affinity of IgG and IgE anti-PIA was studied over the course of honey bee venom therapy. The quantity and relative affinity of IgG4 anti-PIA seem to increase during chronic antigenic challenge while IgGt and IgE antibodies seem to decrease in their relative aflimity 19. ..
GOLDEN DBK, ADDISON BI, GADDE J, KAG!ZY SOBOTKA A, VALENTINE MD,L~CHTENSTEIN LM: Prospective observations on
stopping prolonged venom immunotherapy. J Aller~ Clin Immunol 1989, 84:162Z167. Prospective study of how long venom immunotherapy should be continued to ensure minimal risk of adverse reactions from another sting. Protection from sting reactions was observed 1 year after stopping prolonged immunotherapy, despite decreases in venom-specific IgE and IgG antibodies. 20.
LEE HB, ADmso~
NF JR: Measurement of IgG blocking antibody in human serum: Comparison of ELBA with monoclonal antibody and tluorogenic substrate and StupLyb coccus protein A solid phase RL4. J Allergy Clin Immunol 1988, 82:11-19. Optimization of a non-isotopic fluorescent solid-phase immunoassay for the quantification of allergen-specific IgG antibody. This study demonstrates comparability of this method with the established Protein A solid-phase radioimmunoassay. ??
University Current
School of Medicine, Baltimore,
Opinion
in Immunology
Introduction The field of allergy testing encompasses clinical and laboratory methods that are used in the diagnosis of individuals suspected of having &E-mediated hypersensitivities, in the identification and quantification of offending allergens in the environment, and in the monitoring of immunologic indicators of therapy efficacy. This subject deals with reagents and assays that are constantly moving towards improved standardization, sensitivity and specilicity. An issue of Clinic& Reviews in Allergy was recently devoted to a series of papers on the role of the laboratory in the evaluation of allergic disease [l-7]. These papers examine new developments in skin testing, in vitro assays for IgE, bronchial challenges, nasal challenges, methods for assessing the role of basophils, eosinophils and platelets in allergy, and diagnostic methods and tests for food allergy.
Maryland,
USA
1990, 2:558-564
and house dust mite (Ford et al, JAllergy Clin Immunol 1985, 75:676). Wahl et al. (JAllergy Clin Immunoll989, 84:44&456) applied the Phleum protense IS to the characterization of both in-house and commercially available timothy grass pollen extracts. Using their antiserum, they detected an allergen in their extract that was not described in the initial IS collaborative study. They attribute this failure to differences in the source of the timothy grass pollen. The IS was also useful in validating their in-house grass pollen human serum pool in comparison with that prepared by the World Health Organization.
Standardization
An additional live international allergen standards are being prepared, to extracts of dog dander/hair, birch, bermuda-grass pollen, cat dander/hair and Alternuriu. Iarsen et al. [9] report the characterization of the standard dog (Can& domesticus) hair/dander extract. The total allergenic activity of test dog hair and dander extracts were compared by radioallergosorbent test (RAST) inhibition, skin testing and leukocyte histamine release. Allergen complexity was assessed by electrophoretic separation of the individual extract components and by using IgF antibodies to detect the allergens in crossed immunoelectrophoresis (CIE), rocket immunoelectrophoresis, isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE) with immunoblotting. Twenty-two precipitin lines were detected in the CIE of the proposed IS, 11 that were identified as being of serum origin by precipitation with dog serumspeci6c antiserum. The CIE pattern detected by Ford et al. (Clin Exp Allergy 1989, 19:183-190) in a more recent study with a different dog extract showed 28 distinct antigens, 11 of which were common to dog serum proteins.
Reed et UC [8] examine the major problems in allergy tests that may be associated with the variable qualities of an allergen. Internationally documented allergen stardards are being prepared as ‘yard sticks to compare and validate the allergen content and biological activity of in house or national reference extracts. International standards (IS) have been finalized for timothy grass (Gjesing et al, J Allergy Clin Immunol 1985, 75:258), short ragweed (Helm et al, JAllergy Clin Immunol1984, 73:79(I)
Arntzen et al. [lo] describe the collaborative testing of birch (Bet& verrucasu) pollen extracts for identilication of a suitable IS. Of the six extracts tested, all but one ~fullilledthe desired specifications for an IS: > 0.5mg of protein per ml, > 20 bands by IEF, > 3 detectable al lergens (Ag 34, Ag 19 and Ag 251, > 4Oug of Ag 23 per mg of protein and > 15% of total added radioactivity retained in direct RAST. The chosen standard was shown to be stable for 36 months when stored at 5°C.
Allergens The allergen has been the single most scrutinized component of the allergy test. Allergens are extracted using different solvents and protocols from crude weed, grass and tree pollens, venoms, foods, molds, epidermal scrapings, parasites and insects. Extracts, by their very nature, vary widely in allergen content, biological potency and IgE antibody-binding capacity.
Abbreviations AU--allergy IEF-isoelectric
558
unit; Cl&crossed
focusing
IS--International
immunoelectrophoresis; Standards;
@
FAV-fire
PAC&polyacrylamide
ant venom;
HEP-histamine
gel electrophoresis;
Current Science Ltd ISSN 0952-7915
equivalent
prick;
RAST-radioallergosorbent
test.
Allergy
The most definitive statement this year on allergen and methodOlOgy standardization was presented in the American Academy of Allergy task force report on standardization of old and new technologies used in the diagnosis and treatment of allergic diseases [ 111. These proceedings are divided into four sections relating to percutaneous and intracutaneous skin tests, late-phase reactions, in vitro diagnostic tests, and an overview of diagnostic tests for cell-mediated immune reactions (delayed hypersensitivity). The section on standardization of skin tests examines performance factors, interpretation of test results and biological equivalency tests using 1 mg/ml of histamine dihydrochloride as a reference (e.g. histamine equivalent prick; HEP). Alternatively, biological activity has been expressed in activity units for the allergen extracts without reference to the HEP. The allergy unit (AU) of potency is equivalent to the mean three-fold intradermal dilution of an allergen extract that produces a 50 mm sum of perpendicular eiythema diameters in a patient known to be puncture-test positive. The in vitro diagnostic test section examines technological approaches, reporting schemes, threshold criteria, and quality control. The authors recommend the use of assays with well documented solid-phase allergens and anti-IgE reagents and the use of quantitative reporting methods in which units are proportional to antibody content. Allergen
cross-reactivity
Cross-reactivity among allergens also complicates allergy testing. Hoffman et al. (I Allergy Clin Immunol 1988, 82:81@327) report on the properties of four allergens (Sol i I-N) isolated from fire ant (S0lenopsi.s invictu) venom (FAV). Sol i II (28 kD) and III (26 k~) were shown to be major proteins in FAV, while Sol i I (37kD) and N (20 kD) were only detected in small amounts. All four components were shown to be signilicant allergens that are recognized by human IgE antibodies from FAV-allergic individuals. Hoffman et al. [ 121 used these purified Sol i I-N allergens to study the cross-reactivity of IgE antibodies from patients sensitive to cross-reactive bee and wasp venom. Immunoassays using solid-phase FAV, bee and wasp venoms were used to screen sera from individuals who were: (1) not allergic to venoms; (2) exposed but not allergic to FAV; (3) allergic to FAV; (4) allergic to bee and wasp venoms but not FAV; and (5) allergic to bee and wasp venoms and exposed to FAV. Reactivity with FAVwas 0% (group l), 24% (2),100% (3), 54% (4) and 87% (5). Sol i I was shown by immunoblot analysis to be the major cross-reactive allergen of imported FAV. Commercial sources of FAV vary widely in how much of these components they contain (Butcher and Reed, J Alleqy Clin Zmmunoll!X38, 82:770-777), indicating that more rigorous testing and standardization are needed. Penicillin is one of many antibiotics with a p-lactam structure. Bianca et al. (J Allergy Clin Zmmunol 1989, 83:381-385) challenged a group of patients allergic to penicillin with a new cephalosporin, cephamadole, which contains side-chain structures similar to peni clllin. Two out of 19 penicillin-allergic patients reacted to a cephamadole challenge, supporting previous ob-
testing
Hamilton
servations about the cross-reactivity between penicillin and cephalosporins (Batchelor et al, Immunology 1966, 10:21). Another study examined the possible cross-reactivity between penicillin and imipenem, the first member of new class of jG~tam antibiotics called the carbapenems. Saxon et al. (J Allera Clin Zmmunoll988, 82:213217) found 20 patients who were skin-test positive to one or more penicillin determinants and also reacted to imipenem. These studies indicate the importance of controlled challenge tests in the study of penicillinallergic patients for possible sensitivities to cephalosporins and carbapenems. Allergen
identification
One of the first steps in the preparation of a well documented allergen reference preparation is the identlfication of the major and minor allergens in particular extracts. This year, several studies have enhanced our knowledge of specific allergenic and antigenic components in extracts. Aqueous white oak pollen extracts prepared by Loria et al. (I Allergy Clin Zmmunoll989, 84:!+18) were shown to contain 45 distinct protein bands using sodium dodecyl sulphate polyacrylamide gel electrophoresis. IgE antibodies from 30 out of 38 oak-sensitive patients bound 23 bands from 13.2-108kD. There was no single band that could be recognized by IgE antibody from every patient. Two proteins (74-77.9 k~ and 16.2-17.7 kD) appeared to be important allergens because they were bound by IgE from 71% of patients’ sera. Iranitalab et al. (Allergy 1989, 44:314-321) studied four extracts of Micropolyqora faeni by western blot analysis using serum from farmers with (n = 25) and without ( n = 14) extrinsic allergic alveolitis, all of whom were exposed to hay, and 27 healthy controls. Major antigens of 11,12,25,35 and 60 kD were identiIied with serum from this group. Although farmers with extrinsic allergic alveolitis had higher levels of IgG, IgM and IgA antibodies to these M. faeni antigens than farmers without the disease, there was no correlation between the antibody response and the onset of disease. Variation in antigen content between commercial M. faeni extracts led the authors to conclude that the extracts must have at least the 11, 25 and 60kD components before they can be used in in vitro diagnosis of M. faeni-induced extrinsic allergic alveoli&. Baur et al. (JAllergy Clin Zmmunol1989,83:839+44) examined whether different stages of allergic bronchopulmonary aspergillosis may correlate with variations in the level and specificity of the antibody response. Western blots were performed using @ergillusfumigatus allergen extracts with serum from 28 patients with allergic bronchopulmonary aspergillosis in stages II to V. The pattern of IgG and IgE antibody binding to R jkmigatus did not discrimina te between the stages of disease, as both reacted with the same antigens. Only the level of IgE antibody appeared to be stronger in stages III to V. To identify new allergens, human serum pools from well characterized patients and polyclonal and monoclonal
559
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Atopic allergy
and other
hypersensitivities
antibodies made to the total allergen extract, as well as individual components, are often required. Van Ree et al. (j Allergy Clin Immunoll989, 83:144151) demonstrated the value of both monoclonal and polyclonal antibodies in the identification of new allergens including an as yet unidentified major allergen from grass pollen in the group I molecular weight range. De Grout et al. (I Allergy Clin Immunol1988, 82:778-786) have prepared monoclonal antibodies to Fe1 d I (the major allergen in cat dander) which have been useful in immunoassays that detect the allergen,
-While accurate immunoassays are available for measuring the level of major cat allergens in house dust, the measurement of dog allergens awaits the preparation of specific antibodies. Breed-specific dog dandruff allergens have been identified by Lindgren et al. (I Al/era Clin Immunoll988, 82:196-204) using CIE and serum from patients who were clinically allergic to dogs. Dog breed extracts contained common allergens and also allergenic components that only occur in one or two dog breed extracts. Although the study identified skin-test reactivity to breed-specific allergens in about 15% of individuals allergic to dogs, the degree of reactivity did not correlate with allergic symptoms to that specific dog breed.
Environmental
1988, Schumacher et al. (r Allergy Clin Immunol 82:6Oti16) used immunoblot microscopy to differentiate grass pollen aeroantigens collected in air sam plers. Antigen particle counts were performed by apply ing pollen to adhesive tape, blotting it onto nitrocellulose and detecting the antigen using specific antibodies and a fluorescein-conjugated second antibody. Fluorescence microscopy-derived ‘antigen particle counts’ were compared with light microscopy ‘pollen counts’. This method has potential use in the quality control of source pollen; it may be able to detect contamination of commercially available grass pollen by pollen of other grass types. Moreover, the study showed that the number of airborne particles associated with specific antigens was greater than the number of grass pollen grains, indi eating that morphologically unrecognizable grass pollen antigens were present in such samples.
allergen
testing
One of the most direct methods of allergy treatment is to remove the allergen from the patient’s environment or vice versa. The availability of new monoclonal antibocty reagents and assays has allowed the diagnostic al lergy laboratory to quantify the level of allergen in the home environment. Several recent reviews examine the rationale and practical approaches to the evaluation of allergen levels in the home. Dust containing dust mite, cockroach and animal (cat and dog) and mold allergens is most often found in old houses. Klein and Ziering [ 131 provide a home evaluation questionnaire which is useful in identifying major offending household allergens. Pollart et al. [ 141 focus on house dust mite allergen as a risk factor in allergies and on methods for dust control. The level of dust mite allergen in a specimen of house dust can be used as a bench mark upon which to judge effective removal of allergen by rigorous cleaning techniques (Green et al, Clin Exp Allergv 1989, 19:203_207). Moreover, the level of allergen can be used to assess the relative importance of a particular allergen in the environment to a patient who is skin-test-positive and/or RASTpositive. Assays are available for measuring group I allergens from Dermutopbagoidespteronyssinus (Der p I) and D. farinae (Der f I). Patients exposed to levels of Der p I or Der f I of more than 10 ug per gram of dust often experience allergic symptoms, while those in envi ronments containing < 1 ug/g seem to be symptom-free. While these levels cannot be directly translated into mite counts, 10 gg/g of Der p I is approximately equivalent to 200 mites per gram of dust (Platts-Mills et al, J Allergy RAST inhibition with house Clin Immunoll986,78:398). dust extracts has been used as an indirect approach to estimate the level of D. farinue allergen in house dust (Twiggs et al, Clin Rev Allergy 1988, 6:3542). Wood et al. [ 151 studied the rate of cat allergen reduction in dust specimens collected from 15 homes over a 943.week period after removing the cat. Baseline levels of Fe1 d I in houses with at least one cat were 7-436.7 Food and Drug Administration units per g of dust (median 61.2 units/g). After removal of cats, Fe1 d I levels in most homes declined gradually to < 1 unit/g, which is similar to homes without cats, in 2&24 weeks. This study shows that even when the allergen source (the cat) is removed, elimination of allergen from the environment can be a slow and variable process and monitoring of major allergen levels is useful in documenting allergen removal.
Jensen et al. (Allera 1989, 44:52) studied the use of a high-volume air sampler that was equipped with 0.3 l,trn glass fiber lilters to collect both amorphous air-borne allergens of respirable size and pollen grains. Birch and timothy allergens eluted from lilters were analyzed by an IgG RAST inhibition assay. The amount of airborne allergen collected by the high-volume air sampler and measured immunochemically correlated signiIicantly with pollen counts obtained using a pollen trap located in the same place. 0
Humoral immune responses Allergen-specific
IgE
Diagnostic allergy testing is based on the detection of allergen-specific IgE antibodies by in vivo challenge methods in which the skin, nasal passages and bronchi are exposed to allergen, or by in vitro methods in which .speciIic IgE from serum and nasal secretions is bound to solid-phase allergens in the RAST. Discrepancies have been observed between the levels of allergen-specific IgE detected by skin test and those found in serum by RAST, the most common being a positive skin test with a negative RAST. Van der Zee et al. [ 161 performed paired intracutaneous skin tests and RASTs on 660 adult patients to detect specific IgE to grass pollen, house dust mite and cat dander. Out of 1980 skin test and RAST combinations, there were 140 discrepancies, more than 80%
Allergy
of which were positive skin reactions without detectable allergen-specific IgE antibodies in serum (negative RAST). A second RAST analysis using commercial and in-house reagents and skin tests reproduced > 80% of these discordant results. Thus, discordant skin-test and RAST results are not a consequence of random experimental errors. Histamine was released from the washed leukocytes of 82% of 28 patients with positive skin tests and negative RASTs upon in vitro challenge with the relevant allergen extract. Moreover, low levels of specific IgE in the serum of skin-test-posit-negative individuals were confirmed when a positive RAST was obtained using atlinity-purified IgE from serum of these patients. IgG* antibodies were not detected in most of these preparations. Thus, it was concluded that the majority of positive skin reactions that were accompanied by a negative RAST are caused by IgE antibodies whose levels are too low or whose allergenic components are present on the solid phase in quantities too small to be detected in vitro. Serologists have questioned whether more quantitative estimates of spectic IgE would be useful in monitoring allergy patients. Seasonal exposure to aeroallergens is known to increase serum IgE antibody levels towards the end of the pollination period but the relationship between IgE antibody levels and the patients’ symptoms has remained unclear. Somville et al. [ 171 assessed variations in symptoms and serum IgE antibody levels in 78 grasspollen-allergic patients during the pollen season. They showed that specific serum IgE antibodies are of little use in predicting the clinical symptoms of grass pollen-hypersensitive patients since IgE levels strongly depend on individual immune responses. This conclusion was analogous to that of Reisman and De Masi (Znt Arch Allergy Appl Zmmunol1989, 8967-70) who showed that absolute levels of serum venom-specific IgE in Hymenoptera venom-allergic patients were unrelated to any clinical feature of the disease. These studies suggest that the absolute measurement, in weight-per-volume units, of specific IgE in preseason sera is of minimal value in predicting the extent of clinical symptoms during the season. Technical
Hamilton
82:812) demonstrated the usefulness of solid-phase antiIgE in binding IgE from serum. When it was used with a labeled allergen, isotypic interference of the binding of IgE antibody to limited allergen binding sites by IgG antibody could be minimized. This works best, however, when the ratio of specific IgE to total IgE is high. The value of these modifications is difficult to assess from these reports because many assay and specimen variables alfect the accuracy of an IgE antibody measurement. However, these reports do provide a perspective on trends in in vitro IgE detection methods that relate to: (1) improved quality of the allergen; (2) enhanced binding capacity of the solid-phase allergosorbent; (3) methods for documentation and quality control of the labeled anti-&E; and (4) methods for Improvement of IgE antibody detection in the presence of high levels of IgG antibody.
less common
allergen
specificities
A number of studies examining IgE antibodies to al lergens that are not discussed frequently have been reported this year.
(1)
(2)
(3)
issues
There have been few advances in specific IKE assays during the past year. Several reviews [2,3] and the task force report [ II] continue to recommend that maximum sensitivity is obtained by the modified RAST format with 0.1 ml of serum and an overnight incubation. Some alterations in the solid phase have been proposed to enhance sensitivity of the RAST for detecting IgE antibodies secreted by lymphocytes in culture fluid (Poulsen et al, Allergy 1989, 44:174) and in nasal secretions (Marcucci et al, Clin Exp Allergy 1989, 19:157). A new aluminium hydroxide-adsorbed allergosorbent did not perform as well as the paper disk-insolubilized allergen in the Phadebas RAST (Heinlg et al, Allergy 1989, 44:260). A dipstick enzyme immunoassay for IgE antibodies (Twiggs et al., Ann Al lergy 1989, 63:225) and the multi-allergosorbent chemiluminescent assay (Finnerty et al, Clin Exp Allergy 1989, 19:51) were shown to detect IgE to a number of allergen specificities. Lee et al. (IAllergy Clin Zmmunol1988,
testing
(4)
(5)
(6)
Spaner et al. (I Allergy Clin Zmmunol 1989, 83:1135-1137) presented the case study of an in dividual with hypersensitivity to natural latex who has specific IgE antibodies detectable by skin tests and RASTs. Boxer et al. (r Allergy Clin Zmmunol 1988, 82:622426) presented evidence from experiments in which IgE antibodies to metabisullites from patients with a clinical history and positive skin test were able to sensitize monkeys passively. However, IgE anti-metabisuliite antibod ies could not be detected by immunoassays using metabisulliteprotein conjugates as the solidphase antigen. Clin Zmmunol 1988, Enberg et al. (I Allera 82:795-800) studied the serum of patients with a history of sensitivity to water melon. IKE and IKE antibody responses to extracted and immunoblotted water melon antigens were shown to have extensive heterogeneity. Neither IgE nor IgG antibodies specific for water melon allergens were predictive of clinical symptoms. Kramps et al. (Clin Exp Allergy 1989, 19:509-514) studied individuals who had been exposed to formaldehyde as a result of their occupation. They concluded that formaldehyde may provoke the production of specific IgE antibodies, but rarely does. Although the cross-reactivities of beef, pork and human insulin are well known, Grammer et al. (Clin Exp Allergy 1989, 19:551-553) described a patient with anaphylaxis to beef and pork insulin who did not cross-react with recombinant human insulin. Chloramine T is an oxidative agent used in antiseptics and disinfectants. Wass et al. (Clin Exp AC lergy 1989, 19:463471) examined the specificity
Y
562
Atopic allergy and other hypersensitivities
of IgE antibodies to chloramine T using different ratios of human serum albumin carrier to antigen. No matter what the carrier protein form, IgE antibodies from a chloramine T-sensitive patient remained specific only to the hapten. Interestingly, no IgG antibodies to chloramine T could be detected despite the presence of high, inhibitable levels of IgE antibody.
Allergen-specific
IgC
Allergen-specilic IgG antibodies have been used: (1) as indicators of exposure to a particular antigen; (2) in identification of the offending allergen among a group of related allergenic substances such as the Hymenoptera venoms; and (3) in selected cases, to study the efficacy of an immunotherapy regimen. The levels and specificity of IgG antibodies are highly dependent on the allergen, the patient, and the mode and duration of exposure. The level of IgG antibodies generally correlates with the extent of exposure to the allergen either by accidental or intentional exposure (van Metre et al, J Allergy Clin Immunoll988, 82:10551068; Nordvall et al, Allergy 1989, 44:380-384; Wass et al, J Allergy Clin Immunol 1988, 82679686; Price et al., J Alleqy Clin Immunol 1989, 84:520-528). Allergen immunotherapy is known to induce the formation of IgG antibodies that are highly restricted to IgG subclasses 1 and 4. Because of its compact hinge, inability to activate complement and reported functional monovalency, IgG4 has good properties as a ‘blocking’ antibody. Devey et al. [ 181 used a competitive inhibition enzyme immunoassay to show that the functional aifinities of IgGl anti-phospholipase A2 appear to decrease over the course of honey bee venom immunotherapy, while an increase in the quantity and relative affinity of IgG* antibodies can be observed. Golden et al. [ 193 investigated how long venom immunotherapy should be continued to ensure protection from a systemic allergic skin reaction to a subsequent sting. Thirty patients chose to stop immunotherapy after 5 years of treatment. Skin-test reactivity decreased during the im munotherapy of 18 out of 30 patients, but it remained positive in 23 out of 30 patients. Serum venom-specific IgE was at the lower limit of detection (1 ng/ml) in 11 out of 30 patients. Mean serum IgG anti-venom declined from 5.5 f 0.6 clg/ml to 2.4 f 0.3 cLg/mlby 9 months af ter stopping treatment. One year after stopping, no sys temic reactions were obsetved in 29 patients given a live sting challenge. One month after the sting, the IgG anti-venom increased to 4.1 f 0.5 ug/ml with no signif icant increase in venom-specific IgE. Prolonged venom immunotherapy, therefore, appears to Induce IKE antivenom suppression and may provide persistent protection by mechanisms other than specific IgG antibody. Protection from sting reactions persists for at least 1 year following venom immunotherapy despite a decrease in venom-specific 1gG antibody levels back to baseline and
a persisting venom sensitivity as measured by skin test or RAST. The debate about the ideal method for measuring serum IgG ‘blocking’ antibodies specific for clinically relevant allergens is continuing. One approach has been to mimic the RAST and use a solid-phase allergen to bind antibodies of all isotypes and a labeled anti-human IgG or Protein A to detect bound IgG. Lee and Adkinson [20] describe such a microtiter plate-based enzyme immunoassay for ragweed-specific IgG which uses a fluorescent substrate and performs in a manner comparable to the established Protein A solid-phase immunoassay (Hamilton and Ad122:1073-1079). This assay dekinson, J Immunoll979, sign is constrained by its fixed antigen-binding capacity, which means that some minor isotype antibodies are underdetected. Moreover, some of the antigens on the solid phase are not allergens (they do not recognize specific IgE antibodies). Thus, because this assay detects all IgG antibodies, only some of which are allergen-specific, it may overestimate IgG ‘blocking’ antibody levels which, by definition, must be able to compete with IgE antibody in vitro for the binding of allergen. A second assay design involves the use of a capture antibody to bind all antibody of a given isotype. Labeled allergen is then used to detect bound antibody which is specilic. Ice et al. (IAllergy Clin Immunoll988,82:812-817) report such an assay for ragweed-specific IgG antibodies. While this assay works well for IKE, it has limitations when IgG antibodies constitute only a small percentage of the total serum IgG. Total serum IgE The total serum 1gE has been used as a serological
marker of a predisposition of individuals to develop atopic disease. From a technical perspective, there have been no major advances in total serum IgE quantication methods during the past year. Saryan et al (Ann Allera 1989, 63:323) systematically studied the previously reported problem of the high IgE dose hook effect, which in several immunometric assays leads to falsely low estimates of IgE. The hook-effect-related bias can be idealied and minimized by analysing each test serum at two or more dilutions to conlirm parallelism between the test and reference serum. Cord blood total serum IgE has been used to assess the predisposition of newborn infants to the development of atopic disease. Haus et al. (IAllergy Clin Immunoll988, 82:179) measured cord blood IgE in a multiethnic crosssection of black, white and mixed race newborn infants. After exclusion of infants with an atopic family histoty and maternal ascariasis, mean cord blood IgE levels were signiiicantly higher in the black than in the mixed race or white newborns (0.2 kU/litre, black; 0.06 kU/litre, mixed race; and 0.05 kU/litre, white). They concluded that while cord blood serum IgE in white newborns may reflect a hereditary predisposition for atopy in whites, it appears to have no value in identifying ‘high allergic risk newborn infants in black populations which appear to represent genetically predisposed high I@ responder phenotypes.
Allergy testing Hamilton
The thirtieth reported IgE myeloma patient was identified by McIachlan et al. (Clin Cbem 1988,34:2168-2171). They used zone electrophoresis and IEF in ultra-thin agarose gels in combination with immuno!ixation by antihuman IgE to detect the paraprotein. The IEF spectrotype of this IgE myeloma ranged from p1 5.ti.5. Although this and other IgE myeloma proteins are useful as specificity proteins and invaluable in the study of the IgE structure, Neuberger et al. (Nature 1985, 314:268) reported the most novel human IgE antibody. Their human-mouse chimeric antibody has been engineered by recombinant DNA techniques to contain a mouse V-region specificity for the hapten 4-hydroxy-3-nitro-phenylacetyl, and human IgE constant regions. This reagent has been applied to the quality control of anti-human IgE immunoreagents (Hamilton, Ann Biol Clin 1990, in press).
/n vivo indicators of mast cell degranulation
Improved methods have been reported for monitoring cutaneous mast cell degranulation, especially in patients on drugs that are known to inhibit mast cell and basophil degranulation in viva. Weiss et al. (I Allergy Clin Zmmunol1989, 83:1040) have demonstrated the utility of F(ab’)2 fragments of rabbit anti-human IgE as a universal reagent to evaluate I@-dependent mast cell function in the skin. McBride et al. (JAllergy Clin Zmmunoll988,82:63E&46) reported a monoclonal antibody-based immunoassay for histamine that displayed a linear response from 0.1 to 5ng/ml. Using this assay, they detected 0.193 f 0.08 r&ml histamine in the plasma of 40 healthy individuals. They then examined plasma histamine levels in allergic patients exposed to allergen and observed a detectable increase 5-15 min after antigen challenge; it returned to baseline over a period of 60 min (r Allergy Clin Zmmunoll989, 83:374-380). The suggestion is that plasma histamine may be useful as a direct monitor of mast cell degranulation in allergic individuals.
Annotated
references
and recommended
reading ?? ??.
Of interest Of outstanding interest
1.
GUEIUNB, WATSONRD: Skin tests. Clin Rev Alkrgy 1988, 6:211-227. Review of intradermal and prick skin testing methods with an emphasis on the pathophysiology of the skin response, allergen extract prepara tion and storage and precautions. ??*
2. 00
JOHAN~~ON SGO, YMANL: In vitro assays for immunoglobu-
lin E: Methodology, indications and interpretation. Clin Rev
Allergy 1988, 6:9%139. Comprehensive overview of reagents and in vitro techniques for the quantitation of total IgE and allergen-specilic IgE in human serum. The authors highlight the use of these measurements in the differential diagnosis of atopy and procedures for standardization and quality control of RASTs. HAUJERNGM: Evaluation of in vitro testing to diagnose 3. 0 atopic diseases. Clin Rev A&r&y 1989, 7~23-47. Overview of in vitro methods for the assessment of the allergic patient.
4. CHOI H: Bronchial challenges. Clin Rev Auergv 1988, 6:231-257. 0 Examination of the goals, methods and problems associated with bronchial challenge of the allergic patient. Exercise and direct contract induced bronchial contraction is contrasted to challenge methods using allergen, methacholine and histamine inhalation.
Clin Rev Allergy 1988, PIFKORN U: Nasal provocation. 5. 0 6:285301. Overview of delivery systems, challenge agents and response measurements in diagnostic nasal basophils. HAXXK IB, MA WS: Basophil histamine release: assays and interpretation. Clin Rev Alkrgy 1988, 6:141-165. Summary of the rationale and methods in the study of histamine release from human basophils.
6.
??
BAHNASL: Diagnostic test for food allergy. Clin Rev Alkrgy 1988, 6:25!+283. Review of diagnostic elimination diets, the food/symptom diary, skin testing and in vitro tests used in the assesment of patients with sw petted food allergies.
7.
??
REED CE, YUNGINGER JW, EVANSR Quality assurance and standardization of allergy extracts in allergy practice. JAIkrgy Clin Immunol1989, 84:4-s. Succinct examination of the issues related to allergen standardization and quality control.
8. 0
Conclusions This year has seen advances in our ability to assess the patient’s home environment for the levels of dust mite, animal dander and mold allergens. Furthermore, advances have been made in the standardization of allergens through careful characterization of extracts for specific allergens and the preparation of several nw IS for allergens. The utility of specifk IgE measurements continues to be in diagnosis; semiquantitative (positive/negative) measurements are satisfactory for most applications. Quantitative specific IgG measurements continue to be used to identify specific allergen exposure and for monitoring patients on immunotherapy.
9. 0
LARSEN JN, FORDA, GJEXNGB, LEVYD, PETRUNOV B, SILVESTFU L, LOWENSTEIN H: The collaborative study of the intemation~
standard of dog, Cunis domesticus, hair/dander extract. J AIkrgy Clin Immunol 1988, 82:31%330. Collaborative study of an IS for dog hair/dander. The major analytical methods used to characterize allergens are presented. 10. 0
FC, WIIIIELMSEN TW, ENG A, IIXVENSTEIN H, GJESING B, MA.GCH HJ, STROMBERGR, EINARSSONR, BACHMAN4 MAKINEN-~JNNENS, FORD A The internationalcokbora-
ARNTZEN
study on the first international standard of birch (Betula verrucosa) pollen extract. JAlkrgy Clin Immunoll989, 83:6&82. tive
study used the same methods as in [9]. It pre%ZntspatternS of reactivity for a pollen extract.
This 11. ??*
BERNSTEIN L; Proceedings of the task force on guidelines for standardizing old and new technologies used for the diagnosis and treatment of allergic diseases. J Allergy Clin
563
564
Atopic allergy and other hypersensitivities This report recommends procedures for the standardization of reagents and methods in the diagnosis and treatment of IgEmediated diseases. Four areas were examined: percutaneous and intracutaneous skin tests, late-phase cutaneous reaction methods, in vi&-odiagnostic tests, and tests of cell-mediated immune reactions. 12.
HOFFMANDR, DOVE DE, MOFFITTJE, STAFFORD CT: Allergens in Hymenopteru venom. XXI. Crossreactivity and multiple reactivity between tire ant venom and bee and wasp venoms. J Aller~ Clin hnmunol 1988, 82:82=34. Study of the structural similarity of fire ant and other common Hymenoptera venoms using human serum and immunoblotting methods. ??
KLEINGL, ZIEMNG RW: Environmental control of the home. 13. 0 Clin Rev Allergy 1988, 6~3-22. Principles and rationale for environmental sampling and analysis of house dust for environmental allergens. 14.
POW S, CHAPMAN MD, PIA~-Mms TAE: House dust mite and dust control. Clin Rev Allergy 1988, 6:2%33. Detailed review of methods for monitoring and eliminating dust mite allergen from a house, ??
PA The effect of cat removal on allergen content in household-dust samples. J Alhgy Clin Immunol 1989, 83:73&734. Study of the effect of removing the cat on the level of cat allergen content in the home. Cat allergen elimination from an indoor environment can take 2@24 weeks after removal of the cat. 15. 00
WCOD
16. me
VAN DER ZEE JS, DE GROOT AABERSE RC: Discrepancies
RA, CHAPMAN MD,ADKINSONNFJR,EGGLESTON
H, VANSWIETENP, JANSENHM, between the skin test and IgE antibody assays: studies of histamine release, complement activation in vitro and occurrence of allergen-specific IgG. J Allergy Clin Immunol 1988, 82:27&281. Systematic examination of the causes of discrepancies between skintesting and in vitro testing in aeroallergen disease. Most discordant positive skin test/negative RAST cases studied resulted from the inability of the RAST to detect low levels of IgE antibody or to bind IgE of minor
@ergen specificities onto the solid phase, not because of an absence of speciiic IgE in the patient, 17.
SoMvrLLEMA, ivLic~1~1.5 J, G~u!zs JGG, SAINT~REME JMR: Seasonal variation in specitic IgE antibodies of grass-pollen hypersensitive patients depends on the steady state IgE concentration and is not related to clinical symptoms. J Alkqy Clin Immunol 1989, 83:486494. Study of the relationship between specific IgE antibody levels and symptoms before and after the pollen season, ??
18. ??e
DEVF( ME, LEE SR, RKHARDS D, KEMENYDM: Serial studies on the functional a@Inity and heterogeneity of antibodies of different subclasses to phospholipase A2 (PLA) produced in response to bee-venom immunotherapy. J Allergy Clin Immunol 1989, 84:326330. Functional affinity of IgG and IgE anti-PIA was studied over the course of honey bee venom therapy. The quantity and relative affinity of IgG4 anti-PIA seem to increase during chronic antigenic challenge while IgGt and IgE antibodies seem to decrease in their relative aflimity 19. ..
GOLDEN DBK, ADDISON BI, GADDE J, KAG!ZY SOBOTKA A, VALENTINE MD,L~CHTENSTEIN LM: Prospective observations on
stopping prolonged venom immunotherapy. J Aller~ Clin Immunol 1989, 84:162Z167. Prospective study of how long venom immunotherapy should be continued to ensure minimal risk of adverse reactions from another sting. Protection from sting reactions was observed 1 year after stopping prolonged immunotherapy, despite decreases in venom-specific IgE and IgG antibodies. 20.
LEE HB, ADmso~
NF JR: Measurement of IgG blocking antibody in human serum: Comparison of ELBA with monoclonal antibody and tluorogenic substrate and StupLyb coccus protein A solid phase RL4. J Allergy Clin Immunol 1988, 82:11-19. Optimization of a non-isotopic fluorescent solid-phase immunoassay for the quantification of allergen-specific IgG antibody. This study demonstrates comparability of this method with the established Protein A solid-phase radioimmunoassay. ??