Crossed Radioimmunoelectrophoresis Research Articles

DUST FROM CARPETED AND SMOOTH FLOORS—III. TRIALS ON DENATURATION OF ALLERGENIC PROTEINS BY HOUSEHOLD CLEANING SOLUTIONS AND CHEMICAL DETERGENTS

The effects of detergents on antigens and allergens of birch and timothy grass pollen extracts, codfish, hen egg-white, cat dander and house dust mite were investigated by crossed immunoelectrophoresis (CIE), crossed radio-immunoelectrophoresis (CRIE), radio-allergosorbent test (RAST) inhibition and quantitative precipitation test using laser nephelometry. Nine household cleaning solutions and five chemical detergents were tested. Higher concentrations than recommended for regular cleaning purposes were used as no influence on the antigenic and allergenic activities could be detected using the usual concentrations. Soft soap, guanidine hydrochloride and sodium lauryl sulphate induced most modifications of the antigens and allergens tested. None of the detergents totally destroyed the antigenic and allergenic activities of the selected material, even when used in concentrations up to 10 times that recommended. The materials used for carpet cleaning would not be able to impose any denaturing effects on allergens left on carpets after cleaning.

Comparison of in-house allergen extracts of Phleum pratense (timothy grass) pollen with the international standard and investigation of IgE specificities of a grass-pollen serum pool from West Germany and of the one recommended by the World Health Organization

We compared our in-house reference extract (RE) and a production extract (PE) with the international reference preparation (IRP) of International Union of Immunological Societies of timothy-grass pollen, using various biochemical and immunochemical methods. Furthermore, we compared the IgE composition of our in-house grass-pollen serum pool (West Germany) with the serum pool recommended by the World Health Organization, using the RE for crossed radioimmunoelectrophoresis (CRIE). Our extracts (RE and PE) were nearly comparable with the IRP. Only by CRIE one more allergen could be detected in RE and PE than in IRP and in another extract purchased from an American company. This finding may depend on the region where the source materials were harvested and which source materials were used for the preparation of the extracts. Furthermore, by high-performance liquid chromatography in the low-molecular-weight range, two distinct peaks could be detected in the nondiafiltered IRP, which were detected in the diafiltered RE and PE as traces only. The IgE composition of our in-house grass-pollen serum pool was comparable with the serum pool recommended by the World Health Organization, as detected by CRIE using RE.

Dust from carpeted and smooth floors

Dust samples were collected twice from smooth and carpeted floors in 10 Norwegian schools. The content of antigens and allergens of alder (Alnus incana), birch (Betula verrucosa), timothy (Phleum pratense), cat and dog dander, house dust mite (Dermatophagoides farinae), mould (Cladosporium herbarum), hen egg white and codfish (DIII) were investigated by crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE), radio allergosorbent test (RAST) inhibition and quantitative precipitation inhibition analysis by laser nephelometry. Antigens and allergens of cat and dog dander and hen egg white were most prevalent in the dust samples investigated. With the exception of hen egg white and codfish allergens, no statistically significant differences in mean allergen content were shown in identical quantities of freeze-dried dust extracts from carpeted and smooth floors. RAST-inhibition analyses of identical amounts of dust from either floors showed higher content of allergens of cat, dog, hen egg white, codfish, mould and timothy pollen in classrooms with carpets.

Specific IgE and IgG antibody responses in children to timothy pollen components during immunotherapy.

Fourteen children with timothy grass pollinosis were given immunotherapy (IT) for 3 years with a purified and characterized timothy grass pollen preparation or a crude aqueous timothy pollen extract. Crossed radioimmunoelectrophoresis (CRIE) showed that 75% of the children under 11 years of age developed new specificities of IgE antibodies against timothy antigens, in contrast to older children, where no development of IgE antibodies against new timothy antigens could be detected. IgE antibodies were only detected against antigens formerly known as allergens. Timothy-specific IgG antibodies increased in most children during hyposensitization against the major allergens Ag 19 and Ag 24/25 and several other IgE-binding timothy antigens.

The allergens of dog. I. Identification using crossed radio-immunoelectrophoresis.

The antigens present in an extract of dog hair and dander were examined by crossed immunoelectrophoresis (CIE) and the IgE-binding allergens by crossed radio-immunoelectrophoresis (CRIE), respectively, using sera from 60 British and Finnish animal-allergic subjects. The extract was comprised of a minimum of 28 antigens, 11 of which were common to dog serum. IgE antibody in the sera of the dog-sensitive patients bound to 21 of the 28 antigens at varying frequencies and intensities. Binding of any intensity occurred most frequently to two serum proteins: antigen 23 (IgG) binding IgE in 88% of cases, and antigen 3 (dog serum albumin, DSA) in 77% of cases. Dander antigen 8 bound in 63% and antigen 1 in 42% of the sera. Strong IgE binding, however, was most commonly associated with dander antigen 8 followed by antigens 1 and 23 (IgG) then 3 (DSA). The ranking of the antigens as allergens was similar for the two populations except that DSA was more important for the British than for the Finnish subjects.

Cross-antigenicity between Sarcoptes scabiei and the house dust mite, Dermatophagoides farinae (Acari: Sarcoptidae and Pyroglyphidae).

Sarcoptes scabiei var. canis (L.) (SS) was shown to share a high degree of cross-reactivity with the house dust mite, Dermatophagoides farinae Hughes (DF). Sera from scabies-infested rabbits contained antibodies that recognized at least six antigens in extracts produced from DF body material and three from DF fecal material. Likewise, rabbit antiserum to DF body and DF fecal extracts contained antibodies that recognized SS antigens. Sera from control rabbits reacted with DF body and SS extracts did not show antibodies to antigens in either extract. Six of nine SS antigens were cross-reactive with DF antigens. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS—PAGE) fraction profiles of SS exhibited at least 30 protein and peptide bands. Fifteen SS bands bound DF-specific immunoglobulin E (IgE) following incubation in sera from nine DF skin-test-positive patients with no history of scabies. The IgE binding varied within and among these patients. The sera of these patients also exhibited significant IgE levels to multiple DF antigens, as demonstrated by crossed radioimmunoelectrophoresis (CRIE) and immunoblotting. Control sera from nonallergic control patients who were skin test negative to DF and without scabies exhibited no IgE binding to any SS band.

Olive ( olea europea) pollen allergens—I immunochemical characterization by immunoblotting, crie and immunodetection by a monoclonal antibody

The pattern of reactivity of the Olea europea crude extract antigens was analysed after electroblotting to nitrocellulose from SDS-PAGE. The antigens contained in the 17, 19 and 42 K bands were most reactive with specific IgE from individual sera. Following immunization with a crude extract, one monoclonal antibody ( OL-1) was raised against components which exhibited IgE binding capacity in electroblotting and crossed radioimmunoelectrophoresis (CRIE). Monoclonal antibody OL-1 reacted with the 17 and 19 K antigens and with three arcs of crossed immunoelectrophoresis (CIE), one of which is considered to contain a major allergen by CRIE.

Investigations of Patients Allergic to the House Dust Mite <i>Dermatophagoides pteronyssinus</i> by Crossed Radioimmunoelectrophoresis Using Purified Mite Bodies and Whole Mite Culture Extracts

Sera of 20 patients allergic to the house dust mite Dermatophagoides pteronyssinus were investigated by crossed radioimmunoelectrophoresis (CRIE) using extracts prepared from purified mite bodies (PMB) and whole mite culture (WMC). By CRIE, different allergen patterns and different numbers of allergens were detected in the two extracts. By summarizing the values of the CRIE patterns of the 20 patients' sera into allergograms, 6 out of 16 allergens in PMB and 5 out of 10 allergens in WMC extracts could be identified as major allergens. These diverging results, together with the published literature, emphasize the necessity to reevaluate the International Standard for D. pteronyssinus.

Antigenicity and allergenicity of body and fecal extracts of the mite Dermatophagoides pteronyssinus (Acari: Pyroglyphidae).

Crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) demonstrated that extracts of both bodies and feces of Dermatophagoides pteronyssinus (DP) contained multiple antigens and allergens. DP feces extract contained significantly fewer antigens than did DP body extract. Five DP body antigens and at least 4 DP feces antigens were allergens and bound specific IgE in the 2 mite-sensitive reference sera tested. Several antigens in each extract were potent allergens. Heterologous CIE demonstrated some cross antigenicity between body and fecal extracts, but many of the antigens present in DP bodies were not present in DP feces. All cross-reacting DP body and DP feces antigens were weak allergens for the 2 test sera, based on the long autoradiographic time required for visualization compared to the relatively short exposure time required to visualize IgE binding with the homologous CRIE system. Binding of specific IgE in mite-allergic patient sera to the many antigens varied significantly both in frequency and in strength between and within patients. The results of the study are particularly significant because extracts produced from well-defined, media-free mite body and relatively pure mite feces were used. Inconsistencies in the reported number and potency of mite antigens and allergens are due to the use of poorly defined mite extracts of widely varying quality. Use of highly concentrated, well-defined, natural mite material is important for determining the origin and significance of specific mite allergens.

Investigations of culture medium-free house dust mites: III. Antigens and allergens of body and fecal extract of Dermatophagoides farinae

Crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) were used to characterize antigens (Ags) and allergens derived from Dermatophagoides farinae (DF) culture media-free mite body and mite fecal matter extracts. CIE of DF body and DF feces extracts revealed the presence of 35 and 20 Ags, respectively. CRIE experiments demonstrated IgE binding by 14 and seven DF body and DF feces Ags, respectively, when CIE gels were incubated with reference sera from clinically mite-sensitive patients. Binding of specific IgE to the various Ags in the two extracts varied significantly both in frequency and in strength from patient to patient and within the same patient's serum. Sera from some patients demonstrated IgE binding predilection for specific DF body Ags, whereas other sera exhibited greater binding preference for DF feces Ags. Homologous, heterologous, and intermediate gel CIE and CRIE clearly demonstrated that DF bodies and DF feces share some common Ags or epitopes, but the two different extracts also were quantitatively different. Some Ags and allergens originate from mite body material and are not present in mite feces. These results indicate that only extracts containing high concentrations of both body and fecal allergens should be used in clinical testing and therapy.

Rapid diagnostic methods for aspergillosis

Conventional laboratory methods are always unsatisfactory for the antemortem diagnosis of systemic mycoses, especially aspergillosis, in immunocompromised patients and those with acquired immune deficiency syndrome (AIDS), regardless of their age. There is increasing proof that aspergillosis is not limited to pulmonary, sinus or nasal regions. These facts, plus the high mortality rate, indicate a tremendous need for reliable and rapid methods of diagnosing this infection. Accordingly, refined techniques such as solid-phase radioimmunoassay (SPIRA), crossed-immunoelectrophoresis (CIE), crossed-radioimmunoelectrophoresis (CRIE), radioallergosorbent test (RAST), radioimmunoprecipitation assay (RIPA), paper radioimmunosorbent test (PRIST), computerized enzyme-linked immunosorbent assay (ELISA), biotin avidin enzyme-linked immunosorbent assay and gas-liquid chromatography (GLC) are being considered or used in clinical laboratories for diagnosing aspergillosis. The advantages and limitations of the RIA, ELISA and GLC methods are briefly discussed.

Diagnosis and Immunotherapy of Mould Allergy

Thirty-three adult asthmatic patients suspected of mould allergy were investigated by in vivo and in vitro tests in order to establish a specific diagnosis of asthma caused by the mould species Cladosporium. The patients were evaluated by daily symptom scores in the peak Cladosporium season, bronchial provocation test (BPT), skin prick test (SPT), RAST, histamine release from basophil granulocytes (HIST), and crossed radioimmunoelectrophoresis (CRIE), and the results were scored as negative (score 0), equivocal (score 1) or positive (score 2). Based on daily symptom scores and the result of BPT the patients were classified as being manifest allergic (asthma) to Cladosporium (positive allergy), inconclusive or negative. Positive allergy was defined as asthma symptoms oscillating with the spore concentration and a BPT score 2 (positive at allergen concentration less than 10,000 BU). Negative allergy was defined as no asthma symptoms and a negative BPT (score 0) and inconclusive in the case of symptoms and BPT sum of score 1-3. According to the classification a final diagnosis (positive or negative) could be established in 85% of the patients. "False positive" tests were found: for BPT in 27%, SPT 18%, RAST 0%, HIST 18%, and CRIE 0%. The corresponding figures for "false negative" were: BPT 0%, SPT 0%, RAST 27%, HIST 18%, and CRIE 23%. The relative risk of being allergic in spite of a negative test result was 0% for BPT and SPT, and 25-30% with RAST, HIST, and CRIE. In the case of positive test the risk was 90-100%. Excluding BPT, SPT was found to be the optimal single test to predict/rule out clinical allergy. A stepwise combination of positive SPT and positive RAST was found exclusively in patients clinically evaluated as positive, and does not call for an additional BPT. Using a potent allergenic extract a negative SPT excluded clinically important allergy. The primary conclusion of the study, however, is that the final diagnosis of Cladosporium asthma could not be based on a positive BPT alone (due to "false positive"), but only on a combination of clinical symptoms during the spore season and a positive BPT.

Allergen-specific IgE antibodies against antigenic components in cow milk and milk substitutes.

Crossed radioimmunoelectrophoresis (CRIE) was used to study the presence of serum IgE against antigenic components of cow milk in 21 selected milk-allergic patients. The amount of each IgE specificity was estimated by a scoring system. The milk-allergic children had mainly IgE against alpha-lactalbumin, beta-lactoglobulin, albumin and immunoglobulin, the four major proteins of bovine whey, as well as IgE against three casein components. A serum pool from 1000 normal adults had IgE against the same whey protein, but in smaller amounts, and no IgE against the casein components. Eight cow milk-based formulae, commonly used for infant feeding, and goat milk were studied by the same method. It was found that six of the milk substitutes did not differ significantly from cow milk in antibody binding, but the two hydrolysed casein products, Nutramigen and Pregestimil, consisted of such small molecules that the rabbit antisera could not precipitate the hydrolysed proteins in the gels on the CRIE plates. It was therefore not possible to study their IgE binding, if any, by this method.

Allergy to guinea pigs: II Identification of specific allergens in guinea pig dust by crossed radio‐immunoelectrophoresis and investigation of the possible origin

An extract of dust from the air-vent filters of a room housing guinea pigs was analysed by quantitative immunoelectrophoretic procedures and compared with extracts of various materials derived from guinea pigs. Crossed radio-immunoelectrophoresis (CRIE) of the dust, performed with sera from twenty asthmatic patients who were positive by skin testing and RAST to guinea pig extracts, identified fourteen IgE-binding constituents. Although responses varied, most sera reacted with four particular allergens, antigens 2, 3, 10 and Sl. The numbers of allergens recognized by individual patients correlated with the RAST score, but not with total serum IgE. All seventeen dust constituents detected by crossed immunoelectrophoresis (and all four major allergens), were also present in extracts of guinea pig dander, fur, saliva and urine; several of these components were absent in an epithelial extract, and there were even less in preparations of shaved pelt, serum or faeces. None of the dust extract antigens were detected in materials used in animal husbandry, dust samples from rooms without guinea pigs, or a D. pteronyssinus extract. These findings suggest that inhalant allergens may be derived predominantly from material shed from the guinea pig coat after contamination with saliva, and possibly to a lesser extent, urine.

Purification of two allergens from dermatophagoides pteronyssinus using monoclonal antibodies

Fractionation of soluble extracts from the house dust mite Dermatophagoides pteronyssinus (DP) by SDS-PAGE and crossed immunoelectrophoresis (CIE) revealed at least fifty distinct protein components. Western blotting and crossed radioimmunoelectrophoresis (CRIE) indicated that fewer than one quarter of these components were allergens as determined by their ability to bind IgE from allergic individuals. Following immunization with a crude extract, two monoclonal antibodies were raised against distinct components which exhibited IgE binding capacities in Western blot and CRIE. Affinity chromatography using these monoclonal antibodies yielded components which elicited positive skin test reactions in patients allergic to DP.

Analysis of tobacco leaf allergens by crossed radioimmunoelectrophoresis.

The reactivity of eleven 'tobacco smoke sensitive' and eight 'non-sensitive' individuals to tobacco leaf allergens was tested by Crossed Radioimmunoelectrophoresis (CRIE). All nineteen study subjects had IgE antibodies to tobacco leaf antigens as measured by Radioallergosorbent Test (RAST) and seventeen of the nineteen individuals were atopic. Of the thirty-seven tobacco leaf precipitins detected by Cross Immunoelectrophoresis (CIE), three were identified as allergens by CRIE. All nineteen subjects reacted to at least one of the three allergens detected. However, neither the intensity nor the incidence of reactivity with any of the three allergens correlated with smoking or 'smoke sensitivity'.

Immunochemical and biological characterization of a mugwort (Artemisia vulgaris) pollen extract.

A reference extract of mugwort pollen (Artemisia vulgaris) was characterized by crossed immunoelectrophoresis (CIE), crossed radio immunoelectrophoresis (CRIE) and quantitative skin prick test (QSPT). CIE revealed that the extract contained at least 42 distinct antigens of which 24 migrated towards the anode and 18 towards the cathode at pH 8.6. A CRIE analysis of the crude mugwort pollen extract, performed with sera from 29 mugwort-allergic patients, showed that 10 antigens may be considered allergens; one was classified as a major allergen, five as intermediate allergens, and four as minor allergens. The QSPT performed on the same 29 allergic patients established that 17.4 micrograms lyophilised reference mugwort pollen extract per ml had a biological potency of 1 HEP (histamine equivalent by prick test).

Crossed immunoelectrophoresis and crossed radioimmunoelectrophoresis analysis of "yellow jacket-common wasp" (Vespula spp.).

Crossed immunoelectrophoresis (CIE) pattern of Yellow jacket vespid venom showing 25 immunoprecipitates was used in crossed radioimmunoelectrophoresis (CRIE) analysis with sera from 35 subjects allergic to vespid venom. Phospholipase A and B, hyaluronidase, acid and alkaline phosphatases and a lytic active component were demonstrated in immunoprecipitates using zymography and direct haemolysis of sheep erythrocytes. CRIE analysis proved the allergenicity of these proteins. In addition, another antigen (Ag 17) with no detectable enzymatic or lytic activity was identified as an important allergen. This allergen is most likely identical to the previously described Antigen 5 in vespid venoms. By using CIE/CRIE the presence and complexity of multiple forms of the allergenic protein components were well demonstrated.

Identification of crustacea allergens by crossed radioimmunoelectrophoresis.

Crossed immunoelectrophoresis (CIE) detected 18 precipitating antigens in extracts of shrimp. Of these antigens, crossed-line immunoelectrophoresis (CLIE) of shrimp extract demonstrated that 5 cross-reacted with crayfish, 3 with lobster and 1 with crab extract. Allergens present in the shrimp CIE plates were identified by crossed radioimmunoelectrophoresis (CRIE) using sera from 6 study subjects who were skin-test and RAST positive to shrimp extract. Of the 7 allergens detected, 3 (precipitins 1, 3 and 6) reacted with most of the 6 sera tested from shrimp-sensitive subjects. Precipitins 1 and 6 appear to be common crustacea allergens (present in shrimp, crayfish, lobster and crab) whereas precipitin 3 may be a specific allergen since it is present only in shrimp.

Characterization of IgE and IgG antibody responses to Dermatophagoides farinae in rats.

Rats (Brown Norway/Wistar Fu) that were pretreated or not pretreated with cyclophosphamide were immunized with varying amounts of Dermatophagoides farinae allergen extract with alum as adjuvant. Sera were analysed by RAST and by crossed radioimmunoelectrophoresis (CRIE). The highest IgE antibody responses were recorded in animals pretreated with cyclophosphamide that had received low immunizing doses of antigen. The IgE antibody pattern in CRIE was, however, not influenced by the dose of antigen or by cyclophosphamide pretreatment. The IgG pattern in CRIE closely resembled the IgE pattern, demonstrating a similar specificity of the antibody responses of the two isotypes. This indicates non-class specific control of the specificity of antibody responses.