A reference allergen preparation of the house dust mite D. pteronyssinus, produced from whole mite culture--a part of the DAS 76 study. Comparison with allergen preparations from other raw materials

A D. pteronyssinus whole culture allergen preparation contained 49 antigens as revealed by crossed immunoelectrophoresis (CIE), using polyspecific rabbit antibodies. Crossed radio-immunoelectrophoresis ( CRIE ) with sera from 30 patients revealed nine allergens, antigens 42, X, Y and 23 (in rank order) showing the most frequent and intense IgE-uptake. Nine antigens originated from the culture medium (human dander + yeast), but none of these gave rise to specific IgE-uptake. Extremely few and weak reactions were observed in radioallergosorbent (RAST) with 129 sera, using media extracts on the discs. Purified mite body extract (PMB) contained less ag 42 and more ag Y and ag 23 than whole mite culture extract ( WMC ), whereas an acetone-extracted mite excreta preparation (AML) contained 5 times more ag 42, but was devoid of ag Y and ag 23. Ag X was present in all preparations. The RAST-inhibitory potency of PMB was best correlated with the content of ag X. Preparations with properties similar to WMC and PMB were judged as suitable for clinical application.     

Nonspecific Crossreacting Antigen (NCA) in Human Mammary Carcinoma Extracts

Human mammary carcinoma (HMC) homogenates from 28 patients and mammary hyperplasia (MH) homogenates from 20 patients undergoing reduction mammoplasty were used as antigens in preparing antibodies in rabbits. Crossed immunoelectrophoresis of HMC extracts against the corresponding antibodies revealed one major precipitate not present in MH. This antigen was present in separately prepared extracts of breast tissue from 3 out of 10 patients with HMC. Antibody titre was unchanged following absorption with MH, normal human liver, kidney, skin and serum. Immunochemical crossreaction with pregnancy zone proteins, alpha-1-fetoprotein, ferritin, lactoferrin, plasma, extracts of human placenta and fetal and adult liver did not occur. The antigen could not be demonstrated by rocket immunoelectrophoresis of sera from patients with HMC. Small amounts of the antigen were found in extracts from human spleen and renal carcinoma and larger amounts in extracts from human peripheral granulocytes, but not in preparations of mononuclear leucocytes (98% pure). Tandem crossed immunoelectrophoresis of extracts from HMC and carcino embryonic antigen against antibodies prepared against HMC revealed a reaction of partial identity between the two antigens, and antisera prepared against carcino embryonic antigen contained precipitating antibodies against the HMC antigen. The HMC antigen was eventually identified as nonspecific crossreacting antigen (NCA) described by other authors.     

Detection and Quantitation of Dermatophagoides Antigens in House Dust by Immunochemical Techniques

Rabbit antibodies against D. pteronyssinus whole culture extract were rendered specific to mite antigens by immunoabsorption with extract of human dander. Using the absorbed antibodies, an extract of a mite culture gave rise to 15 peaks in crossed immunoelectrophoresis and to a titer of 512 in counter-immunoelectrophoresis. When applied to dust samples from the homes of allergic patients, counterimmunoelectrophoresis gave reproducible results within an error of 1 titer-step. Mite antigens were detected in 64% of 105 dust samples investigated. The titer correlated well (P less than 0.005) with the concentration of Dermatophagoides bodies, found in 13 mattress dust samples by microscopy. Advantages and limitations of immunochemical quantitations of house dust mites in dust samples is discussed in relation to investigations by microscopy.     

Quantitative Immunoelectrophoretic Analysis of Extract from Cow Hair and Dander

Quantitative immunoelectrophoresis used for the analysis of a diabased, centrifuged and freeze-dried extract from cow hair and dander revealed 17 antigens. Five of these were identified as serum proteins. Partial identity to antigens of serum and extract from hair and dander of goat, sheep, swine, horse, dog, cat and guinea pig, and to antigens of house dust was demonstrated. Sera from 36 patients with manifest allergy to cow hair and dander selected on the basis of case history, RAST, skin and provocation test, were examined in crossed radioimmunoelectrophoresis (CRIE); sera from five persons with high serum IgE, but without allergy to cow hair and dander, and sera from five normal individuals were controls 31/36 of the sera contained IgE with specific affinity for two of the antigens of the extract. Further, two major and six minor allergens were identified. The control sera showed no specific IgE binding. A significant positive correlation was found between RAST and CRIE for the first group of patients. The approximate molecular weights of the four major allergens obtained by means of gel chromarography were: 2.4 × 10⁴, 2 × 10⁴, 2 × 10³ dalton, respectively. Using Con-A and Con-A Sepharose in crossed immunoaffinoelectrophoresis, eight of the antigens were revealed to contain groups with affinity for Con-A.     

Characterization of Bovine Whey Proteins by Crossed Immunoelectrophoresis

A reference pattern obtained by crossed immunoelectrophoresis of bovine whey proteins from mature milk, using rabbit antibovine whey antibodies, has been worked out. Out of the total of 29 easily recognizable precipitates, 5 milk-specific proteins and 21 serum proteins were identified. Furthermore, whey proteins which also occur in bovine milk fat globule membrane preparations and in extracts of bovine hair and dandruff were identified. The use of the obtained reference pattern for the identification of milk allergens and the determination of the specificity of human milk precipitins against bovine whey proteins is outlined.     

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.     

Activation of the classical pathway of human complement in vitro by house-dust extracts is caused by IgM antibodies to polysaccharide antigen(s) and is not related to atopy

The mechanism of activation of the classical pathway of human complement by house-dust extracts and its relevance to atopic disease was studied. Our results confirm that for most sera of adults, house-dust extract is, on a weight basis, a more potent C-activator than aggregated human IgG or lipopolysaccharide (endotoxin). Naturally occurring IgM antibodies directed against ubiquitous polysaccharides appeared to be the dominant factor in the C1 activation by house-dust extracts in human sera. Large variations were found between sera with respect to the concns of these IgM antibodies as measured by C1 activation or fixation of haemolytic complement. The IgM antibody titre was, however, not associated with atopic disease. Consequently, we do not support the hypothesis put forward by Berrens et al. (1978) (Allergol. Immunopath. 6, 45-54) that there might be a relation between atopy and enhanced reactivity of serum complement with allergenic extracts. More than 90% of the C-activating potential of allergen extracts like house dust was found in the fractions with high mol. wt material (mol. wt greater than 100 K). Therefore, these antigens are easily separated from the known IgE-binding major allergens of house-dust mite and cat dander.     

Crawfish and lobster allergens: identification and structural similarities with other crustacea

Antigenic and allergenic components in crawfish and lobster extracts were studied using crossed immunoelectrophoretic techniques. Crossed immunoelectrophoresis with rabbit antisera revealed 23 antigens in crawfish and 17 antigens in lobster extracts. Both extracts exhibited structural similarities in antigens mutually and with other crustacea in cross-line immunoelectrophoresis. Crossed radioimmunoelectrophoresis (CRIE) demonstrated 6 crawfish and 4 lobster allergens when individual or pooled sera from radioallergosorbent test (RAST)-positive crustacea-sensitive subjects were used. Since radiostaining was also observed with sera from RAST-negative nonsensitive subjects, specificity of IgE binding was tested using CRIE-inhibition. Preincubation of RAST-positive sera with crawfish or lobster extract decreased radiostaining in CRIE, while no changes occurred when using control sera. These results confirmed the presence of IgE-mediated mechanisms in seafood allergy and demonstrated a number of shared antigenic determinants among crustacea allergens.     

Allergen profiles of dog hair and dander, body fluids and tissues as defined by immunoblotting

The sera from 25 patients with clinical type I allergy against dogs were investigated by means of immunoblotting, using extracts of dog hair/dander, skin, hair, saliva, salivary gland, serum and liver. 96% of the patients' sera showed IgE antibodies reactive with 19- and 23-kilodalton (kDa) proteins in the hair/dander extract. The 23-kDa IgE-binding protein was preferentially detected in the hair extract and saliva but not in skin, salivary gland, serum and liver extracts. The 19-kDa band was strongly expressed in skin, but not in hair, serum and liver. Inhibition experiments using the 23-kDa containing extract prepared from hair and the 19-kDa containing extract prepared from skin revealed that these two proteins are likely to be immunologically independent allergens.     

Antigenic/Allergenic Composition of Poodle/Alsatian Dandruff Extract

Poodle and Alsatian dog dandruff extracts were characterized by crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE) using sera from 24 individuals clinically sensitive to dogs. By using a system with intermediate gel in immunoelectrophoresis, the content of dander-specific and serum-specific allergens was established. 29 antigens (18 dander-specific and 11 serum-specific) were identified in the mixed breed Poodle/Alsation dandruff extract. Of these, 24 antigens were radiostained in CRIE. 16 allergens were dander specific and the remaining eight were serum specific. Positive dog dander RAST (e5 and Poodle/Alsatian dandruff extract) results were observed in the tested dog hypersensitive subjects. Our results suggest that the mixture of Poodle/Alsatian dandruff extract may be a suitable preparation for the diagnosis and treatment of dog allergy.     

Allergy to rabbits

Crossed immunoelectrophoresis and crossed radio-immunoelectrophoresis techniques have been employed for analysis of extracts of various rabbit source materials to identify rabbit allergens in addition to the already described Antigen R1 (AgR1). Urine and dander extracts were found to contain only low levels of AgR1 and its presence in urine was as a contaminant due to mode of collection--it was not present in urine collected directly from the bladder. Other allergens were only recognised by highly rabbit-sensitive individuals, one in particular (Ag2) being present in several source materials. Serum albumin proved to be of minor allergenic importance and except for dander its presence was only in minimal amounts. As both AgR1 and Ag2 are significant components in extracts of fur and dust these extracts are therefore most recommended for use in investigations of individuals sensitive to rabbits.     

Allergen Content in Aqueous and Hydrophobic Extracts from Cow Hair and Dander

Rabbit antibodies were raised separately against an aqueous and a detergent extract of row hair and dander. It is shown by means of quantitative immunoelectrophoresis that the extracts contain the same major allergens and that these are equally potent in RAST-inhibition experiments. The extraction with a detergent did not lead to the appearance of new allergens.     

Crossed Immunoelectrophoresis Analysis of Bee Venom

Bee venom obtained by electrical stimulation has been analysed by crossed immunoelectrophoresis against high-titer rabbit antibodies. The antigenic analysis of bee venom revealed that the extract contained 17 antigens, which were detected in die crossed immunoelectrophoresis pattern after staining with Coomassie Brilliant Blue. The crossed immunoelectrophoresis pattern indicated heterogeneity in several of the antigens, as most of the preceipitates (exhibiting enzyme and hemolytic activities) represented multiple forms of phospholipase A, hyaluronidase, acid phosphatase and melittin.     

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 lour particular allergens, antigens 2. 3, 10 and SI. The numbers of allergens recognized by individual patients correlated with the RAST score, but not with total scrum 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.     

Allergen extract of horse hair and dandruff. Quantitative immunoelectrophoretic characterization of the antigens.

Freeze-dried extract of horse hair and dandruff was obtained by extraction, centrifugation, dialysis and freeze-drying. Quantitative immunoelectrophoresis using rabbit antibodies revealed the extract to be composed of 25 antigens of which some were mutually partial identical; 4 were serum-specific and non showed partial identity to solubilized hair proteins. Partial identity to antigens to serum and extract from hair and dandruff of cow, dog, cat, guinea pig, and of extract from house duct was demonstrated. After subjecting the extract to dialysis, ultrafiltration, freeze-drying and storage below 37degreesC for not more than 24 h the antigenic stability and the allergenic activity were unaffected. The effect of enzymatic degradation of the individual proteins with regard to antigenic stability and allergenic activity was also examined.     

Antigenic Analysis of Treponema pallidum: Cross-reactions between Individual Antigens of T. pallidum and T. Reiter

Seven antigens were demonstrated in the Nichols pathogenic strain of Treponema pallidum when tested by crossed immunoelectrophoresis against rabbit antibodies raised by immunization with T. pallidum sonicate. Indirect evidence was obtained for the presence of two more T. pallidum antigen of the nine antigens six reacted with antibodies in a human syphilitic serum pool. Cross-reactions between individual T. Reiter antigens and the seven directly demonstrated T. pallidum antigens were studied by different immunoelectrophoretic techniques. using rabbit anti- T. Reiter Ig, rabbit anti- T. pallidum Ig, and human syphilitic serum pool. of the seven T. pallidum antigens three were not found in T. Reiter, three had epitopes identical to corresponding antigens in T. Reiter, and one had both cross-reacting and T. pallidum -specific epitopes. Human syphilitic serum had antibodies against two of the T. pallidum -specific antigen and against four T. pallidum antigen cross-reacting with antigen of T. Reiter.     

Immunological Investigation of Possible Structural Similarities between Pollen Antigens and Antigens in Apple, Carrot and Celery Tuber

Crossed line immunoelectrophoresis (CLIE) was used to show that a minor allergen present in birch pollen and another present in timothy pollen shared common epitopes with antigens in apple, carrot and celery tuber. Major pollen allergens were not involved. Structural similarities were also noticed in some mugwort pollen antigens and antigens in apple, carrot and celery, but none of these mugwort antigens acted as an allergen. In crossed immunoelectrophoresis (CIE) of samples of apple, carrot or celery extracts towards antibodies against birch pollen, grass pollen or mugwort pollen, four to 14 distinct precipitates were observed and shown to be specific. In crossed radioimmunoelectrophoresis (CRIE) none of the precipitates could be shown to act as an allergen. Using allergen detection on nitrocellulose paper, the apple, carrot and celery extracts were shown to possess IgE-binding capacity.     

Antigens Common to Scabies and House Dust Mites

Twenty-five antigens were demonstrated in a crude Dermatophagoides farinae (DF) extract by means of crossed immunoelectrophoresis (CIE), using a rabbit anti-DF serum. With the same antiserum, four antigens were demonstrated in a scabies mite preparation, indicating an immunological relationship between these scabies antigens and some of the DF antigens. In tandem CIE experiments the DF antigens were found to be partly identical to three of the scabies antigens. No IgE activity against any of these scabies antigens was evident in pooled sera from patients with house dust mite allergy by crossed radioimmunoelectrophoresis. However, one such patient had IgE antibodies to a DF antigen and a partial identical scabies antigen.     

Crossed radioimmunoelectrophoretic analysis of cultivated rye (Secale cereale) pollen allergens

Cultivated rye pollen extracts were characterized by means of crossed immunoelectrophoresis (CIE) and crossed radioimmunoelectrophoresis (CRIE). 32 antigens were identified in CIE and among these 16 were radiostained in CRIE analysis of a rye pollen sensitive patient panel. Crossed line immunoelectrophoresis revealed that some of the rye pollen antigens were immunological partially identical with antigens of wheat flour and rye flour.     

Detection of Rickettsia rickettsii antibodies in human sera by crossed immunoelectrophoresis

To identify Rickettsia rickettsii antigens of immunological importance, we examined sera from patients with serologically confirmed cases of Rocky Mountain spotted fever by crossed immunoelectrophoresis for antibodies to antigens extracted from the R strain of R. rickettsii with the detergent Triton X-100. Sixteen antigens were identified in the detergent extract by crossed immunoelectrophoresis with a hyperimmune rabbit serum raised against whole rickettsiae. When the rabbit antiserum was placed in the reference gel and patient sera were placed in the intermediate gel, antibodies to one or more antigens were detected in 61 of 71 North Carolina sera, all of 7 Oklahoma sera, and 9 of 10 Montana sera obtained from 1 day to 40 years after onset of Rocky Mountain spotted fever. Antibodies to antigens 1 and 16 were found as early as 1 day after onset of illness, and antibody to 16 was found in 20 of 29 sera obtained within the first 7 days of illness. Antibodies to antigens 2 and 3 generally did not appear until the third week of illness but were found in six of seven serum samples collected 4 to 40 years after onset of Rocky Mountain spotted fever. Antibodies to R. rickettsii antigens 1, 7, 8, and 16 were found in sera from patients with illnesses caused by other etiological agents. Four of the Oklahoma and Montana sera from Rocky Mountain spotted fever patients, but none of the North Carolina sera, had antibodies to antigen 12. Sera containing antibodies against antigens 3 and 14 prevented death of mice challenged with two 50% lethal doses of R. rickettsii.