Immunoglobulin D in serum, body fluids and lymphoid tissues

Measurements of serum IgD concentrations at various ages and in a number of diseases are reported. IgD was not detected in plasma obtained during the first 6 months of life and was only very rarely found in cord plasma. Older children and adults showed a wide range of levels. IgD levels were not increased in three examples of `autoimmune'' disease, but increases were noted in some patients having chronic infections. IgD was usually absent in hypogammaglobulinaemia. IgD was not detected in a variety of body fluids.Lymphoid cells containing IgD were rare in lymph nodes, spleen and intestinal mucosa compared with cells containing other classes of immunoglobulins. In adenoid tissue IgD containing cells occurred more frequently and were often similar in numbers to those containing IgM.     

Biopterin derivatives in human body fluids and tissues.

Levels of biopterin derivatives in urine, serum, milk, cerebrospinal fluid, brain, and liver have been measured with the Crithidia fasciculata assay. Normal levels in serum and urine have been given and compared with those in a number of benign and malignant proliferative disorders, phenylketonuria, kidney disease, Parkinson's disease, schizophrenia, controlled epilepsy, rheumatoid arthritis, and pernicious anaemia. The active component of Crithidia factor in serum was 7,8-dihydrobiopterin. Tissue, urine, and some serum samples contained two active materials, the principal one being 7,8-dihydrobiopterin; a minor constituent was probably tetrahydrobiopterin. Serum biopterin levels following methotrexate administration were raised and subsequent administration of folic acid and 5-formyltetrahydrofolic acid further increased serum levels of biopterin derivatives; this was in contrast to the total absence of response to oral folates without prior methotrexate and to 5-methyltetrahydrofolic acid either with or without methotrexate being given.     

Carbohydrate epitope defined by an antitumor monoclonal antibody detected on glycoproteins and a glycolipid by immunoblotting

The murine monoclonal antibody (MAb) MOv2 was found to be directed against the carbohydrate moieties of different kinds of molecules expressed on a human ovarian cystoadenocarcinoma. To define further the glycoconjugates carrying the MOv2-defined epitope, different procedures were used to analyze materials from surgical specimens and carcinoma cell lines. SDS-PAGE and immunoblotting showed glycoprotein molecules migrating in the gel as high and intermediate molecular weight components. A low-molecular-weight band, migrating approximately with the dye front, was also immunostained by MOv2. On the other hand, the immunostaining of high-performance thin-layer chromatography (HPTLC) of the total glycolipid extract and its neutral and acid fractions, after DEAE chromatography, showed selective reactivity with a neutral glycolipid. Reanalysis by immunoblotting of this glycolipid band scraped off the HPTLC plate indicated that it corresponds to the low-molecular-weight component. Periodate oxidation and Pronase digestion further demonstrate the saccharide nature of the determinant on both types of glycoconjugates. In conclusion, we report evidence that with a single analytical procedure, i.e., immunoblotting, it is possible to recognize the same carbohydrate determinant carried on both protein and lipid molecules.     

Optimal conditions for the removal of house dust mite, dog dander, and pollen allergens using mechanical laundry

BACKGROUND: Mechanical laundry is an effective tool for the environmental control of allergens, but the optimal conditions for removing allergens are not yet clear. OBJECTIVE: To evaluate the optimal conditions of mechanical laundry for the removal of house dust mite (HDM), dog dander, and pollen allergens. METHODS: The 4 washing modes of 30 degrees C (86 degrees F), 40 degrees C (104 degrees F), 60 degrees C (140 degrees F), and steam water (SW) with detergent were evaluated. Allergen removal performance was assayed using a 2-site enzyme-linked immunosorbent assay (ELISA) or an ELISA inhibition test. RESULTS: Using the 30 degrees C and 40 degrees C washing modes, only 6.5% and 9.6% of Dermatophagoides farinae, respectively, were killed. However, using the 60 degrees C and SW washing modes, all HDMs were killed. The amounts of Der f 1 remaining after the 30 degrees C, 40 degrees C, 60 degrees C, and SW washing modes were 26.8%, 2.4%, 1.3%, and 0.6%, respectively, with unmanipulated contaminated sheets. The effects of rinse on Der f 1 levels after the 30 degrees C washing were greater compared with those after the 40 degrees C, 60 degrees C, and SW modes. The amounts of Can f 1 in the extractions after washing were 0.3% to 1.3% for all modes, and all extracts, even without a rinse, did not inhibit specific IgE binding to dog allergens according to ELISA. The remaining pollen allergen levels after washing were lower in the 60 degrees C and SW modes than in the lower temperature modes. However, the levels did not differ among the various washing modes after rinsing once. CONCLUSION: Water temperature and number of rinses are critical factors for the removal of HDM, dog dander, and pollen allergens.     

Allergy to fruits and vegetables in pollen‐sensitive patients: Allergen characterization by IgE immunoblotting and peroxidase staining

Allergies to several plant foods, i.e. apples, nuts, stone fruits, celery and spices, are highly associated with pollen allergies. The observed crossreactivities are due to structural similarities between food and pollen allergens. Using horseradish peroxidase (HRP) as the marker enzyme we have further developed and optimized a sensitive and specific immunoblotting procedure for the detection of human IgE antibodies specific to food and pollen proteins separated by SDS‐PAGE and immobilized on nitrocellulose blots. Our optimization studies involved four colour substrates and six buffer systems. The best results were obtained when 0.3% Tween 20 and no protein was used for membrane blocking, whereas blocking with proteins yielded high backgrounds. Immuno‐ detection amplification was accomplished by the following incubation steps: (a) sample (human serum); (b) secondary antibody; (c) biotinylated tertiary antibody; and (d) streptavidin‐HRP. Staining was performed with 3,3',5,5'‐tetramethylbenzidine which is an Ames test‐negative, alternative substrate for HRP. The quality of several commercially available anti‐IgE antibodies was examined. For some of these reagents very high non‐specific binding to food and pollen proteins occurred. The application of our method to apple and birch pollen allergen characterization is described. Results of IgE‐ and IgG‐immunoblotting and immuno‐detection of allergens following two‐dimensional‐ electrophoresis, as well as cross‐reactivity studies by means of immunoblot inhibition, are presented.     

Comparison of IgE-binding antigens in horse dander and a mixture of horse hair and skin scrapings

Extracts of horse dander (HD) and horse hair and skin scrapings (HHSS) have been compared with respect to their content of proteins and carbohydrates. The protein content of HD is more than double that of HHSS, while the carbohydrate content is of the same order. SDS-PAGE and IEE, both combined with immunoblotting, and CIE/CRIE showed the IgE-binding ability of the proteins/glycoproteins present in the two extracts. SDS-PAGE/immunoblotting showed the presence of mainly the same IgE-binding bands in the two extracts. Nine were detected in HD, and seven in HHSS. Four of these were glycoproteins. The most important allergen was a glycoprotein with molecular mass of 27 kDa showing IgE-binding ability to the 16 patient sera available. Another important allergen with molecular mass of 67 kDa is probably horse albumin, IEF/immunoblotting showed the presence of a glycoprotein with IgE-binding ability at pH 3.2 in both extracts. Tandem CRIE showed that both extracts contained the same four allergens as major allergens. In addition, as we observed when analyzing both extracts in CRIE against 16 different patient sera, each extract contained allergens not present in the other extract. We may conclude from this investigation that commercial allergen extracts to be used in diagnosis and immunotherapy should include both horse dander and horse hair and skin scrapings in the starting material for the preparation of extracts. If not, allergens which may be of importance for some patients will be missing.     

The allergens of dog II. Identification and partial purification of a major dander allergen

A dog hair and dander (DHD) extract was prepared from hair obtained from mixed breeds. By SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and immunoblotting, using sera from 32 dog-allergic subjects, a number of IgE radio-staining bands could be seen. In 78% of sera a protein of molecular weight (MW) of 21 000 daltons, designated Ag X, was found to bind IgE and in 34% it did so strongly. This allergen was isolated from DHD by size-exclusion and ion exchange chromatography. The final product was a single allergen of MW of 21 000 and an isoelectric point of approximately 5.2. An additional protein-staining band could still be seen of MW of 24 000 daltons. Using a serum which contained IgE antibodies only to Ag X, this allergen was found only in DHD extract and dog saliva and was absent from dog serum and urine. It was the same dog allergen that we [1] reported as Ag 8 using crossed radio-immunoelectrophoresis (CRIE) and that Blands et al. [2] and Løwenstein [3] described as Ag 13. We propose that this major dog allergen be given the title Can f I according to the new allergen nomenclature.     

Detection of an allergen in dog dander that cross-reacts with the major cat allergen, Fel d 1

BACKGROUND: A considerable proportion of animal-allergic patients are sensitized to both cat and dog allergens but knowledge about cross-reactive allergens in cat and dog dander is limited. OBJECTIVE: To investigate whether dog dander contains an allergen that cross-reacts with the major cat allergen, Fel d 1. METHODS: Recombinant Fel d 1 with the same immunological properties as natural Fel d 1 was used for quantitative (CAP) IgE competition experiments performed with sera obtained from cat-allergic patients (n=36). A Fel d 1 cross-reactive dog allergen was characterized by one- and two-dimensional immunoblotting using rFel d 1 for IgE inhibition experiments and with monospecific, polyclonal rabbit anti-recombinant Fel d 1 antibodies. RESULTS: In 25% of Fel d 1-reactive cat-allergic patients, more than 50% inhibition of IgE reactivity to dog allergens was achieved with recombinant Fel d 1. An Fel d 1 cross-reactive 20 kDa allergen with a pI of approximately 3.4 was detected in dander extracts of several different dog breeds. CONCLUSION: This is the first report demonstrating the presence of an Fel d 1-like allergen in dog dander extracts, which may be responsible for double positivity to cat and dog in serology. However, the clinical relevance of this cross-sensitization needs to be confirmed. These results are important for the diagnostic and therapeutic use of dog dander allergen extracts.     

Determination of kallikrein by radioimmunoassay in human body fluids

A sensitive and specific radioimmunoassay for human urinary kallikrein was developed, which allows tissue kallikrein determination in human urine, saliva, pancreatic juice, bile and sweat. In several body fluids a kallikrein-like antigen was found, but not in gastric juice and breast milk. According to gel filtration studies, complex formation of kallikrein with serum proteins or different molecular weight forms of kallikrein in serum and urine may be assumed. Pancreatic kallikrein secretion follows the same pattern after stimulation with secretin and cholecystokinin as trypsin and chymotrypsin in normal individuals. In chronic pancreatitis the kinetic behaviour remains unchanged with respect to the enzyme secretion, but the secretion of kallikrein is reduced to about 20%.     

Mannose in body fluids as an indicator of invasive candidiasis.

Using gas-liquid chromatography, we measured five mannose in the serum of six nondiabetic patients with autopsy-proven invasive candidiasis. In all patients serum mannose concentrations were higher than mannose levels found in serum from normal adults and children or from patients with catheter-associated candidemia, mucosal candidiasis, and other mycoses. Spinal fluid from two patients with Candida meningitis showed increased free mannose as compared to seven non-inflammatory spinal fluid samples. However, free mannose in the serum of poorly controlled diabetics (blood glucose of greater than or equal to 300 mg/dl) did overlap concentrations in patients with invasive candidiasis. In vitro culture of Candida albicans demonstrated increasing concentrations of mannose associated with growth of the organism. We conclude that physical and chemical assay for mannose in body fluids may be a useful technique to assist in the diagnosis in invasive candidiasis.     

Comparison of IgG antibody profiles by immunoblotting in patients with acute and previous Toxoplasma gondii infection.

The IgG antibody profile to Toxoplasma gondii proteins of less than 37 kilodaltons in sera from patients with acute and previous infection was studied by immunoblotting. Bands at 28, 29, and 36 kilodaltons were more common in acute infection (10 out of 10, nine out of 10, and nine out of 10, respectively) compared with previous infection (five out of 10, four out of 10, and one out of 10, respectively). The 6 kilodalton band was present in 10 out of 10 sera from patients with acute infection and five out of 10 sera from those with previous infection. A new observation is a doublet of bands of 22-25 kilodaltons present only in sera from patients with acute infection. This doublet may be a more reliable indicator of acute infection than the 6 kilodalton band.     

Cortisol in hair, body mass index and stress-related measures

Hair cortisol concentrations (HCC) are assumed to reflect integrated cortisol secretion over extended periods of time and may provide a sensitive marker for stress-associated endocrine changes. Here, we report data from two independent studies of 155 (study I) and 58 participants (study II) in which HCC associations with different stress-related measures and body mass index (BMI) were investigated. Consistent evidence for positive associations between HCC and BMI was seen across both studies (study I: r=.33, p<.001; study II: r=.42, p=.001). On the other hand, findings failed to reveal reliable HCC associations with psychosocial variables, showing only a positive relationship with self-reported social overload in study II (r=.29, p=.03) but not with other stress-related measures.     

Identification of allergens in dog dander extract. I. Clinical and immunological aspects of allergenicity activity

Allergenic activity of mixed dog dander extract was compared with dog serum albumin by skin testing, RAST testing and RAST inhibition. Whereas albumin is a potent allergen, additional lower molecular weight allergens determined by sucrose density gradient centrifugation and immunoelectrophoresis were found. Identification of the most potent allergens and standardization of extract used for testing and treatment is a necessary prerequisite in understanding dog dander hypersensitivity.     

Virus-specific serum IgG, IgM, and IgA antibodies in cytomegalovirus mononucleosis patients as determined by immunoblotting technique

The immune response to individual human cytomegalovirus (CMV) structural polypeptides was studied in paired sera from 15 adult CMV mononucleosis (CMV-MN) patients and healthy controls by immunoblotting technique (IB). IgM and IgG antibodies to at least 11 structural polypeptides with molecular weights of 28K, 49K, 55K, 57K, 66-70K, 82K, 87K, 110K, 150K, 205K, and 235K were detected in the patients' sera in the serum sample obtained in the acute phase of the disease. IgA antibodies to polypeptides with molecular weights of 66-70K, 82K, 110K, and 150K were also detected in these sera. In healthy seropositive adults, IgG antibodies with the same molecular weight polypeptides, excluding the 205K and 235K polypeptides, were detected as in convalescent CMV-MN patients. A prominent reactivity of IgM and IgA antibodies to the 66-70K and 150K polypeptides was noted in the acute sera from all the CMV-MN patients examined, but not in a number of late convalescent sera. The potential implications of these findings in the development of specific serological tests are discussed.