Identification of some rabbit allergens as lipocalins.

Rabbits are frequently used as laboratory animals or kept as domestic pets. Rabbit serum albumin and a 17-kDa protein referred to as Ory c 1 have previously been reported as allergens. Several other allergenic proteins have been recognized by crossed immuno-electrophoresis but have not been characterized. The aim of this study was to characterize the allergenic proteins present in rabbit saliva, urine and fur on the basis of molecular size and, where possible, to determine their amino acid sequences. Extracts from the male New Zealand white rabbit were used for developing specific direct RAST and RAST inhibition assays. Proteins in the extracts were separated by SDS-PAGE and the individual allergens identified by immunoblotting with serum from rabbit-allergic individuals. The N-termini of four allergens were sequenced. Saliva was the most potent extract. In total, 26 protein bands were recognized as allergens in the three extracts: 12 in saliva, seven in urine and seven in fur. Their molecular weights ranged from an 8-kDa species in saliva to an 80-kDa protein in urine. The N terminal sequences of an 18 kDa and a 21-kDa species in saliva, were identified as lipocalins with sequence similarity to a recently described odourant binding protein. This is the first evidence that allergens from the rabbit are members of the lipocalin superfamily of proteins, suggesting that similar mechanisms may be involved in eliciting the allergic response to rabbits. The 18 kDa allergen from saliva may be the previously named rabbit allergen, Ory c 1.

Meniere's disease: evidence of an immune process.

OBJECTIVE: This study aimed to present further evidence of specific antibodies reactive against bovine inner ear proteins found in patients with Meniere's disease. BACKGROUND: There are a growing number of experimental and clinical features of Meniere's disease that indicate an immune association. Circulating antibodies directed against inner ear proteins have been detected in patients with bilateral progressive hearing loss and Meniere's disease. METHODS: A total of 36 patients with classical Meniere's disease were studied noting the presence of active or inactive, bilateral or unilateral disease. Bovine membranous labyrinth was used as inner ear extract and separated to molecular weights using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blotting included the addition of serum buffer (casein, Tween, and bovine serum albumins) and the use of alkaline phosphatase labeled anti-human immunoglobulin antibodies. RESULTS: The findings represent strong evidence of antibodies reactive to inner ear proteins. The incidence of these antibodies was correlated significantly with disease activity. CONCLUSIONS: The findings are further evidence of an immune process that is involved in Meniere's disease.

Allergy to laboratory animals among animal handlers.

OBJECTIVE: To determine the prevalence among laboratory animal handlers of allergy to laboratory animals and of asthma and the factors associated with their development. DESIGN: A cross-sectional survey. SETTING: Teaching and research institutions in Sydney, between January 1989 and December 1992. PARTICIPANTS: Laboratory animal handlers (teaching and research staff, animal house workers and animal husbandry students and teachers). MAIN OUTCOME MEASURES: Duration of exposure to laboratory animals, allergic symptoms on contact, skin reactivity to laboratory and domestic animal allergens and evidence of current asthma. RESULTS: 228 subjects were surveyed. Allergy symptoms occurred in 73 (56%) of the subjects exposed to laboratory animals for three months or more. This group also had significantly higher prevalences of skin reactivity to laboratory animal allergens (62%) and bronchial hyperresponsiveness (21%) than those with shorter exposure (14% and 8%, respectively). Atopic subjects exposed to laboratory animals (particularly those sensitised to domestic animals) and animal attendants (with a high intensity of exposure to laboratory animals) had significantly higher frequencies of skin reactivity to laboratory animals and asthma than other subjects (77% and 30%, respectively, among exposed atopic subjects and 84% and 33%, respectively, among animal attendants). CONCLUSIONS: Allergy to laboratory animals is an occupational hazard among laboratory animal handlers, especially for those who are atopic and sensitised to domestic animals, and may lead to the development of asthma. Screening for atopy and skin reactivity to laboratory animals before and during employment would enable those at risk to take precautions.

Specificity of two-site immunoassays.

When cross-reaction in two-site immunoassays was investigated both theoretically and experimentally it was found that such systems do not always result in enhanced specificity. Computer simulation studies indicated that substances which display negligible cross-reaction in a radioimmunoassay could produce an assay response identical to that of the analyte in a two-site immunoassay using excess antibody. Cross-reactivity in two differing two-site immunoassays was compared to that obtained in radioimmunoassays using the same monoclonal antibodies for human chorionic gonadotrophin. In addition to the effects of excess antibody, cross-reactivity was observed in one of the two-site immunoassays which could not have been predicted from the specificity of the antibodies or cross-reactivity in the radioimmunoassays. The unexpected cross-reaction of the beta subunit of human chorionic gonadotrophin in the assay resulted from an apparent alteration in the specificity of one of the antibodies following binding of the beta subunit to the second antibody. These studies emphasise the complexity of binding reactions in two-site immunoassays.

Heterophilic antibodies: a problem for all immunoassays.

We verified that antibody-binding substances in serum that interfere in two-site immunoassays involving murine antibodies are heterophilic antibodies. Incubation of serum containing heterophilic antibodies and a murine monoclonal antibody to human choriogonadotropin (hCG) leads to formation of a series of soluble immune complexes. We investigated the recognition of hCG by reagent antibody in the presence of heterophilic antibodies and found this recognition to be diminished. Consequently, about 30% of serum samples containing heterophilic antibodies falsely appear to contain increased concentrations of hCG. The effect on analyte recognition probably results from steric inhibition of hCG binding to complexed antibody. Heterophilic antibodies detected with a murine antibody also bound immunoglobulin from several other species but did not bind all of those tested.

Incidence and specificity of interference in two-site immunoassays.

Using a modified "two-site" immunoradiometric assay, termed an "interference assay," we have demonstrated the occurrence of non-analyte antibody-binding substances in approximately 40% of serum samples. These substances multivalently bind antibodies from any of several species at a site other than the antigen-binding site. Using a two-site immunoradiometric assay for human choriogonadotropin, we have investigated their effect on analyte quantification. In this system these antibody-binding substances mimic the presence of analyte; when analyte is actually present, they can also cause over- or underestimates. Addition of excess nonspecific immunoglobulin from an appropriate species eliminates this interference. However, the amount of nonspecific immunoglobulin added to an assay system may not always be enough to block interference in all samples.

Covert cross reactants in a two-site immunoassay studied with monoclonal antibodies.

A one-step two-site immunoradiometric assay for the measurement of free beta subunit of human chorionic gonadotrophin (beta-hCG) was developed using monoclonal antibodies. The immobilized antibody was specific for free beta subunit and the radiolabeled antibody recognized both intact human chorionic gonadotrophin (hCG) and free beta subunit. Although the level of hCG "cross-reaction" was low when studied using conventional techniques, the apparent beta-hCG content of samples was found to be inversely proportional to the hCG level. From both experimental evidence and computer simulation studies this was found to be due to the binding of hCG to the limited amount of 125I-labeled antibody present. The term covert cross reactants has been introduced to describe substances which bind to only one of the antibodies in a two-site immunoassay. When establishing such an assay the effect of covert cross reactants on the response of an analyte should be investigated.

Use of immunoaffinity chromatography for purification of 125I-labeled human prolactin.

We assessed a simple method for purifying 125I-labeled human prolactin, taking advantage of the abundant supplies of monoclonal antibodies available. 125I-Labeled human prolactin purified by immunoaffinity chromatography is compared with that purified by gel filtration on Sephadex G-100. We used monoclonal antibodies to prolactin to prepare the affinity chromatography columns. Prolactin was radiolabeled by the Chloramine T method, purified by each of the above procedures, and the binding and displacement characteristics were studied in radioimmunoassays in which either monoclonal antibodies or a rabbit anti-prolactin serum was the first antibody. A nonimmune fraction of 125I-labeled prolactin that co-eluted with the immunoreactive hormone from Sephadex G-100 was removed by affinity chromatography, which increased the antibody binding of 125I-labeled prolactin in the radioimmunoassay in the absence of unlabeled antigen (B/T0, in percent) twofold or more, increased the assay sensitivity, and increased the slope of antigen displacement measured by the 50% intercept. Several advantages make this the purification method of choice.

A monoclonal antibody suitable for the radioimmunoassay of prolactin in human serum.

Monoclonal antibodies directed toward human PRL (hPRL) have been produced by fusion of mouse myeloma cells (Sp2/0-Ag 14) with spleen cells from mice immunized with hPRL. Total immunizing doses of 20 microgram and 64 microgram hPRL resulted in the production of three highly specific hPRL antibodies. The high affinity antibody, with a Ka value of 0.23 X 10(10) M-1, was used to establish a RIA highly suitable for the measurement of hPRL levels in human serum. The correlation of serum hPRL levels measured using the antibody and those in a conventional rabbit anti-hPRL assay was 0.99 (y = 1.16 - 7.2). These results demonstrate that using the mouse hybridoma technique, it is possible to produce high affinity monospecific monoclonal antibody suitable for the measurement of hPRL in human serum.