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- Material and methods
Potential factors influencing antigen detection in immunoassays for measuring rat or mouse aeroallergen (i.e., assay setup, antigen specificities, standard extracts used, and antigen decay) were investigated in a three-country study (the UK, The Netherlands, Sweden). An inhibition enzyme immunoassay (EIA) setup gave nominal rat urinary allergen (RUA) sample values seven times higher than a sandwich EIA setup utilizing identical antibodies and standards. In immunoblotting experiments, pooled patient serum and polyclonal rabbit antibodies partly detected different rat antigens; monoclonal antibody specificity could not be determined. Immunoblot detection of mouse urinary antigens (MUA) by the polyclonal rabbit antibodies from all laboratories was similar. In both the RUA and the MUA assays, urinary antigen standards were detected with similar potency, except purified Rat n 1, which was an inefficient inhibitor in the RUA RAST inhibition. In the sandwich EIA RUA assays, a rat room-dust extract was detected with 700–800-fold less sensitivity than rat urine, whereas in the RAST RUA assay, dust inhibited equally with rat urine. Simulated decay did not decrease the potency of urinary antigen in any assay. Thus, assay setup and choice of detection antibodies strongly influence the nominal allergen levels. We recommend the use of standardized and characterized antibodies and standard extracts in sandwich EIAs to measure airborne rodent urinary allergens.
Abbreviations: NHLI: National Heart and Lung Institute, London, UK; WAU: Wageningen Agricultural University, Wageningen, The Netherlands; NIWL: National Institute for Working Life, Solna, Sweden; Ab: antibody; mAb: monoclonal antibody; EIA: enzyme immunoassay; RAST: radioallergosorbent test; RIA: radioimmunoassay; MUA: mouse urinary allergen; RUA: rat urinary allergen; BSA: bovine serum albumin; HSA: human serum albumin; PBS: phosphate-buffered saline; PTFE: polytetrafluoroethylene (Teflon); TBS: Tris-buffered saline.
Airborne allergens from sources such as cat, dog, and laboratory animals are collected on filters, eluted, and measured by a wide variety of methods. Patient serum ( 1), polyclonal antibodies ( 2), monospecific antibodies ( 3), or monoclonal antibodies (mAb) ( 4) have been used for allergen detection. Furthermore, the immunologic methods differ, including inhibition assays ( 5) or sandwich assays ( 6) utilizing radioactive ( 7), fluorescence ( 8), or enzymatic ( 9) visualization, and quantification systems. Thus, it is not surprising that reported allergen values vary greatly between studies on the same species. To our knowledge, there have been few efforts to compare or evaluate (5, 10) and none to standardize different methods. Recently, in a preliminary study, two methods for rat urinary aeroallergen measurement were compared ( 11). In 40 samples, exchanged between the UK and Sweden and analyzed in each country, the values obtained correlated (t2=0.72, P<0.0001), but differed by several orders of magnitude.
Thus, one goal of a European Union Concerted Action program, “Epidemiology of occupational allergic asthma and exposure to bioaerosols”, was to evaluate rodent aeroallergen measurement methods and the need for standardization. As described in a companion paper ( 12), a large variation in nominal allergen levels was found in triplicate samples, eluted and analyzed by the methods of the National Heart and Lung Institute (NHLI), the UK; Wageningen Agricultural University (WAU), The Netherlands; and the National Institute for Working Life (NIWL), Sweden, respectively. In the rat urinary allergen (RUA) assays, the nominal RAST inhibition (NHLI) levels were 2–3 orders of magnitude higher than in the polyclonal (WAU) and monoclonal (NIWL) sandwich enzyme immunoassay (EIA) methods, respectively. In the mouse urinary allergen (MUA) assays, in which all institutes used rabbit polyclonal antibodies, values were more similar, within an order of magnitude, despite one assay being a competitive inhibition RIA (NHLI) and the others sandwich EIAs (WAU and NIWL). In this paper, we explore some possible effects of the different assay setups (inhibition vs sandwich EIA), antibody specificities, and standard extracts, and of antigen decay, on the ability to detect antigen.
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- Material and methods
In this three-country study comparing different methods for measuring rodent aeroallergens, large differences in values were found in parallel air samples, especially between RAST inhibition and sandwich EIA measurements. Differences between EIA assay values could partly be attributed to extraction practices, and partly to type of immunoassay (companion paper ). We have now examined how assay setup, type and specificities of the antibodies, standard extract differences, and antigen aging may affect the ability to detect antigen.
When the same antibody and standard were utilized in an inhibition and a sandwich EIA setup, respectively, inhibition gave sevenfold higher aeroallergen sample values. This may have been due to differences in availability of antigen epitopes in solution compared to the bound state, which changes the three-dimensional structures of the molecules ( 21). The decreasing ratio of inhibition setup to EIA setup with increasing allergen concentration was also observed in the air-sample comparisons ( 12); however, the reason for the decrease is not known. The inhibition standard curves are considerably less steep than the sandwich EIA curves. A small difference in inhibition will thus give a large difference in nominal allergen level, and larger measurement variability. This may contribute to the r2 values for air samples being smaller between inhibition and sandwich methods than the r2 values between sandwich methods (companion paper ). Similarly, in a comparison between EIA methods for measuring swine aeroallergens, a sandwich setup was found to give better reproducibility than an inhibition design ( 10).
Immunoblotting experiments showed several differences in the specificities of the antibodies used, especially in the binding to rat room dust. While the specificity of the mAbs could not be shown here, they have previously been shown to bind selectively to the Rat n 1 isoallergens ( 16). In the MUA blots, all rabbit Ab bound to 15–18-kDa proteins present in urine and dust (probably Mus m 1 , to which most mouse-allergic patients react ). The WAU and NIWL Ab also bound lightly to 44–50-kDa proteins whose identity is not known. The ability of the Mus m 1 affinity-purified Ab (NIWL) to detect these allergens suggests that these may be aggregate forms of Mus m 1.
In agreement with a previous comparison ( 11), all urine extracts were detected similarly in the RUA and MUA assays. However, purified Rat n 1 (used as standard in the mAb assay) was inefficient as an inhibitor in the RAST-inhibition assay, indicating that a significant proportion of the patient IgE is specific for other rat urinary proteins, as confirmed in the Western blots. In the mAb RUA assay, differences in detection reflected the relative amount of Rat n 1.02 (α2u-globulin) in each extract. All RUA and MUA assays were specific; binding to other allergens would be of little, if any, practical significance.
The most important difference between RUA assays was in their detection of the rat room-dust extract (containing material from bedding, food, feces, hair, etc.). When added at the same total protein concentrations as the urine reference (as measured with the Pierce BCA method), dust was 700–800-fold less potent in the EIAs, whereas it was detected with equal potency by the pooled patient serum in the RAST-inhibition assay. If the dust extract approximates air samples, this could explain a major part of the observed differences ( 12). Perhaps IgE binding to rat dander or hair allergens plays a part; food and mouse room-dust extracts were not detected. In contrast, all rabbit polyclonal MUA assays detected mouse room dust with about 30–50-fold less sensitivity than mouse urine. The antibodies used derive from different types of immunization: human IgE produced after occupational inhalation of dust, and Ab from rabbits or mice serially immunized with mouse or rat urinary proteins, respectively. Although the latter antibodies capture the urinary allergens, the clinical allergenic potency of an air sample may be underestimated.Swanson et al. ( 5) similarly observed, when measuring airborne mite allergens using mAbs, rabbit polyclonals, or human sera as detection antibodies, that human sera gave about 20-fold higher values.
Using the artificial “aging” protocol, we could not find evidence that differences in detection of dust might be due to antigen degradation. However, it is possible that the protocol used was not representative of natural antigen breakdown.
To summarize, we have shown that several factors contribute to differences in nominal levels of allergen measured in air samples. The most important is source and type of antibodies, whose specificities contributed to up to 800-fold of the differences. Assay design contributed, with inhibition giving about sevenfold higher values. We found only a minor effect of using urine standards from different sources. In the companion paper ( 12), we concluded that the use of Tween and BSA enhanced extraction efficiency up to about 10-fold and improved stability during storage.
By definition, the use of patient serum for allergen detection has clinical relevance. However, monoclonal sandwich assays specific for major allergens have important advantages, both for the purpose of standardization, and because of high sensitivity, specificity, and reproducibility ( 12). The disadvantage is that they may not detect other present allergens. Standardization of polyclonal EIA sandwich assays may be achieved by the acquisition of large amounts of reagents and an agreed assay protocol.
However, it is important to appreciate that what is measured by immunoassays is a marker of allergen load, not the actual dose of airborne allergen inhaled and potentially reacting with the immune system.