490 J Clin Pathol 1992;45:490-493 Comparison of and enzyme

linked immunosorbent assay in the detection of J Clin Pathol: first published as 10.1136/jcp.45.6.490 on 1 June 1992. Downloaded from abnormal in pigeon breeder's disease

C Simpson, P V Shirodaria, J P Evans, D I H Simpson, C F Stanford

Abstract antibodies against antigens in pigeon drop- Aims: To compare the sensitivity of two pings or pigeon serum. It has also been repor- methods for the detection of serum ted that the pigeon droppings are probably the antibodies to pigeon faecal antigens in principal source of the major antigens to which patients with pigeon breeder's disease. these patients develop antibodies.3 The cir- Methods: Serum samples stored at - 20°C culating antibodies in the sera of these patients from 50 patients with pigeon breeder's are usually detected by Ouchterlony's double disease, 50 control samples from patients diffusion method (immunodiffusion).4 Other with other respiratory diseases, and 50 methods such as immunoelectrophoresis,5 pas- healthy blood donors were examined for sive haemagglutination,5 indirect immuno- the precipitating antibodies and IgG fluorescence6 and immunoradiometric assay7 antibodies to antigens present in extract have also been used. Several recent reports of pigeon droppings by immunodiffusion have detailed that enzyme linked immunosor- and enzyme linked immunosorbent assay bent assay (ELISA) has been successfully (ELISA), respectively. applied to the detection ofantibodies to pigeon Results: Both antigen preparations of antigens in patients with pigeon breeder's pigeon dropping extract were equally disease."'0 One of the problems with an effective. A positive immunodiffusion immunodiffusion test used in the diagnostic reaction gave one or more precipitin lines laboratory is that precipitating antibodies to and these antibodies were detected only in pigeon antigens cannot always be detected in undiluted sera from 80% of the patients the sera ofpatients with a history ofexposure to with pigeon breeder's disease. In the birds and clinical symptoms suggestive of ELISA the sera were tested at a starting pigeon breeder's disease.5 We therefore com-

dilution of 1 in 100 because positive re- pared the sensitivity of ELISA and immuno- http://jcp.bmj.com/ actions were observed with sera from diffusion for the detection of IgG antibodies to healthy blood donors at lower dilutions. pigeon antigens in the sera of patients with All sera which gave optical density read- pigeon breeder's disease and control groups. ings above 3 SD of the control value were considered to have IgG antibodies. These antibodies were detected in sera from all Methods the patients with pigeon breeder's dis- Serum samples included in this study were on September 28, 2021 by guest. Protected copyright. ease. The titres were much from: (i) sera sent to our Regional Allergic higher in those patients who had Alveolitis Laboratory for a precipitin antibody precipitating antibodies (range 800- test to pigeon antigens (All these patients had a 51 200) than those without (range 100- history of exposure to pigeons and clinical 800). The antibodies were not detected in symptoms such as fever, cough, and breathless- Department of the sera of patients with respiratory dis- ness suggestive of pigeon breeder's disease.); Microbiology and sera conditions Immunobiology, The eases or healthy blood donors by either (ii) from other respiratory Queen's University of method. which included asthma, bronchopulmonary Belfast Conclusions: Antibodies to pigeon drop- allergic aspergillosis, asbestosis, bronchiectasis C Simpson and mycetoma; and (iii) control sera from P V Shirodaria ping antigens were detected by immuno- D I H Simpson diffusion and ELISA in sera from patients healthy blood donors. Regional Mycology with pigeon breeder's disease but not in Fifty subjects were included in each group, Laboratory, Royal control sera. ELISA was a more sensitive six women and 44 men, with a mean age as Victoria Hospital, method for antibodies and follows: (i) 45-7 years (range 14-69 years); (ii) Belfast detecting J P Evans therefore seems to have considerable 45-5 years (range 15-69 years); and (iii) 44 2 D I H Simpson potential as a routine technique in the years (range 16-70 years). The subjects in the Royal Victoria serological diagnosis of pigeon breeder's three groups were matched for sex and age Hospital, Belfast disease. within two years. The sera were collected over C F Stanford two years and stored in small aliquots at - 200C Correspondence to: Caroline until tested under code. Simpson, Department of Microbiology and Pigeon breeder's disease, a hypersensitivity Two preparations of pigeon dropping Immunobiology, The extracts were initially used as antigens for Queen's University of pneumonititis, is caused by the inhalation of Belfast, Grosvenor Road, pigeon derived antigens.' The clinical picture comparison in gel diffusion test and ELISA. A Belfast BT12 6BN, Northern comprises chills, fever, cough and dyspnoea commercial preparation of pigeon faecal Ireland extract was purchased from Mercia Diagnos- Accepted for publication several hours after exposure to pigeons.' The 1 November 1991 disease is associated with circulating IgG tics Ltd, Guildford, Surrey, England. The Comparison of immunodiffusion and ELISA for detecting antibodies to pigeon breeder's disease 491

second extract was prepared in our laboratory 3-0% H202 and 0-4 mg/ml orthophenylene- by disrupting fresh pigeon faeces (obtained diamine dihydrochloride) was added to each from a pet shop) in distilled water with glass well. The reaction was stopped after 20 min-

beads. The suspension was centrifuged at 2000 utes by adding 100 Ml IMH2SO4 to each well. J Clin Pathol: first published as 10.1136/jcp.45.6.490 on 1 June 1992. Downloaded from x g for 10 minutes and the supernatant was The optical density (OD) ofthe final colour was further clarified by filtration through a 0-45 gm read at 490 nm and 410 nm in a dual wavelength filter (Millpore Corporation, Massachusetts, Dynatech MR600 microplate reader. USA). The supernatant was stored at - 70°C in small aliquots and a single extract preparation Absorption ofsera was used in the gel diffusion test and ELISA The specificity ofthe ELISA was confirmed by throughout the study. The concentra- absorption of positive pigeon breeder's disease tion of commercial pigeon droppings extract sera (diluted 1 in 10) with either a washed pellet was 11 0 mg/ml and fresh ex-tract was 10-3 mg/ of pigeon droppings, or a pellet of hen drop- ml. pings, or a pellet of human faeces (30% v/v). The absorption was carried out for one hour at SEROLOGICAL METHODS 37°C, followed overnight at 4°C. The sera were Precipitins clarified by centrifugation at 10000 x g for 20 Precipitating antibodies against pigeon drop- minutes at 4°C before testing. ping antigens were detected and semiquant- itatively evaluated by immunodiffusion. The gels were prepared in 15% ion agar (Oxoid) in McIlvane's buffer (pH 7-0) contain- Results ing 0-2% sodium azide in 5 cm plastic Petri IMMUNODIFFUSION dishes (Cellcult). A cutter giving a central well Similar results were observed with the im- (12-5 mm in diameter) for serum and six munodiffusion test when a commercial peripheral wells (4 mm in diameter) for preparation or our laboratory preparation of antigens at a distance of 6 mm from the central pigeon dropping antigens were used. Sera with well was used. The pigeon dropping extract precipitating antibodies gave between one and used had a final concentration of 39 jig/ml. The four precipitin lines after incubation of the gel- sera were tested undiluted and also at dilutions diffusion dishes for four days at room tem- of 1 in 5, 1 in 10, and 1 in 100. The diffusion perature. The precipitin lines were only time was two to seven days at room temperature observed with undiluted sera and were not and the gels were observed daily for the observed at serum dilutions of 1 in 5, 1 in 10, appearance of precipitin lines. Weak reactions and 1 in 100 even after the gels had been stained were further analysed after staining with with Azocarmine B. Azocarmine B (Edward Gurr Ltd, London). STANDARDISATION AND SPECIFICITY OF ELISA ELISA To determine the optimal dilution of human http://jcp.bmj.com/ The solid phase indirect ELISA used for the sera to be used in the ELISA, 18 sera positive detection of IgG antibodies to pigeon dropping for precipitating antibodies from patients with antigens was based on the method described pigeon breeder's disease and 18 age and sex by Voller et al." ELISA was performed in matched sera without precipitating antibodies flat-bottom wells of a microtitre from healthy blood donors were tested at polystyrene dilutions of 1 in 10, 1 in 50, and 1 in 100. Figure

plate (Sterilin Ltd; Middlesex, England). The on September 28, 2021 by guest. Protected copyright. optimal concentration of pigeon dropping 1 shows that all pigeon breeder's disease sera at antigens used in the ELISA was determined by 1 in 10, 1 in 50, and 1 in 100 dilutions gave high chessboard titration using precipitin antibody OD readings. With control sera, strong and positive sera from patients with pigeon moderate reactivity was observed at dilutions breeder's disease and negative control sera of 1 in 10 and 1 in 50, respectively. The OD from blood donors. Both antigen preparations values at the 1 in 100 dilution were much lower; (commercial and our own) were used at an therefore, a dilution of 1 in 100, which yielded a optimal concentration of 1 0 pg/ml in the ELISA. For coating wells of the microtitre A B C > 20 rn-mm plate, the antigen preparation was diluted in E 1 8 0 bicarbonate-carbonate buffer, pH 9-6, and 0 * S 100 Ml ofthe dilution was added to each well for 0) 16 two hours at 37°C. After five washings with CD 14 *. phosphate buffered saline containing 0-05% 12 J) C)1 a) Tween-20 (PBS-T), 100 jil of serial two-fold V 1.0 dilutions of patient serum in PBS-T-BSA '0 08 ._ (bovine serum albumin) were added to appro- 0 06 G, O 00 priate wells and incubated for two hours at 0-4 o~ So 0 0 n 0 o% oo 37°C. After five washes with PBS-T 100 pl of -i 02 LU goat antihuman IgG coupled to horseradish I ______- - peroxide and diluted 1 in 1000 (ICN Immuno- Figure I Optical density readingsfor serum IgG biologicals, Lisle) was added to each well and antibodies to pigeon dropping antigens by ELISA: (A) incubated for one hour at 37°C. This was pigeon breeder's disease sera ( 0 ) and control sera ( 0 ) followed by five further washes with PBS-T diluted I in 10; (B) pigeon breeder's disease sera (0) and control sera (0) diluted I in 50; (C) pigeon and 100 pl of freshly prepared substrate solu- breeder's disease sera ( 0 ) and control sera ( 0 ) diluted tion (0 1 M phosphate-citrate buffer, pH 5-0, 1 in 100. 492 Simpson, Shirodaria, Evans, Simpson, Stanford

Incidence ofantibodies to pigeon dropping antigens shown by immunodiffusion and were found in the sera of those patients who ELISA in patient and control sera also had precipitating antibodies. The range of titres in these was between No of sera positive No of sera positive antibody patients Study group by ELISA by immunodiffusion 800 and more than 51200; sera of pigeon J Clin Pathol: first published as 10.1136/jcp.45.6.490 on 1 June 1992. Downloaded from breeder's disease patients who lacked Pigeon breeder's disease 50/50 40/50 Other respiratory diseases 0/50 0/50 precipitating antibodies had lower antibody Blood donor controls 0/50 0/50 titres and the range was between 100 and 800 (fig 2). much lower reactivityy with control sera, was used in all subsequenit tests. The standardisa- Discussion tion of the ELISA vvas further achieved by The mechanisms by which exposure to pigeons routinely including positive serum with produces pigeon breeder's disease is not precipitating antibod.ies from patients with known. But it is generally accepted that pigeon breeder's disease and negative serum precipitating antibodies and activated T lym- without precipitating antibodies from blood phocytes to pigeon material are involved in the donors in each plate. Thus OD readings of pathogenesis of pigeon breeder's disease.'2"" colour change for po;sitive serum at 1 in 100 The presence of precipitating antibodies alone dilution was always mlore than 2-0; at 1 in 800 to pigeon antigens is not diagnostic of pigeon dilution it was betwee-n 0 9-1 1. The mean of breeder's disease as these antibodies are also the OD readings us;ed as a reference was found in the sera of asymptomatic pigeon calculated from the 00D values obtained from breeders.8 But the presence of antibodies 50 normal sera at 1 in 100 dilution. This together with a clinical history is considered to reference value of 0-0832 (3 SD) was considered be one of the major diagnostic criteria for the evidence ofthe present ce ofantibodies to pigeon disease.9'1 dropping antigens. Differences in IgG In this study we have shown that IgG antibody titres to pilgeon dropping antigens antibodies to pigeon antigens are present in the were not observed wihen 24 pigeon breeder's sera of patients with pigeon breeder's disease disease sera with preciipitating antibodies were using the immunodiffusion test and ELISA. As titrated by ELISA us;ing either a commercial it was difficult to obtain sera from asymp- preparation or our laiboratory preparation of tomatic members of a local pigeon racing club, pigeon dropping extracct as antigen. A standard- only sera from symptomatic patients with ised preparation of poigeon dropping antigen pigeon breeder's disease were analysed. The prepared in our labor,atory was therefore used pigeon dropping extract was used as a source of in all subsequent ELI SAs. antigens because it is generally accepted that The absorption c)f 30 positive pigeon pigeon dropping extract contains a full "com-

breeder's disease sera with a pellet of pigeon plement" of major antigens against which http://jcp.bmj.com/ droppings completelyy removed the positive antibodies are formed in patients with pigeon reaction of these sera iin the ELISA. However, breeder's disease.3 The commercial prepara- positive reaction to piigeon dropping antigens tion and our laboratory preparation of pigeon was not removed after absorption of these sera dropping antigens were adequate for use as with a pellet of hen droppings or human faeces antigens in the immunodiffusion test and (results not shown). ELISA.

The precipitating aritibodies to pigeon drop- Precipitating antibodies to pigeon dropping on September 28, 2021 by guest. Protected copyright. ping antigens were d(etected by the immuno- antigens were detected by immunodiffusion diffusion test in 40 ssera from patients with test only in the undiluted sera of pigeon pigeon breeder's diseaise. Antibodies were not breeder's disease patients. Moreover, the detected in 10 sera fr(om patients with pigeon antibodies were found in 80% of the patients' breeder's disease, sera from patients with other sera. These antibodies were not detected in sera respiratory diseases, a]nd in those from healthy of patients with other respiratory diseases or blood donors (table) Moreover, antibodies control blood donors. In the positive immuno- discovered by double gel-diffusion test were diffusion test of pigeon breeder's disease sera only detected when se:ra were used undiluted. one or more precipitin lines were detected. The IgG antibodies to pigeon dropping These results are similar to those reported by antigens were detectedI by ELISA in all 50 sera Elgefors et al5 and Andersen et al,8 who found from patients with p)igeon breeder's disease precipitating antibodies in 60% of pigeon (table). Moreover, the highest antibody titres breeder's sera and 100% of pigeon breeder's disease sera, respectively. The higher incidence Figure 2 Reciprocal >51200 observed by Andersen et al may have been due titres of serum IgG 51200 so to the analysis of a small number of sera from antibodies to pigeon 25600 6664MMMOD dropping antigens in Lo 12800 pigeon breeder's disease patients.8 patients with pigeon * 6400 0400 The positive reactions with pigeon breeder's breeder's disease with 3200 .e disease antigens were observed in the ELISA precipitating antibodies o 1600 es" ( * ), patients without O. 800 000 when control sera were tested at lower dilu- precipitating antibodies 400 0 tions, although OD readings obtained were ( 0 ), patients with other cc 200 00000 much lower than those observed with sera from respiratory diseases ( A ) 100 00 and control blood donors < 100 pigeon breeder's disease patients at similar dilutions. At dilutions in (A ). Pigeon breeder's Other Control higher (1 100) pigeon disease respiratory blood donors breeder's disease sera gave much higher OD diseases readings than control sera. Whether positive Comparison of immunodiffusion and ELISA for detecting antibodies to pigeon breeder's disease 493

reactions observed with control sera at lower We thank Mr J J McAlister, principal MLSO ofthe Department ofMicrobiology and Immunobiology, and Dr M McClelland of dilutions are due to specific antibodies to The Northern Ireland Blood Transfusion pigeon dropping antigens or to crossreacting Service.

antibodies is not clear from the present study J Clin Pathol: first published as 10.1136/jcp.45.6.490 on 1 June 1992. Downloaded from and needs to be further investigated. Antibodies to pigeon dropping antigens were detected in all the sera from pigeon breeder's disease patients and all sera gave OD readings 1 Reed CE, Susman A, Barbee RA. Pigeon breeder's lung. above 3 SD of the control value. These results JAMA 1965;193:261-5. confirm that ELISA is more sensitive than 2 Fink JN, Sosman AJ, Barboriak JJ, Schlueter DP, Holmes RA. Pigeon breeder's disease. Ann Int Med 1968;68: immunodiffusion in detecting antibodies to 1205-19. pigeon dropping antigens, and as such is of 3 Edwards JH, Barboriak JJ, Fink JN. Antigens in pigeon breeder's disease. 1970;19:729-34. great potential as a routine laboratory tech- 4 Longbottom JL, Pepys J. Pulmonary aspergillosis: Diag- nique for the serological diagnosis of pigeon nostic and immunological significance of antigens and C substance in Aspergillus fumigatus. J Pathol Bacteriol breeder's disease. It was also interesting to note 1964;88:141. that the highest antibody titres were found in 5 Elgefors B, Belin L, Hanson LA. Pigeon breeder's lung. Clinical and immunological observations. Scand J Respir those patients who also had precipitating Dis 1971;52:167-76. antibodies. Similar observations have been 6 Eade OE, Hodges JR, Berrill WT, Lang C, Lloyd RS, Wright R. Immunofluorescent antibodies in patients with reported by Andersen et al.8 The reasons for bird-fancier's lung. Clin Exp Immunol 1978;32:259-62. these differences in antibody responses in 7 Patterson R, Wang JLF, Fink JN, Calvanico NJ, Roberts M. IgA and IgG antibody activities of serum and bron- patients with pigeon breeder's disease are choalveolar fluid from symptomatic and asymptomatic unclear. Factors such as variation in the quan- pigeon breeders. Am Rev Respir Dis 1979;120:1113-8. 8 Andersen P, Christensen KM, Jensen BE, et al. Antibodies tity of antigen during exposure, length of time to pigeon antigens in pigeon breeders. Eur J Respir Dis of exposure, or influence of host factors may 1982;63:1 13-21. 9 Bourke S, Anderson K, Lynch P, et al. Chronic simple have a role. Studies on the genetic predisposi- bronchitis in Pigeon fanciers. Chest 1989;95:598-601. tion to produce increased concentrations of 10 Sandoval J, Banales JL, Cortes JJ, Mendoza F, Selman M, Reyes PA. Detection of antibodies against avian antigens precipitating antibodies in pigeon breeder's in bronchoalveolar lavage from patients with Pigeon disease patients have proved inconclusive.8 14 breeder's disease: usefulness of enzyme-linked immuno- sorbent assay and enzyme immunotransfer blotting. J Clin Further studies using techniques such as Lab Analyt 1990;4:81-5. immunoblotting to ascertain whether antibody 11 Voller A, Bidwell DE, Barlett A. Enzyme immunoassays in diagnostic medicine. Bull WHO 1976;53:55-65. responses to various polypeptides of pigeon 12 Boyd G, McSharry CP, Banham SW, Lynch PP. A current in these would review of pigeon fancier's lung. Clin Allergy 1982;12 dropping antigens vary patients (suppl):53-9. be rewarding. Inclusion of asymptomatic 13 Costabel V, Bross KJ, Marxen J, Matthys H. T- in such a would also to shed lymphocytosis in bronchoalveolar lavage fluid of hyper- patients study help sensitivity pneumonitis. Chest 1984;85:514-8. light on the antibody response in patients with 14 Rittner C, Sennekamp J, Vogel F. HLA-B8 in pigeon pigeon breeder's disease. fancier's lung. Lancet 1975;ii:1303. http://jcp.bmj.com/ on September 28, 2021 by guest. Protected copyright.