Eleven patients with CFA (six male, five female), eight with sarcoidosis (five male, three female) and one with systemic lupus erythematosus (SLE; female) were recruited from the relevant clinics at the Sir Charles Gairdner Hospital, Perth, Western Australia. The diagnosis of CFA was based on the following: presence of diffuse mid-, late or pan-inspiratory crackles, with or without clubbing; the absence of clinical, radiological or laboratory evidence of any other ILD; no exposure to agents that are known to be able to induce ILD (ie dusts, such as asbestos; drugs, such as nitrofurantoin, amiodarone and bleomycin; and biological antigens, such as pigeon droppings and inhaled fungi); diffuse reticular shadowing on chest radiography without pleural or hilar disease; a high-resolution thoracic computed tomography scan pattern consistent with CFA; and lung function tests that revealed the presence of restriction with reduced gas transfer.
The index case had clubbing and crackles, the radiological pattern referred to above, negative antinuclear antibodies and negative rheumatoid factor, a transfer factor that was 56% of the predicted value and an arterial partial oxygen tension of 63 mmHg. All patients with sarcoidosis had a clinical pattern that was consistent with sarcoidosis plus histological evidence of noncaseating granulomata in the absence of any evidence for other granulomatous disease, such as tuberculosis or fungal disease. The patient with SLE had typical subacute cutaneous lupus with breathlessness and linear atelactases, but borderline interstitial disease. She exhibited high levels of circulating anti-SSA, but was negative for anti-DNA antibodies.
Blood was obtained by venipuncture from patients attending the respiratory clinic at the Queen Elizabeth II Medical Centre in Perth, Western Australia. Patients included 11 with CFA, eight with sarcoidosis, three with ILD and one with SLE. In addition, sera were obtained from four healthy laboratory volunteers aged between 29 and 58 years. Blood was allowed to clot at room temperature, and the clots were allowed to retract overnight at 4°C. Sera were clarified by centrifugation and stored in aliquots at –20°C. These procedures were all approved by the Human Rights Committee of the University of Western Australia, fulfilled National Health and Medical Research Council of Australia Guidelines on Human Experimentation, and informed consent was obtained from all patients before enrollment in the study.
Construction of λ expression library
cDNA was prepared from a tumour cell line, AB1, using Xho I tagged oligo dT as a primer. cDNA was blunted, capped with Eco RI adapters and ligated into the λ zap vector (Stratagene, La Jolla, CA, USA). The library, with a complexity in excess of 1 × 106, was amplified and the phage stored at 4°C at a concentration of 1 × 1010 pfu/ml. The production and characterization of this library has been described in detail elsewhere .
Screening of the library
The index serum was used at a dilution of 1/100. In order to remove any background reactivity to bacterial proteins, antibodies from the serum were absorbed onto an Escherichia coli lysate by admixture followed by centrifugation. The process was repeated three times. A total of 5 × 105 plaques were screened using the serum, and positives were identified using an alkaline phosphatase conjugated antihuman IgG (Promega, Madison, WI, USA) and the picoBLUE immunoscreening kit (Stratagene). The positive clones from the primary screen were picked and replated over several rounds until they were monoclonal. In vivo excision of the purified plaques as the pBluescript phagemid was carried out using the ExAssist helper phage, as described by the manufacturer (Stratagene). Inserts were sequenced by standard dideoxy chain termination.
Clonal phage preparations were titrated and applied to a bacterial lawn using a plate replicator to achieve a plaque density of approximately 100/cm2. Individual filter lifts containing plaques from each of the clones of interest were then tested with each of the sera and developed as indicated above. A reaction was scored on a scale from negative to +++ within each filter.