Rapid and sensitive method for quantitation of Legionella pneumophila serogroup 1 antigen from human urine

J A Mangiafico, K W Hedlund, A R Knott
1981 Journal of Clinical Microbiology  
A reversed passive hemagglutination test was developed to assay relative concentrations of soluble antigen of Legionnaires disease (Legionella pneumophila serogroup 1) in human urine samples. The test is highly sensitive, being able to detect as little as 0.0002 ,ug of total antigen. Preliminary results with this test on serial urine and serum samples from a patient with legionellosis show that measurable amounts of antigen are present in urine during the course of the illness. However, no
more » ... s. However, no antigen could be detected in the serum of the patient. The reversed passive hemagglutination (RPHA) test has been successfully used in the past for detection or assay or both of tetanus toxin (2) and staphylococcal enterotoxin B in culture fitrates and in food samples (5) . The test is rapid, reliable, and highly sensitive and does not require extensive use of reagents. With the reported success of detecting Legionnaires disease (LD) antigen in sputum and urine samples (1, 6), we believed that an RPHA test might be an easy and rapid method for accomplishing this aim. We describe in this report the development of an RPHA test for the detection and quantitation of soluble Legionella antigen. The report also includes results obtained (with this method) from serial urine and serum samples obtained from a patient with LD. MATERIALS AND METHODS Erythrocyte preparation. Sheep erythrocytes (SRBC) were collected in an equal volume of Alsever solution and stored at 4°C for at least 3 days before use. SRBC were washed three times with 0.9% NaCi and suspended to 2.5% in 0.15 M phosphate-buffered saline (PBS), pH 7.2. The cells were tanned by mixing equal volumes of 2.5% SRBC and 1:20,000 tannic acid in PBS (pH 7.2) and incubating the mixture in a 37°C water bath for 15 min. The tanned cells were then washed three times with PBS (pH 7.2) and resuspended to 2.5% in PBS (pH 6.4). Tanned ceils were sensitized by mixing with an equal volume of optimally diluted goat anti-Legionella globulin; the mixture was incubated for 15 min at 37°C. Sensitized cells were washed twice with PBS (pH 7.2) with 1% normal rabbit serum (PBS-NRS) and resuspended to 0.7% in PBS-NRS for use in the microtiter test system. Soluble antigen preparation. Cultures of the Seattle 1 strain of Legionella pneumophila on modified Mueller-Hinton medium (3) were originally obtained from J. E. McDade and C. C. Shepard (Centers for Disease Control, Atlanta, Ga.). Soluble antigen was prepared from organisms grown on Mueller-Hinton medium as previously described (4). The purified final product was lyophilized and stored at -20°C. Globulin preparation. Anti-Legionella globulin was prepared from goats hyperimmunized with the Seattle 1 strain of L. pneumophila. Goat serum was saturated with (NH4)2SO4 to 50%. The precipitate was washed several times with saturated (NH4)2SO4, dialyzed against 0.9% NaCl, and stored at -20°C. This globulin preparation was found to contain approximately 820 gg of antibody nitrogen per ml by the Kjeldahl test. Normal goat immunoglobulin G was obtained from Cappel Laboratories, Inc., Cochranville, Pa. In other studies not described here, rabbit anti-Legionella globulin gave less satisfactory results than the goat anti-Legionella globulin. The concentration of globulin required for optimal sensitization of tanned SRBC was determined by titrating known quantities of standard soluble antigen, using tanned SRBC with various concentrations of globulin. Based on these box titrations, goat anti-Legionella globulin containing ap^proximately 80 gg of antibody nitrogen per ml was used in the sensitization procedure. Test samples were diluted 1:8 by combining 0.1 ml of sample with 0.7 ml of PBS-NRS diluent. The mixture was heat inactivated for 30 min at 60°C, absorbed with 0.1 ml of packed SRBC for 30 min, and then centrifuged. The test was performed in Titertek Uplates (Linbro Scientific, Hamden, Conn.). All but the first column of wells received 0.05 ml of PBS-NRS diluent. To the first column of wells was added 0.1 ml of the 1:8 dilution of test sample, and serial twofold dilutions were then prepared with 0.05-ml microtiter loops. Finally, each well received 0.025 ml of 0.7% tanned, sensitized SRBC. Plates were incubated at room temperature for 2 to 3 h and read for agglutination patterns. Included in each test run were several standard antigen titration controls. Serial twofold dilutions of standard antigen at various known starting concentrations were prepared as described above. Antigen concentrations in test samples were calculated by multiplying the reciprocal of the greatest dilution that re-843 on May 8, 2020 by guest
doi:10.1128/jcm.13.5.843-845.1981 fatcat:gb6dsdzlhvc67cnt3sel2i4pj4