137 different parts of India (1979-1982). Zndiun 3oumul of Medical Research, 79, 47-78. Smith, J. T. (1%9). R factor gene expression in gram negative bacteria. Journal of General Microbiology, 55, W-120. Young, H. K., Jesudason, M. V., Kosbi, G. & Amyes, S. G. B. (1986). Trimetboprim resistance among urinary pathogens in South India. 3oumal of Antimicrobial Chemotherapy, 17, 615-621. Sundaram, S. P. & Murtby, K. V. (1984).Occurrence of transferable multi-drug resistance in Vibrio cholerae 01 in an endemic area. ZndianJownal of Medical Research, 79, 722-727. Received 20 June 1989; revised 25 July 1989; accepted for publication 14 September 1989 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPJCAL MEDICINE AND HYGIENE (1990) 84, 137-138 Caution when standardizing serum antibody competition assays Javed N. Agrewala, Sudhii Sinha and U. Sengup- ta Department of Intmutwlogy, CentralJALMA Insti- tute for Leprosy, Agra-282001, India Serum antibody competition (SAC) assayis highly specific and sensitive. Recently, this test has been used for serodiagnosis of disease such as leishmaniasis (JAFFE & MCMAHON-PRATT, 1987), tuberculosis (IVANYI et al., 1983), leprosy (SINHA et al., 1983), and schistosomiasis /MITCHELL et al., 1981). In the present study ‘* I-labelled rabbit anti-dapsone anti- body was used in an SAC assay on 140 leprosy patients who were being given dapsone (DDS) orally, 100 mg/d, for at least 3 months. Thirty leprosy patients who had been released from chemotherapy and had not taken dapsone during the preceeding 12 months or more, and 8 normal control subjects, were also included in the study. Not only the antibody but also the drug competed simultaneously in this dap- sone anti-dapsone binding assay. Sera from leprosy patients, normal controls and leprosy patients released from chemotherapy were subjected to globulin precipitation with saturated ammonium sulphate solution (HUDSON & HAY, 1980). Anti-dapsone antibody was measured by the SAC assay. Briefly, polyvinyl chloride (PVC) micro- titre plates (Dynatech, USA) were coated with 100 ~1 per well of DDS-casein (80 @ml). After washing and blocking with bovine serum albumen (BSA, l%), untreated sera, sera dialysed for 24 hours, or globulin fractions were added together with 50 pl of 1251- labelled anti-DDS antibody (2x lo5 cpm) and incu- bated for 2 h at 37°C. After washing, y-radiations were counted. Mean binding of 1251-anti-DDSanti- body to wells coated with casein alone was taken as zero; this count was subtracted from each of the test binding values, and relative values for each serum dilution were then calculated. Binding of 1251-anti- DDS antibody was taken as 100%. The results were expressed in terms of ID25 values, which represent the serum dilution causing 25% inhibition of ‘251-anti- Address for reprints and correspondence: U. Sengupta, Central JALMA Institute for Leprosy, Taj Ganj, Agra- 282001, India. DDS antibody binding to the antigen-coated wells. Presence of dapsone in globulin and serum was determined by direct radioimmunoassay. Brielly, 50 ~1of standard DDS-BSA solution (DDS concen- trations 0.1, O-2, 0.3, 0*4, O-5, l-0, l-5, 2.0, 2.5 and 3.0 &ml), 50 fl of serum or globulin eluted from the sera of normal human subjects, leprosy patients on dapsone therapy and patients released from chemotherapy, were coated on the PVC micro&e plates. These plates were incubated overnight at 4”C, washed, dried, blocked (SF+, 1%) and kept at 37°C for 2 h. The plates were aFam washed and dried by blotting; 50 ~1 of ‘* I-anti-DDS antibody 1.3- 1.2 - l.l- Fig. 1. Direct binding assay for demonstration of dapsone in standard dapsone (DDS) solution (0) and in serom samples of patients (-+); 3=serum samples from three patients; 5=serum samples from five patients. Serum samples from 8 normal conrrols, and globulin fractionsfrom 54 leprosy patients and 30 patients released from chemotherapy, were negativefor dapsone. (2X lo5 cpm) were added to each well and incubated at 37°C for 2 hours. The plates were washed, dried and the wells were cut out for y-radiation counting. Using different concentrations of DDS solution, dapsoneup to a minimum level of 0.3 &ml inhibited in the SAC assay. Globulin fractions from leprosy patients were found to be devoid of dapsone by direct