The results of this experiment are shown in Fig. (urine or cerebrospinal fluid [CSF]) and as a convenient screening method to assay MSH production by mutant strains. MATERIALS AND METHODS Reagents and materials. Immulon-4 HBX 96-well flat-bottom microtiter plates and plate-sealing tape were purchased from Dynex Technologies, Inc. (Chantilly, Va.). Centricon-100 spin filters were obtained from Amicon, Inc. (Beverly, Mass.). Deglycosylated avidin (ImmunoPure NeutrAvidin) was purchased from Pierce (Rockford, Ill.), and Tween 20 was purchased from Bio-Rad Laboratories (Hercules, Calif.). Goat anti-rabbit immunoglobulin G (IgG) (whole molecule) secondary antibody [F(ab)2 fragments conjugated to bovine intestinal alkaline phosphatase], soluble protein A (Sigma P 6031), avidin (ExtrAvidin) alkaline phosphate conjugate, bovine serum albumin portion V (Sigma A 4503), fish skin (Teleostean) gelatin, mc26 and mc2155 were kindly provided by J. Davies (University or college of British Columbia, Vancouver, British Columbia, Canada). Mutant strains I64 and 49 are chemical mutants of mc2155 shown by monobromobimane labelling and HPLC analysis (12, 14) to produce 0.05 and <0.004 mol of MSH per g (residual dry weight), respectively, of mycothiol. NJH 9141 was obtained from the University or college of California at San Diego (UCSD) Medical Center. was produced at 37C in Middlebrook 7H9 (broth or agar) supplemented with 0.05% (wt/vol) Tween 80 and 0.4% (wt/vol) glucose or on Middlebrook 7H10 agar supplemented with 0.05% (wt/vol) Tween 80 and 0.4% (wt/vol) glucose with or without OADC supplementation. was produced in Middlebrook 7H9 broth supplemented with OADC and 0.05% (wt/vol) Tween 80. Body fluid specimens. All cerebrospinal specimens were excess samples from routine clinical specimens obtained at the UCSD Medical Center. Urine samples POLB were obtained from a healthy donor. Antibody preparation. The primary antibody to MSH was prepared as explained previously (21). Briefly, purified MSH from was conjugated to keyhole limpet hemocyanin by treatment with maleimidobenzoyl-for antigen-antibody equilibria in answer. Affinity-purified rabbit polyclonal anti-MSH antibody prepared as explained above was diluted to 0.2 M in phosphate-buffered saline (PBS; pH 7.2), and Dimethyl trisulfide two additional 10-fold dilutions in PBS were made from this to give 0.02 and 0.002 M solutions. The MS-MPB standard was similarly diluted to 0.2, 0.02, and 0.002 M in PBS. Three microfuge tubes each received 400 l of anti-MSH answer and 400 l of MS-MPB answer of equivalent molarity to produce final concentrations of 10?7, 10?8, and 10?9 M, respectively. In parallel, control samples were made made up of MS-MPB and PBS instead of antibody answer. The tubes were vortexed well and incubated at room temperature to allow the antigen-antibody conversation to occur; at 10 min, 1 h, and 3 h, duplicate 100-l aliquots were removed from each tube, transferred to prechilled Centricon-100 (100-kDa molecular mass cutoff) spin filters, and centrifuged for 15 min at 1,000 cells in body fluids. was harvested at early- to mid-log-phase growth and diluted in new medium to give concentrations ranging from 3 103 to 3 104 CFU in a volume of 10 l. Human CSF (several pooled samples) or urine was filter sterilized through a 0.45-m-pore-size filter prior to experiments. Sterile-filtered CSF was divided into two portions, one of which (referred to as enriched CSF) received the addition of 1% (vol/vol) glycerol and 0.5% (wt/vol) glucose. To each microfuge tube was added 10 l of cell suspension and 990 l of sterile-filtered urine, CSF, or enriched CSF. The tubes were capped, vortexed, and centrifuged for 10 Dimethyl trisulfide min at 13,000 cells were produced to early logarithmic phase in Middlebrook 7H9 medium supplemented with 0.4% (wt/vol) glucose and 0.05% (vol/vol) Tween 80 and diluted in fresh medium to an initial concentration of 108 CFU/ml; the cells were then further diluted in series as required. To each well of an Immulon-4 microtiter plate was added a 100-l aliquot of diluted cell suspensions. A 10 mM answer of MPB in dimethyl sulfoxide was prepared shortly before use. This was diluted immediately before the reaction to 6 M in room heat CH3CN. Additions of the reagents to the microtiter plate were made by means of a multichannel pipetter. To each well was added 20 l of 0.1 M Na2HPO4 (pH unadjusted), followed by 120 l of 6 M MPB in CH3CN. The plate was covered and incubated in a water bath at 60C for 15 min. To block Dimethyl trisulfide unreacted MPB, 20 l of 60 M aqueous 2-mercaptoethanol was added to each well to give a final volume of 260 l per well; the plate was covered, incubated a further 5 min.