New dip-and-read test for determining leukocytes in urine.

A new reagent strip for the determination of leukocytes in urine (LEUKOSTIX; Ames) is described. The test is based on the esterase activity in leukocytes as a marker. Upon contact between the reagent matrix and a urine containing leukocytes, an amino acid ester is hydrolyzed by the esterase to its corresponding alcohol. The free alcohol then couples with a diazonium salt to produce a purple azo dye. The relative concentration of leukocytes in the urine is obtained by visually comparing the strip reaction with a color chart. Performance of the strip was evaluated in a clinical study involving eight different sites and 867 urine specimens. The comparison method was sediment microscopy; specimens containing five cells or more per high-power field were considered to be positive. Sensitivity was 76.3%, specificity 80.8%. Performance was comparable with that of the CHEMSTRIP LN (Boehringer-Mannheim Diagnostics, Inc.) leukocyte test, which we evaluated concurrently.

A control system for monitoring the performance of leukocyte strip tests.

The control system described here is used to monitor the performance of urinary reagent-strip tests for leukocytes. The presence of leukocyte esterase in the urine is used as a marker for leukocytes in urine. The control system is based on sonicated leukocytes, isolated from whole blood. The esterase activity of this sonicate is determined by spectrophotometry with the N-tosyl-L-alanine ester of 5-phenyl-3-hydroxypyrrole as substrate. The assay result is used to determine the amount of sonicate needed to prepare buffered esterase-containing solutions. Such control solutions mimic leukocytic urines and are stable for 6 h at room temperature. The variability of the control system was tested by preparing it five times in a day on five separate days. The overall CV for Leukostix Reagent Strips (Ames Division, Miles Laboratories, Inc.) when tested with these solutions and analyzed with a reflectance spectrophotometer was 8%; for visual readings it was 10%. The overall CV for Chemstrip LN Reagent Strips (Biodynamics, Indianapolis, IN) was 10%.

Adaptation of Prostatic-Group-Label Homogeneous Immunoassay to reagent-strip format.

The Prostatic-Group-Label Immunoassay (PGLIA) technique has been incorporated into a reagent-strip format. We report use of flavin N6-(N'-2,4-dinitrophenyl-6-aminohexyl)adenine dinucleotide (DNP-FAD) as the prosthetic group derivative and 6-N-(2,4-dinitrophenyl)aminohexanoic acid (DNP-caproate) as the competing ligand. DNP-FAD not bound by antibody combines with glucose oxidase apoenzyme, which then reacts with glucose and oxygen, and gives color through a peroxidase-linked system. The rate of color generation is thus a function of the DNP-caproate concentration. PGLIA reagent strips are prepared by sequential impregnations of filter paper with an acetone solution of indicator (3,3',5,5'-tetramethylbenzidine); an aqueous solution containing glucose oxidase apoenzyme, the rest of the color generation system, stabilizers, and antibody to DNP; and a solution of DNP-FAD in n-propanol. This preparation permits effective antibody binding, and prevents premature interaction of immunoassay components. A quantitative color response to concentrations of DNP-caproate in the range of 1 to 8 mumol/L was demonstrated with these reagent strips. Prototype PGLIA reagent strips for theophylline and phenytoin have been successfully developed by substituting the appropriate FAD derivative and antibody for the corresponding reagents in the DNP model system.