Creatine kinase kit formulations compared for suitability in development of creatine kinase isoenzymes on cellulose acetate.

To the Editor: The last step in electrophoretic procedures for creatine kinase (CK; EC 2.7.3.2) isoenzyme separation on cellulose acetate is to incubate the plate with a reagent that will develop either fluorescence or color for each isoenzyme. In our laboratory we routinely use fluorescence to detect the isoenzymes. The reagent used must be highly sensitive, so that low proportions of the isoenzyme of heart origin can be detected. The fluorescence must be stable because results may be reviewed as much as three days later. Finally, the reagent itself must be stable. We examined five commercial creatine kinase reagent formulations: CK-F16001 Lot 8811 (Sclavo Diagnostics); Fast Chem CK-NHC Lot 65302101 (BMC); Spin Chem Lot 62491 (Smith Kline Instruments); CPK SVR Lot 853056 (Calbiochem); N-16 Lot 78J053 (Worthington). These reagents were used on cellulose acetate plates (Helena Laboratories; Titan III iso Plates) on which the following samples were electrophoresed: Dade CK isoenzyme control, a preparation derived from fish heart prepared in isotonic saline solution, serum from a patient with abovenormal skeletal muscle CK, and a hemolyzed patient sample with heart (MB), brain (BB), and muscle (MM) isoenzyme bands. All procedures were run simultaneously and exactly the same except for the developing reagents. All reagents were diluted as directed by the manufacturers. Densitometric readings of the plates showed that the Sciavo reagent is the most sensitive toward all three isoenzyme bands. For example, the peak areas measured for the MM band of the hemolyzed patient sample were: 86 units for the Sciavo reagent, 35 for Spin Chem, 33 for Calbiochem, 24 for BMC, and 12 for Worthington. With heart (MB) isoenzyme the Sclavo reagent produced from two to four times the peak areas of fluorescence of the next most sensitive reagent. During the following eight days I found the fluorescence on the plates to be the most stable for the Sciavo and BMC reagents if the plates were stored in the dark. Repeat densitometric readings of all the plates gave results after eight days that were proportionately equal to those on the first day. However, the Worthington reagent plate showed extensive fading, and I made no readings after the second day for that reagent. As for reagent stability, the reconstituted Calbiochem and BMC reagents could be used for two to three days, but the Sciavo reagent consistently produced strong fluorescent patterns with results equal to those of freshly reconstituted reagent for as long as eight days. The reasons for the greater sensitivity and fluorescent pattern stability of the Sciavo reagent are not apparent from information on the ingredients as listed on the package insert by the manufacturer. However, repeated use of the reagent during the last seven months has confirmed the above findings.