Method and apparatus for quantitating enzyme activity

Claim 2

In a system as claimed in claim 2, said discs severally having selected mutually incompatible in storage reagents required for a given test impregnated on separate said discs, said reagents contained in said discs being freeze-dried for separation and storage prior to test use.

Claim 3

In a system as claimed in claim 3, said upper glass fiber disc containing Cysteine hydrochloride, said lower glass fiber disc containing a composite of reagents required for a Creatine phosphokinase assay excluding N-methylphenazonium methosulfate and enzymes, said membrane filter disc containing N-methylphenazonium methosulfate, and said cellulose acetate disc containing Creatine phosphokinase, Hexokinase and Glucose-6-phosphate dehydrogenase.

Claim 4

In a system as claimed in claim 4, said composite of reagents comprising in concentration (mg./ml.): Reagents Concentration (mg./ml.) Cystein hydrochloride 1.0 Adenosine diphosphate 1.0 Nicotinamide adenine dinucleotide phosphate 1.0 Glucose 3.0 Adenosine-5''-monophosphate 4.0 Nitro blue tetrazoleum 1.0 Creatine phosphate 5.0 Magnesium acetate 2.0 N-Methylphenazonium methosulfate 0.1 Hexokinase 0.1 IU Glucose-6-phosphate dehydrogenase 5.0 IU Creatine phosphokinase 5.0 IU; and said reagents excluding Cysteine hydrochloride, being dissolved in glycylglycine buffer, adjusted to pH 6.8.

Claim 5

In a system as claimed in claim 5, a blood test specimen placed on said upper glass fiber disc in a reagent containing array taking up cysteine hydrochloride, reagents from said lower glass fiber disc, and N-methylphenazonium methosulfate from said membrane filter disc, delivering both filtered plasma and reagents to the enzymes on said cellulose fiber disc where the reaction takes place.

Claim 6

In a system as claimed in claim 5, each said area including a said stacked array, each said area comprising a spot, three said spots containing CPK standards representing normal, elevated, and high enzyme levels, respectively, a fourth said spot being a blank containing all reagents except creatine phosphate and the fifth said spot being adapted for assay of CPK and containing reagents for a complete reaction sequence.

Claim 7

In a method for quantitating enzyme activity in a system as claimed in claim 7, applying a drop of water to the upper sides of said three standards containing spots, applying a drop of whole blood to the upper sides of said fourth and fifth spots, inverting the slides mounting said stacked arrays constituting said spots, observing development of color and comparing intensity of color developed in said fifth spot with that in said three standards containing spots for a direct reading of CPK activity.

Claim 8

An enzyme CPK assay test unit for visual determination of enzyme content of whole blood comprising: A. a support; B. means constituting a plurality of test spots mounted on said support: i. at least first ones of said spots containing reagents for assay of CPK activity in blood serum including standards representing normal, elevated, and high enzyme levels; ii. an additional said spot being a blank containing all reagents in the first said spots except creatine phosphate; and iii. a further said spot containing reagents activatable by whole blood for a complete reaction sequence; iv. said reagents being mutually incompatible in storage and separated by impregnation on a plurality of discrete separate laminae which collectively constitute each said spot; v. said laminae in each said spot being in a rigidly confined stacked array and some said laminae constituting prefilters to remove amorphous matter including white cells from blood to prevent such matter from subsequently clogging pores of others of said laminae in a said stacked array.

Claim 9

A test unit as claimed in claim 9, said reagents being severally applied to discs constituting said laminae forming said spots to separate reagents incompatible in storage and being in a freeze-dried storage condition, the reagents in the first said spots being activated by water, activation of reagents in all said spots presenting an indicating color therein whereby enzyme content in whole blood is visually determinable by color comparison with the colors of the standard indicators.