RAPID METHODS FOR ASSAY OF ENZYME SUBSTRATES AND METABOLITES

• US 3,791,933 A
• Filed: 02/25/1971
• Issued: 02/12/1974
• Est. Priority Date: 02/25/1971
• Status: Expired due to Term

• Current Assignee: Cambridge BioScience Corp. (Agenus Inc.)
• Original Assignee: Donald J. Sibbett, Rudolph H. Moyer
• Inventors: Moyer, Rudolph H., Sibbett, Donald J.
• Primary Examiner(s): Tanenholtz, Alvin E.
• Assistant Examiner(s): Hensley, Max D.
• Application Number:
  US05/118,903
• Time in Patent Office: 1,083 Days
• Field of Search: 195/13.5R,127 23/253TP
• US Class Current: 435/287.8
• CPC Class Codes: B01L 2300/028 Graduation
• CPC Class Codes: B01L 2300/0681 Filter
• CPC Class Codes: B01L 2300/0816 Cards, e.g. flat sample carriers usually with flow in two horizontal directions
• CPC Class Codes: B01L 2400/0406 capillary forces
• CPC Class Codes: B01L 3/5023 with a sample being transported to, and subsequently stored in an absorbent for analysis
• CPC Class Codes: B01L 3/5085 for multiple samples, e.g. microtitration plates
• CPC Class Codes: C12Q 1/32 involving dehydrogenase
• CPC Class Codes: C12Q 1/54 involving glucose or galactose
• CPC Class Codes: C12Q 1/60 involving cholesterol
• CPC Class Codes: C12Q 1/62 involving uric acid
• CPC Class Codes: Y10S 435/805 Test papers
• CPC Class Codes: Y10S 435/81 Packaged device or kit

Abstract

Method and apparatus for quantitating the amounts of enzyme substrates and metabolites present in biological fluids using spot test techniques. The tests utilize both fixed, preselected color standards and substrate and metabolite standards freezedried on transparent membranes or in porous pads for comparison. Other reagents including enzymes, dyes, cofactors are also freeze-dried in absorbent pads. The assembly may utilize any or all of the following in various combinations: porous discs of materials such as glass fibre filters to store reagents, to serve as liquid reaction volume measurement, and to assist in removing cells from blood samples; microporous membranes to act as barriers to blood cells; a protein enrichment membrane or a dialysis membrane to restrict passage of large molecules to the indicator zone; and a transparent window which may be a second microporous membrane or impermeable plastic on which indicator dyes are dried. In assembled format, the test plates, slides or discs fulfill all requirements for conducting the selected test: no added instrumentation, controls or other measurements are required. These test devices may assist in the diagnosis of a number of pathological conditions which give rise to abnormal levels of metabolites or enzyme substrates. Methods for rapid assay of serum cholestrol, uric acid, testosterone, androsterone and galactose have been indicated as examples. A wide variety of other compounds found in biological fluids may also be assayed by similar technology.

Claims

2. In a system for quantitating metabolite and enzyme substrate concentrations in body fluids such as whole blood, urine, cerebrospinal fluid and the like:

A. a support;

B. means constituting a plurality of separate restricted test zone areas in a rigid array mounted on said support;

C. at least one of said areas including as a stacked array a plurality of superposed test reagent impregnated members in a rigidly confined column which is adapted for placement of a fluid test media thereon, said stacked array including in descending sequence;

i. a porous upper glass fiber disc;

ii. a porous lower glass fiber disc;

iii. a membrane filter disc;

iv. a porous glass fiber filter disc; and

v. a transparent membrane;

D. said upper and lower glass fiber discs constituting filters to remove amorphous matter including white blood cells and to partially remove red blood cells to prevent such matter from subsequently clogging pores of said membrane filter disc;

E. said upper and lower glass fiber discs constituting dried reagent storage reservoirs for elution therefrom by cleAr filtrate passing therethrough;

F. said membrane filter disc constituting a unit for removal of red blood cells, platelets, soluble proteins and remaining particles from a test specimen;

G. said upper and lower porous glass fiber discs and said porous glass fiber filter disc constituting fluid volume control units and said porous glass fiber filter disc additionally functioning to assist in drawing liquid through said membrane filter disc and to distribute reacting liquid consisting of the eluted filtrate and reagents across said transparent membrane which constitutes a viewing window;

H. said support including;

i. upper and lower slides;

ii. a top retaining ring mounted on the top of said upper slide;

iii. a lower retaining ring mounted on the top of said lower slide;

iv. the stacked array mounted between said upper and lower retaining rings;

v. means securing together corners of said slides; and

vi. spring clamps operatively engaged over and covering the edges of said slides for maintaining effective contact among the members constituting the stacked array confined between said retaining rings.

3. In a system as claimed in claim 2, calibrated and shaded color test comparison means associated with the rigid array for visual comparison with color developed in a test zone area.

4. In a system as claimed in claim 3, said comparison means including a movable color shaded slide and a coacting calibrated scale.

5. In a system as claimed in claim 3, said comparison means including a revolvable color shaded disc and a coacting calibrated scale.

6. In a system as claimed in claim 3, said comparison means including a fixed color shaded strip and a coacting calibrated scale.

7. In a system as claimed in claim 2, said upper and lower glass fiber discs impregnated with a blood uric acid test composition comprising dry bovine serum albumin, glycine-borate buffer, uricase and the upper surface of the transparent membrane having thereon a dry deposit of O-Dianisidine Hydrochloride-Peroxidase mixture and dextran.

8. In a system as claimed in claim 2, said upper and lower glass fiber discs and said porous glass fiber filter disc impregnated with a testosterone and androsterone test composition comprising dry human serum albumin, Nicotinamide adenine dinucleotide Hydroxysteriod Dehydrogenase, N-methyl phenazonium metho sulfate and the upper surface of the transparent membrane having thereon a dry deposit of a solution of Nitro Blue tetrazolium and dextran.

9. In a system as claimed in claim 2, said discs impregnated with a galactose test composition comprising dry human serum albumin and Galactose oxidase, and a dry deposit of a solution of O-dianisidine, Peroxidase and Dextran on the upper surface of the transparent membrane.

10. In a system as claimed in claim 2, said discs for cholesterol screening including on the upper and lower glass fiber discs dry serum albumin, a dry deposit of an enzyme solution containing B-glucuronidase in acetate buffer, and the porous glass fiber filter disc being impregnated with a dry deposit of a substrate solution containing phenolphthalein glucuronide in acetate buffer and the upper surface of the transparent membrane having thereon a precipitated base reactible to obtain anionic red color from released phenolphthalein.

11. In a system as claimed in claim 2, said glass fiber filter disc having a freeze dried deposit of a first selected reagent thereon and said upper and lower glass fiber discs each having a freeze dried deposit of a second selected reagent required for a given test impregnated thereon.