CYSTIC FIBROSIS DETECTION METHOD

Abstract: A method of diagnosing cystic fibrosis, identifying carriers for cystic fibrosis, and non-carriers or "normal" persons. Heretofore, no carrier or prenatal detection method procedure for cystic fibrosis existed. The detection is based on the discovery of the genetic abnormality or biochemical defect in cystic fibrosis, which occurs in mitochondria, minute bodies found in the cytoplasm of most cells which are the principal energy source of the cell and contain the cytochrome enzymes of terminal electron transport. The method comprises an assay carried out on preparations derived from human cells possessing mitochondria. The assay may be a kinetic assay of the enzyme complex of the energy conserving site of the mitochondrial electron transport system of the cells or an assay of mitochondrial activity governed by the enzyme complex. The assay is then evaluated by comparison with standards established as the result of similar assays of cells of other subjects of known condition. The assays provide determination of characteristics which differ in the three types of individuals (normal, carrier, and affected) and provide a basis for their distinction.

Description: CYSTIC FIBROSIS DETECTION METHOD -1- Technical Field The present invention relates to the field of medical diagnostic techniques, more particularly, an un- equivocal method of diagnosing cystic fibrosis, identi¬ fying carriers for cystic fibrosis, and non-carriers or "normal" persons. Background of the Invention Cystic fibrosis (CF) is an inherited disease of the exocrine glands, affecting most characteristically the pancreas, respiratory system, and sweat glands. It is transmitted within families as an autosomal recessive trait, affected individuals possessing a double dose of the mutant gene. Biological parents of subjects with CF each possess a single dose of the mutant gene and by definition are obligatory carriers for the condition. Carriers are, however, clinically no ' rmal and their detec¬ tion prior to the birth of an affected child has been precluded by the absence of detectable effects of the gene in single dose. The disease has a 1:4 chance of occurring in a child of either sex if both parents are carriers. It primarily involves Caucasians and usually begins in in¬ ancy. The disease is typified by chronic respiratory infection, pancreatic insufficiency, and susceptibility to heat prostrations. It is a major cause of death in child¬ ren. It is estimated that there are between ten and twelve million carriers for cystic fibrosis in the United States. Each year between two and three thousand children are born in the United States who are affected by cystic fibrosis. The cost of therapy for cystic, fibrosis patients is estimated at between about $15,000 to $20,000 per year per patient. Of patients diagnosed in early childhood, fewer than fifty percent reach adulthood. Prior to this invention, no carrier or prenat detection procedure for cystic fibrosis existed (Dennin C.R., et al: "Cystic Fibrosis: Projections into t Future", Mangos, J.A. , Editors, Stratton Corporation, N York, pages 141-143, 1976). Diagnosis of the diseas state was based on clinical signs and increased swe salinity. No specific protein or enzyme assay for cyst fibrosis existed. (Nadler, H.L., et al: "The Metabol Basis of Inherited Disease", Stanbury, J.B. et a Editors, McGraw-Hill, New York, pages 1683-1710, 1978). Summary of the Invention The detection method of this invention is bas on the discovery of the genetic abnormality or biochemic defect in cystic fibrosis. It has been discovered th increased calcium uptake by mitochondria occurs i patients with cystic fibrosis and obligate heterozygot (HZ = carriers) . Mitochondria are minute bodies or stru tures found in the cytoplasm of most cells. Mitochondr are- the principal energy source^'of- the cell and conta the -* dehydrogeriase and cytochrome " enzyme systems -of te minal electron transport. The genetic abnormality cystic fibrosis is expressed in the first energy co serving site of mitochondrial electron transport [nicoti amide adenine dinucleotide dehydrogenase (NADH dehydroge ase), (complex 1, NADH: ubiquinone oxidoreductas.e, NAD ferricyanide oxidoreductase) (E.C.I.6.5.3)]. Kineti characteristics of this enzyme system differ in the. thr types of individuals (normal, carrier, and affected) a provide a basis for their distinction. Mitochondri activity governed by the enzyme system, such as calci and oxygen uptake, also differs and is also distinguis able among the three types of individuals. Broadly stated, the cystic fibrosis detecti method according to the present invention comprises assay carried out on preparations derived from hum fibroblasts, lymphocytes, amniotic cells, or any oth cell type possessing mitochondria. A homogenate is pr pared of the cells. An assay is then performed on t sample of mitochondrial activity of the cells. This m ( O be a kinetic assay of the enzyme complex of the energy conserving site of the mitochondrial electron transport system of the cells or an assay of mitochondrial activity governed by the enzyme complex. The assay is then eval- uated by comparison with standards established as the re¬ sult of similar assays of cells of other subjects of known condition. Examples of kinetic parameters of the enzyme complex of the energy conserving site of the mitochondrial electron transport system of the cells include (1) affini¬ ty of the enzyme for its substrate (Km), (2) pH optimum for enzyme activity, (3) temperature optimum for enzyme activity, (4) inhibition (K τ ) by a number of inhibitors of the enzyme, and (5) differential electrophoretic mobility ' of the enzyme. Mitochondrial activity governed by the enzyme of the energy conserving site include (1) calcium uptake as indicated by intracellular calcium pool size differences, (2) oxygen consumption, and (3) differential inhibition of oxygen consumption: Brief Description of—the Drawings - The invention is illustrated in part by .the ac¬ companying drawings in which: FIGURE 1 shows graphically the correlation between pH and optimum enzyme activity in fibroblast cell ' homogenates from cystic fibrosis patients, carriers, and controls; FIGURE 2 shows graphically the results of a blind experiment in which cell strains from five controls and five known carriers were assayed for enzyme activity at pH 8.0 and 8.3; FIGURE 3 shows graphically the results of assays at various pH of enzyme activity in isolated mitochondrial preparations from patients, carriers, and.controls; FIGURE 4 shows graphically the results of car- rier detection by assay of enzyme activity in lymphocyte cells from a random sample of thirty-one individuals; and FIGURE 5 shows graphically the inhibitory effect of rotenone on oxygen consumption by fibroblast cells from cystic fibrosis patients, carriers, and controls. Detailed Description of the Preferred Embodiments The invention is directed to a relatively simple and inexpensive test in the form of a mitochondrial assa for detecting clinically normal carriers for cystic fibro- sis and for diagnosing individuals with the disease prio to the onset of symptoms. Heretofore, no method has " existed for detection of carriers and affected individuals are diagnosed by physical signs only. No prenatal tests existed. Now carriers for cystic fibrosis can be identi- fied prior to the birth of an affected child. Potential parents of cystic fibrosis children can be counseled as to the risk of having a cystic fibrosis child. The detection test is based upon the recognitio of the fundamental biochemical abnormality in the disease and the application of this knowledge to individual, fami¬ ly and population testing through assay of mitochondrial conditions or activities determined or governed by the enzyme system of the first energy conserving site of the mitochondrial electron transport system of sample cells. The assay tests may be carried out on preparations derived from human fibroblasts, lymphocytes, amniotic cells, or any other cell type possessing mitochondria. Although cystic fibrosis is considered to be a generalized disease of exocrine glands, skin fibroblasts have been used as a model system for cystic fibrosis studies. Although not exocrine cells, fibroblasts do secrete their products by mechanisms very similar to those which occur in exocrine cells. In addition, the donor's genotype is retained in cultured fibroblasts for many generations. For these reasons, skin fibroblasts were used in the initial studies in connection with the present detection method. It was found that skin fibroblasts from subjects with cystic fibrosis in.culture express premature 2- senescence. Increased intracellular calcium (Ca ) which occurs in cells from cystic fibrosis patients and obligate carriers was related to this precocious aging. The site of the altered intracellular calcium pool was traced to mitochondria. Accumulation of increased calcium by mitochon¬ dria is a highly complex and incompletely understood process. One of the systems related to mitochondrial Ca 2+ influx consists of the terminal steps of oxidation of metabolites since the uphill accumulation of Ca 2+ in mitochondria is thought to be driven by a proton gradient generated during electron transport. It was reasoned that the increased sequestration of Ca 2+ m mitochondria from subjects with cystic fibrosis and carriers might be a reflection of increased electron transport. If this was the case, oxygen consumption would be expected to be in¬ creased in these cells. Oxygen consumption in cells was examined from cystic fibrosis patients and age- and sex-matched controls. It was found that cells from cystic fibrosis subjects and obligate carriers do consume signi¬ ficantly more oxygen than do their respective control cells. Following treatment of the cells with the electron transport site 1 inhibitor, rotenone, cells from cystic fibrosis subjects and carriers consume equal amounts of oxygen as do controls. That is, cells with the different genotypes respond differentially to the inhibitor. Also, mitochondrial NADH dehydrogenase energy conserving site 1 of the electron transport system, the target of rotenone, has different properties in cells from cystic fibrosis subjects, carriers and controls. Later studies were based on the use of lymphocytes which are more readily obtained and isolated from venous blood and more readily assayed for enzyme activity. Assays are carried out using standard methods and procedures well understood in the art. NADH is the preferred substrate for the kinetic assays of the enzyme by NADPH (nicotinamide adenine dinu- cleotide phosphate) may be used. Numerous electron accep¬ tors are suitable for determination of enzyme activity including soluble metal ferricyanide salts, the ubiqui- nones and the tetrazolium salts. Any or all combinations of substrate, buffer constituents and electron acceptors may be used to distinguish the different types of indivi- duals by any or all of the enzyme kinetic characteristic enumerated. Assay of inhibition of the NADH dehydrogenas may be used to distinguish the three types of individuals CF affected, carriers and controls. Inhibition is ob tained by the use of any of a number of inhibitors of th enzyme including rotenoids, piercidin A, rhein and othe quinone analogs, thiol reagents, Demerol, iron chelators NAD (nicotinamide adenine dinucleotide, oxydized form) AMP (adenosine monophosphate), ADP (adenosine diphos phate), ATP (adenosine triphosphate), guanidinium salts NADH and the general class of barbituates. For quick, easy identification of CF subjects carriers, and normal individuals by physicians or hospi tals or public health services, the determination o optimum pH for NADH hydrogenase activity is most feasible The detection system is easily packaged in the form of kit of three vials, each of which has identical consti tuents in the form of a liquid mixture including an enzym substrate and an electron acceptor.- Each vial " also con tains a buffer to establish least alkaline, more alkalin and most alkaline conditions in the respective vials. Fo example, the vials may be at pH 8.0, 8.3, and 8.6. suitable sample of cell extract, lymphocytes for example may then be added to each vial. Depending upon the elec tron acceptor used, a colorimetric, spectrometric o spectrophotofluorometric assay can be used to determin relative enzyme activity. Although the specific activit optima may vary, the relative pH optima among the thre groups, CF subjects, carriers, and normal individuals retain the same positions. The invention is further illustrated by th following examples: Example 1 Preparation of Fibroblast Cell Cultures Skin fibroblast strains from the upper arm wer obtained from subjects with CF and age- and sex-matche controls and from obligate CF heterozygote parents an O their age- and sex-matched controls. Cells were stored in liquid nitrogen at early passages and thawed for use as needed. In all but a few experiments, strains were pas¬ sage matched. For O- consumption studies and mitochon- drial preparations, monolayers w„ere grown in 285 x 115 mm roller bottles (Bellco Glass, N.J.). For NADH dehydrogen- ase studies, monolayers were grown in 75 cm flasks (Falcon, Oxnard, Ca.). Monolayers were cultured in Eagle's minimal essential medium with Earle's salts plus L-glutamine (K.C. Biologicals, Inc., Lenexa, Kansas), supplemented ' with 10 per cent fetal calf serum, 100 units per ml of penicillin, and lOO/ g/ml of streptomycin (Gibco, Grand Island, N.Y.). The cultures were maintained at 37° C in 95 per cent air - 5 per cent CO^ atmosphere. Cells were harvested with trypsin-EDTA [(1:250 trypsin + 0.2 g EDTA/1 (Gibco, Grand Island, N.Y.)]. Example 2 Assay of Fibroblast NADH Dehydrogenase Activity <— —- Monolayers of fibrobl-ast cells were— grewn to confluence. At the time of assary--they were rinsed with 3 ml of trypsin-EDTA. Five ml of trypsin-EDTA were ' added and the monolayer incubated at 37° C for 10 min. Cells were removed by gentle shaking and transferred to a cen¬ trifuge tube containing 1 ml of fetal calf serum (Gibco, Grand Island, N.Y.). An aliquot of lOo l was removed and suspended in 9.9 ml of Isoton II balanced electrolyte solution (CMS., Minneapolis, Mn. ) for cell counting. The cell suspension was centrifuged at 410 g for 5 min, the supernatant discarded and the pellet washed twice with 0.03 M potassium phosphate buffer (pH 7.6) at 0°. The cells were suspended in 2 ml of 0.12 M potassium phosphate buffer (pH 7.6) at 0°. After removal of a 150 / • ! aliquot for protein analysis the cell suspension was homogenized at 0° at approximately 10,000 rpm in a PT-10 Polytron (Brinkman, Westbury, N.Y.). The resulting homogenate was centrifuged at 1400 x g for 10 min and the supernatant used for assays. Individual flasks yielded between 1.5 x 6 6 10 and 2.5 x 10 cells, the cell and enzyme activity yields from the three genotypes being equivalent. The oxidation of NADH by homogenats was assaye in an Aminco-Bowman spectrophotofluorometer using potas sium ferricyanide as an artificial electron acceptor. Th excitation and emission wave-lengths for NADH were 350 n and 470 nm, respectively. The reaction mixture consiste of 200/7 * 1 of 0.01 M potassium ferricyanide (Sigma, St Louis, Mo.), 100/ 1 of NADH (Sigma) in 0.002 M potassiu phosphate buffer, and 2.6 ml of 0.12 M potassium phosphat buffer. NADH concentrations varied between 2.5 and 25-^ M Reaction mixture pH varied between 7.9 and 9.0. Non-enzy matic reaction was recorded first and IQo l of whole cel extract was added to the reaction mixture to assay th oxidation of NADH. The reaction was assayed at room tem perature. Activity of NADH dehydrogenase was expressed a Δ fluorescence (F) min/mg protein or cell. In some ex periments, NADH dehydrogenase obtained from isolated mito chondria was assayed. Determinations of pH optima fo -NADH dehydrogenase revealed that optimum enzyme activit in cell homogenates occurred at pH 8.6 in CF strains an pH 8.0 in control strains (FIGURE 1). The clear and con sistent difference in pH optimum for NADH dehydrogenase i CF and control strains occurred at all passages teste (Table 1). We examined pH optima for this enzyme syste in strains from obligate heterozygotes and their controls Optimum pH occurred at pH 8.3 in the carriers and at p 8.0 in their controls (FIGURE 1). The pH optimum of NAD dehydrogenase was recorded on individual cell strains o the three genotypes. Each curve represents a differen cell strain. pH optima were repeatedly different in the thre genotypes. In a blind experiment carriers could be dis tinguished from their controls based on this paramete alone (FIGURE 2). Oxidation of NADH by NADH dehydrogenas from whole cell extracts of five control and five hetero zygote cell strains was assayed in a blind experiment a pH 8.0 and 8.3. Each line in the graph represents different cell strain assayed at pH 8.0 and 8.3. Enzym activity is expressed as --- fluorescence/min/cell. With U O out exception enzyme activity was greater at pH 8.3 than. at pH 8.0 in carrier strains and at pH 8.0 than at pH 8.3 in controls. Each strain could be diagnosed blindly as to control or CF carrier status by these criteria. Each line represents a different cell strain. The effect of pH on NADH dehydrogenase activity in relatively pure mitochondrial preparations is shown in FIGURE 3. A sharp peak occurred at pH 8.0 in the control strains and at pH 8.6 in the CF strain. Peak activity in the heterozygote strain was at pH 8.3, but the peak was broad. These experiments were repeated several times with different cell strains with consistent results. Under the conditions used Vmax was equal in the three genotypes. Example 3 Assay of the Affinity of the Enzyme for its Sub¬ strate Apparent affinity of the enzyme NADH dehydrogen¬ ase for its substrate (Km) was determined for each cell line at pH 8.0 (the control Optimum) and at 8.6 (the CF optimum). No difference in Km between control and HZ strains occurred at either pH. At pH 8.0 KM for the CF strains was significantly greater and at pH 8.6 signi¬ ficantly lower than in the HZ and control strains (Table Example 4 Assay of Lymphocyte NADH Dehydrogenase Activity Five to 10 cc samples of venous blood are ob¬ tained and lymphocytes isolated. Cells are counted, a homogenate is obtained and after centrifugation the rela- tive activity of NADH dehydrogenase determined. The substrate is NADH, the electron acceptor in this example is ferricyanide. Enzyme activity is determined at e.g. pH 8.0, 8.3 and 8.6. Change in fluorescence is recorded and expressed as Δ fluorescence/minute/cell. When assayed in this fashion, with or without variations of ingredients or conditions, individuals with the three genotypes can be distinguished unequivocally. NADH activity is always greatest at pH 8.0 in "normal" individuals, at 8.3 in Table 1 Fibroblast strain source and NADH dehydrogenase kinetics (a pH 8.6 @ pH 8.0 Donor pH Km Km Group- ■ Strain Sex Age optimum passage! Cs) passage passag CF 46 M 16 8.6 5, 6, : 10 9.8 12 27.9 6 • CF 69 M 17 8.6 4, 10 8.5 4 25.3 6 CF 70 M 21 8.6 4 9.8 12 34.3 6 CF 71 M 7 8.6 4 11.7 12 37.2 6 CF 72 F 20 8.6 10, 11 11.4 4 36.3 6 HZ .73 F 54 8.3 10 19.2 13 18.9 14 HZ 75 F 35 8.3 9, 11 20.8 13 22.7 14 HZ 76 F 30 8.3 9, 11, ' 12 14.5 13 22.6 14 HZ 77 M 60 8.3 10, 12 17.2 13 19.9 14 HZ 78 F 48 8.3 11, 12 19.1 13 17.9 14 C(H) 81 F 32 8.0 9, 11 20.8 13 22.6 14 C(H) 82 F 30 8.0 10, 12 19.2 13 18.1 14 C 83 F 22 8.0 4, 10, 11 17.7 12 23.2 7 c 84 M 20 8.0 4, 10 18.3 4 21.2 7 c 85 M 6 8.0 5, 10 20.2 4 18.4 7 C(H) 87 F 54 8.0 10 16.7 13 20.7 14 C(H) 88 F 43 8.0 9, 11 20.0 13 18.7 14 C(H) 89 M 58 8.0 10, 11 18.2 13 21.7 14 C 90 20 8.0 4 17.1 12 24.2 7 c 91 M 20 8.0 4 17.0 12 25.9 7 carriers and at 8.6 in subjects with CF. These pH optima may differ depending on reagents and assay conditions. Nevertheless, when various pH's are used it is clear that for CF preparations enzyme activity is greatest at more alkaline conditions, HZ optimum activity at less alkaline conditions and control optimal enzyme activity at rela¬ tively the least alkaline conditions. The results of experiments involving a random sample of thirty-one individuals are illustrated in FIGURE 4. Each line represents a different individual. Based on pH optima, twenty-nine of thirty-one individuals are hon-carriers. Two of thirty-one (6 per cent) are con¬ sidered carriers for CF based on the assay. The theoreti¬ cal carrier frequency is 5 per cent. Examples 5 and 6 Assays of 0_ Consumption and Rotenone Inhibition Confluent monolayers of fibroblasts as prepared in Example 1 were harvested by trypsin-EDTA digestion. Cells were pelleted by centrifugation at 600 x g for 5 min, washed with 10 ml of cold Hank's Balanced Salt Solu¬ tion (HBSS) containing 0.1 mg/ml bovine serum albumin and 10 mg/ml dextrose, pH 7.4. 100 ^1 aliquots of the washed cell suspension were taken for cell counting using a ZBI Coulter Counter. The washed cells were again pelleted by centrifugation and resuspended in 1 ml cold HBSS. 0_ con¬ sumption was measured polarographically on a cell suspen- sion of 1 x 10 7 cells in a final volume of 3 ml by an 0 2 electrode system (Clark model 53, Yellow Springs Instru- merits, Ohio). In additional experiments, rotenone (Sigma, St. Louis, Mo.) was used to specifically inhibit site 1 of the electron transport system. In these experiments the baseline 0 2 uptake rate was determined for each cell line. Prior to removing the cells from the electrode chamber, 100 ^1 of 100/ M rotenone were added and 0 ? consumption recorded. Preliminary experiments included the addition of 100' 1 of 0.1 per cent KCN to assess the extent of inhibition caused by this mitochondrial electron transport inhibitor. KCN completely abolished measurable 0 uptak in all cell lines tested. 0_ uptake (n atoms O_/min/10 cells) was greater in fibroblasts from CF subjects (N 6): 328.7 + 28.2 than in their controls (N = 6): 199.5 22.8 and in those from heterozygote subjects (N = 8) 227.7 + 17.7 than in their controls (N = 8): 156.3 + 15. (P<.005 by Student's t-test in both comparisons). At the dosage used, rotenone inhibited 0_ con sumption in all cell strains tested but the inhibitor effect was CF > HZ _ controls. The inhibitory effect o rotenone on 0 ? consumption in the three genotypes is show in FIGURE 5. The change in 0 2 consumption rate caused b rotenone was recorded as per cent of initial 0~ consump tion rate. Each point represents per cent inhibitio measured on one cell strain. It is apparent that many modifications an variations of this invention as hereinbefore set forth ma be made without departing from the spirit and scope there of. The specific embodiments described are given by wa of example only and the invention is limited only by th terms of the appended claims.

Claims: WHAT IS CLAIMED IS: 1. A method for the diagnosis of cystic fibrosis, the detection of non-symptomatic carriers of the disease, and the identification of non-affected persons, which method comprises: A) obtaining a sample of cells possessing mitochondria from a human subject, B) preparing a homogenate of the cells, C) performing an assay of the mitochondrial activity of the cells, and D) evaluating the assay by comparison with standards established as a result of similar assays of other subjects of known condition. 2. A method according to claim 1 wherein said assay is a kinetic assay of the enzyme complex of an energy con¬ serving site of the mitochondrial electron transport sys¬ tem " of the cells. '— ' - — 3. A method according to claim 2 wherein said enzyme is the .enzyme of the first energy conserving site, NADH dehydrogenase. ■ 4. A method according to claim 1 wherein said assay is an assay of mitochondrial activity governed by the en¬ zyme complex of an energy conserving site of the mitochon¬ drial electron transport system of the cells. 5. A method according to claim 2 wherein said assay is a determination of the affinity of the enzyme for its substrate. 6. A method according to claim 2 wherein said assay is a determination of pH optimum for enzyme activity. 7. A method according to claim 2 wherein said assay is a determination of temperature optimum for enzyme activity. OMPI 8. A method according to claim 2 wherein said assa is a determination of differential inhibition of enzym activity. 9. A method according to claim 2 wherein said assa is a determination of differential electrophoretic mobili ty of the enzyme. 10. A method according to claim 4 wherein said assa is a determination of calcium uptake by the mitochondria. 11. A method according to claim 4 wherein said assa is a determination of oxygen consumption by the mitochon¬ dria. 12. A method according to claim 4 wherein said assa is a determination of inhibition of oxygen consumption b the mitochondria. 13. A method according to claim 5 wherein enzyme affinity for its substrate is measured at at least two o three pH values representing least alkaline conditions, more alkaline conditions and most alkaline conditions. 14. A method according to claim 13 further charac- terized in that said pH values are about 8.0, 8.3, an 8.6. 15. A method according to claim 6 wherein enzyme activity is measured at at least two of three pH values representing least alkaline conditions, more alkaline conditions and most alkaline conditions. 16. A method according to claim 15 wherein said pH values are about 8.0, 8.3 and 8.6. ' 17. A method for the diagnosis of cystic fibrosis, the detection of non-symptomatic carriers of the disease and identification of non-affected normal individuals, which method comprises: A) obtaining a sample of venous blood from a human subject, B) isolating the lymphocytes from said blood . sample, C) preparing a homogenate of the lymphocyte cells, D) performing a kinetic assay of the NADH de- hydrogenase enzyme complex of the first energy conserving site of the mitochondrial electron transport system of the lymphocyte cells by determination of the pH optimum for activity of said enzyme at pH values of about 8.0, 8.3 and 8.6, and E) evaluating the assay by comparison with standards established as the result of similar assays of other subjects of known condition. 18.— *~ A kit for the diagnosis 1 -of cystic fibrosis, the detection of non-symptomatic carriers of the disease, and identification of non-affected normal individuals by kinetic assay of the pH optimum for enzyme activity of the NADH dehydrogenase enzyme complex of the first energy conserving site of the mitochondrial electron transport system of cell samples from a human subject, said kit comprising: A) a plurality of three vials, each adapted to receive a sample of cell extract, and B)_. a liquid mixture within each of said vials including 1) an enzyme substrate, 2) an electron acceptor, and 3) * a buffer, the buffer in each vial dif- fering to establish least alkaline, more alkaline and most alkaline conditions in the respective vial. 19. A kit according to claim 18 wherein the liquid mixtures within said vials are at about pH 8.0, 8.3 and 8.6.

Inventors: SHAPIRO B
Applicants: UNIV MINNESOTA, REGENTS UNIVERSITY MINNESOTA
Application year: 1980
Main classification: C12Q1
IPC codes: C12Q1, G01N33