ENZYME REFERENCE COMPOSITION

Abstract: A stable enzyme reference composition having an excel­ lent shelf-life. The composition comprises at least one enzyme of known value; about 20 to about 40 weight percent of at least one alkylene polyol having from 2 to 5 carbon atoms; about 3 to about 8 grams (gm) per deciliter (dl) total protein present in a human serum albumin matrix; and about 60 to about 80 weight percent water.

Description: ENZYME REFERENCE COMPOSITION Cross References to Related Attlication This application is a continuation-in-part of application Serial No. 17,871, filed March 5, 1979. Background of the Invention 1. Field of the Invention This invention relates to a laboratory material and, more particularly, to a stable enzyme reference composition. 2. Description of the Prior Art Various compositions capable of being employed in conjunction with the analysis of enzymes to either calibrate G instrument or to periodically verify that the instrument is still operating within the tolerances desired are known to those skilled in the art. For examples U.S. Patent 3,466,249 (hereinafter referred to as Anderson) discloses a blood serum reference standard comprising a first container of freeze-dried blood serum and a second container of aqueous ammonium bicarbonate Tre final pH of Andersongs reconstituted serum is 7.5tO5. This pP range is felt by Anderson to be within an acceptable range for test systems as well as for stability of the reconstituted serum's components, particularly the enzymes. U.S. Patent 3,629,142 (hereinafter referred to as Narbach) discloses a freeze-dried serum reference standard comprising blood serum and a carbonate or bicarbonate of tris (hydroxymethyl) aminomethane which is reconstituted by the addition of distilled water. The tris carbonate imparts a normal pH to the serum so as to provide for the stability of its constituents, and particularly the enzymes. (For normal serum and plasma that are collected under routine conditions, the normal pH range is generally considered to be about 7.3 to about 7.45. See Tietz, Fundamental of Clinical Chemistry, W.E Saunders Co., Philadelphia, FA. (1970), pg. 634; and Henry et al, Clinical Chemistry, Principles and Technics, 2nd Edition, Harper & Row, New York, NY (1974), pgs. 774-778.) U. S. 3,875,375 and 4,121,905 (hereinafter referred to Maurukas I and Maurukas II) disclose a biological reference control composition comprising in its non-biological compound from about 60 to about 80 weight percent water and from about 20 to about 40 weight percent of at least one alkylene polyol having from 2 to 5 carbon atoms, the remainder being chiefly at leas one natural biological material selected from the group consisting of blood serum, enzyme, metabou lites, electrolytes, and hormones. Although the pE of this composition is not disclosed in either Maurukas I or Maurukas ZI, experiments have shown the pH to be from about 8.3 to about 8.5. One short coming of the above prior art composition is that their enzyme constituents are highly unstable. For example, the manufacture of a commercial composition believed to be within the scope of Narbach, supra, recommends that determinations of enzyme constituents oi its distilled or deionized water reconstituted serum be made only on the day that the serum is reconstituted. Similarly, the manufacturer of a commercial composition within the scope of Anderson also recommends that the determinations of its reconstituted composition be made only on the day that the serum is reconstituted. Both of the above reconstituted compositions are recommended by their manufacturer to be stored between 20 and 8 C. Summary of the Invention The instant invention encompasses an enzyme reference composition having an improved shelf life. The composition comprises at least one enzyme of known value; about 20 to about 40 weight percent of at least one alkylene polyol having from 2 to 5 carbon atoms; about 3 to about 8 gm/dl total protein present is a human serum albumin matrix; and about 60 to about 80 weight percent water. The enzyme constituents of a composition within the scope of this invention retain 90% of their initial value, based upon an Arrhenius Plot, for the periods set forth in Table I. Table I ARREENIUS PLOT FOR 90% LIFE, AT 40C STORAGE Enzyme Constitution Shelf-Life Amylase 6.8 Years y-Glutamyl Transpepitidase 206.9 Years Lactic Dehydrogenase 1.0 Years Creatine Kinase 145.9 Years Alkaline Phosphatase 9.9 Months Aspartate Amino Transferase 2.4 Years Alanine Amino Transferase 6.6 Years The above Arrhenius Plot shows that the enzyme constituents of the enzyme reference compositions within the scope of this invention are stable for periods of time far exceeding the shelf life of enzyme constituents present in prior art reference compositions. Description of the Preferred Embodiments The enzyme reference composition of the instant invention comprises at least one enzyme constituent of known value. Any enzyme of clinical signifi cance can be present therein. Typical enzyme which are currently assayed in clinical laboratories include acid phosphatase (ACP); aldolase; alkaline phosphatase (ALP); amylase; cholinesterase; creatine kinase (CK; also known as creatine phosphokinase (CPK)); a-glutamyl transpeptidase (Y-GT; GGT); a-hydroxybutyric dehydrogenase ( D; B 35; isocitric dehydrogenase (ICD); lactic dehydrogenase (LDH); leucine aminopeptidase (LAP); lipase; alanine amino transferase (SGPT; ALT; GPT; glutamic-pyruvic transaminase); and aspartate amino transferase (SGOT; AST: GOT: glutamic-oxalacetic transaminase). The activity of each enzyme present in the enzyme reference composition is not critical. Preferably, the activity of each enzyme should be within a range covering both normal and abnormal values The source of each enzyme is also not critical. However, the preference source of several enzymes are set forth in Table II. Table II Enzyme Constituent Preferred Source ACP Human Seminal Fluid Calf Intestine Amylase Pig Pancreas CK Monkey Muscle SGOT Pig Heart SGPT Pig Heart y-GT Pig Intestine LDH Chicken Heart The enzyme reference composition of the instant invention also comprises from about 20 to about 40 weight percent of at least one alkylene polyol having from 2 to 5 carbon atoms. Preferably, the alkylene polyol comprises from about 30 to about 34 weight percent of the enzyme reference composition Suitable alkylene polyols which can be used include, but are not limited to, ethylene glycol, propylene glycol, butylene glycol, pentanediol, and glycerol. The alkylene polyol material is preferably selected from a group consisting of ethylene glycol, propylne glycol, glycerol, and mixtures thereof. The enzyme reference composition of the instant invention also comprises from about 3 to about 8 gm/dl total protein. Preferably, from about 4 to about 5 gm/dl total protein is present in the enzyme reference composition. The total protein is present in a human serum albumin matrix. The enzyme reference composition of the instant invention also comprises from about 60 to about 80 weight percent water. Preferably, water comprises from about 66 to about 70 weight percent of the enzyme reference composition. - The water can be part of the human serum albumin material and/or can be added as a separate constituent. The pH of enzyme reference composition of the instant is not critical. The optimum pH of the enzyme reference composition will vary depending upon the particular enzymes present therein. In general, the pH can be from about 5.0 to about 8.7. Preferably, the pH can be from about 6 to about 8.5. More preferably, the pH can be from about 6 to about 7. Usually, the preferred pH will be from about 6.4 to about 6.6. However, ACP has an optional pH of about 6.0. The pH can be adjusted by any conventional means employed by those skilled in the art, e.g., by the addition of hydrochloric acid (HC1) or sodium hydroxide (NaOH) to the composition. The enzyme reference composition of the instant invention can also further comprise metabolites, electrolytes, and hormones of known value Preferably, the enzyme reference composition of the instant inventlon will further comprise sodium, potas siv-Ln, calcium, phosphorous, magnesium and chloride in amounts of interest to the clinical chemist. The enzyme reference composition of the instant invention can be stored in any suitable container, such as a glass ampule. In general, the enzyme composition of the instant invention can be prepared via the following procedure A solution having a desired percentage of albumin therein is made by dissolving an appropriate amount of albumin in distilled or de-ionized water. To this albumin solution is then added a sufficient amount of at least one alkylene polyol having 2 to 5 carbons so that the alkylene polyol-albumin solution comprises from about 20 to about 40 weight percent alkylene polyol, from about 60 to about 80 weight percent water, and from about 3 to about 8'gms/dl total protein in a human serum albumin matrix. The pH of the alkylene polyol-albumin solution is then adjusted to a desirable level. After adjusting the PHj the alkylene polyolalbumin solution is assayed for enzyme activity and salt content to obtain baseline values for each consti tuent of each of the solution's desired constituents. An amount of each of the constituents is then added to the solution so that the enzymes and salts are present in their desired amount. If necessary, the pH of the solution is then readjusted to its optimum level with respect to the enzymes present therein. The stable enzyme reference composition of the instant invention can be employed as an enzyme reference standard or as an enzyme reference control, i.e., the composition can be employed to either calibrate an instrument or can be employed to periodically verify that the instrument is still operating within the tolerances desired. For the above uses, the enzyme reference composition of the instant invention can contain at least one enzyme in amounts typical of those of interest to those in the clinical laboratory. The following examples are provided for the purpose of further illustration only and are not intended to be limitations of the disclosed invention. Example 1 Table III sets forth one preferred embodiment of the enzyme reference composition of the instant invention wherein the enzyme activity is measured at 370C.: TABLE III Test Specification ACP 0-20 IU/I ALP 0-350 IU/I Amylase 0-400 IU/I CK 0-600 IU/I SGOT 0-200 IU/1 SGPT 0-200 IU/I Y-GT 0-300 IU/I LDH 0-500 IU/1 Sodium 125-157 mEq/l Potassium 3.5 - 4.5 mEq/l Calcium 8.4 - 10.6 mg/dl Phosphorous 1.6 - 2.4 mg/dl Magnesium 1.6 - 2.4 mg/dl Chloride 88 - 112 mEq/l pH . 6.5 i 0.1 EBsAg-B* Negative Total Protein (Human Albumin) 4.5 f 0.5 gm/dl Ethylene glycol 33-1/3% by weight Water 66-2/3% by weight *Hb 5Ag-3 denotes Hepatitis-Surface Antigen Type E Example 2 Preparation of Albumin Base Enzyme Reference Composition Add albumin (Human Cohn Fraction V) slowly to a 33-1/3% by weight aqueous solution of ethylene glycol with continuous mixing. The albumin is preferably dissolved at room temperature, but extended mixing at 2 to 80C. is acceptable. The amount of albumin added is such that the total protein content is about 4.5 + 0.5 gm/l . The pH of the resulting solution is adjusted, if necessary, to 6.5 + 0.1 with 6 N HC1 or 6 N NaOH. Next, filter the solution through an ethylene glycol impervious filter material having a final filter porosity of 0.4 - 0.6p. Assay the filtered solution for enzyme acti vity and salt content to obtain baseline values for each constituent of the composition. Add an amount of each constituent so that the enzymes and salts are present in amounts within the levels set forth in Table III, supra. If necessary readjust the pH of the enzyme reference composition to 6.5 + 0.1 with 6 N HC1 or 6N NaOH. Example 3 One lot of enzyme reference composition within the scope of the instant invention was formulated according to the procedure set forth in Example 2, supra, and stored in plastic vials. These compositions were incubated at various temperatures (-15 , 25 , 320, 370, and 410C.) and assayed at 370C. at designated time intervals. The data obtained are set forth in Tables IV-X. TABLE IV ALP 41 C. 37 C. 32 C. 25 C. -15 C. Hour A A/Ex100% B B/Ex100% C C/Bx100% D D/Ex100% E 0.0 445.0 100 445.0 100 445.0 100 445.0 100 445.0 4.0 462.0 104 449.0 101 455.0 102 448.0 101 445.0 8.0 471.0 100 474.0 101 479.0 102 464.0 99 470.0 24.0 433.0 94 445.0 96 460.0 100 483.0 105 462.0 48.0 415.0 89 426.0 92 448.0 97 463.0 100 464.0 72.0 400.0 86 432.0 93 444.0 95 463.0 99 466.0 96.0 382.0 84 415.0 91 435.0 95 460.0 101 457.0 120.0 391.0 85 431.0 94 445.0 97 454.0 99 460.0 144.0 372.0 82 409.0 90 420.0 93 453.0 100 454.0 312.0 322.0 69 375.0 80 415.0 88 444.0 95 469.0 480.0 281.0 60 353.0 75 398.0 85 426.0 90 471.0 648.0 264.0 38 326.0 72 378.0 84 417.0 92 452.0 TABLE V AMYLASE 41 C. 37 C. 32 C. 25 C. -15 C. Hour A A/Ex100% B B/Ex100% C C/Bx100% D D/Ex100% E 0.0 420.0 100 420.0 100 420.0 100 420.0 100 420.0 4.0 412.0 98 405.0 96 402.0 96 412.0 98 420.0 8.0 380.0 95 397.0 100 397.0 100 382.0 96 398.0 24.0 361.0 95 368.0 96 368.0 96 381.0 100 382.0 48.0 362.0 91 372.0 94 381.0 96 379.0 95 397.0 72.0 354.0 89 373.0 94 391.0 98 -- N/A -- 397.0 96.0 319.0 86 357.0 96 369.0 99 -- N/A -- 371.0 120.0 329.0 84 -- N/AÚ -- 377.0 96 386.0 99 391.0 312.0 295.0 75 377.0 96 389.0 99 394.0 101 392.0 480.0 291.0 74 375.0 95 407.0 103 382.0 97 394.0 648.0 306.0 75 381.0 94 408.0 100 414.0 102 406.0 1. N/A denotes not available. TABLE VI CK 41 C. 37 C. 32 C. 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 559.0 100 559.0 100 599.0 100 559.0 4.0 543.0 97 551.0 99 558.0 100 533.0 99 559.0 8.0 515.0 93 544.0 98 550.0 99 559.0 101 554.0 24.0 417.0 75 514.0 92 544.0 98 549.0 99 557.0 48.0 300.0 54 474.0 86 529.0 95 554.0 100 554.0 72.0 207.0 38 450.0 83 517.0 95 524.0 97 543.0 96.0 -- N/AÚ -- 410.0 76 499.0 93 534.0 99 539.0 120.0 -- N/AÚ -- 408.0 73 519.0 92 527.0 94 562.0 144.0 -- N/AÚ -- 377.0 68 507.0 92 552.0 100 551.0 312.0 -- N/AÚ -- 259.0 46 476.0 85 543.0 97 559.0 480.0 -- N/AÚ -- -- N/AÚ -- 445.0 80 538.0 96 558.0 648.0 -- N/AÚ -- -- N/AÚ -- 415.0 74 503.0 90 558.0 1. N/A denotes not available TABLE VII SGOT 41 C. 37 C. 32 C, 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 464.0 100 464.0 100 464.0 100 464.0 100 464.0 4.0 463.0 100 458.0 99 467.0 101 460.0 99 464.0 8.0 454.0 99 459.0 100 458.0 100 462.0 100 460.0 24.0 439.0 96 449.0 98 454.0 99 453.0 99 457.0 48.0 421.0 92 435.0 95 449.0 98 459.0 100 460.0 72.0 418.0 88 456.0 96 466.0 98 467.0 99 474.0 96.0 389.0 82 428.0 90 451.0 95 472.0 100 474.0 120.0 368.0 79 413.0 89 439.0 94 448.0 96 466.0 144.0 347.0 75 395.0 85 426.0 92 455.0 98 462.0 312.0 238.0 51 321.0 69 388.0 83 439.0 94 466.0 480.0 153.0 33 254.0 55 346.0 74 423.0 91 465.0 648.0 -- N/AÚ -- 290.0 45 313.0 68 408.0 88 462.0 1. N/A denotes not available. TABLE VIII SGPT 41 C. 37 C. 32 C. 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 522.0 100 522.0 100 522.0 100 522.0 100 522.0 4.0 509.0 98 517.0 99 517.0 99 519.0 99 522.0 8.0 473.0 91 478.0 92 493.0 95 511.0 98 521.0 24.0 438.0 84 475.0 91 488.0 94 511.0 98 521.0 48.0 418.0 78 458.0 86 488.0 92 514.0 96 533.0 72.0 369.0 74 418.0 84 466.0 94 -- N/AÚ -- 496.0 96.0 357.0 68 418.0 80 474.0 90 508.0 97 525.0 120.0 329.0 66 411.0 83 453.0 91 487.0 98 497.0 1. N/A denotes not available. TABLE XI y-GT 41 C. 37 C. 32 C. 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 139.0 100 139.0 100 139.0 100 139.0 100 4.0 139.0 100 136.0 98 139.0 100 143.0 103 139.0 8.0 138.0 101 140.0 102 137.0 100 139.0 101 137.0 24.0 133.0 96 137.0 99 133.0 96 139.0 101 138.0 48.0 135.0 98 136.0 99 138.0 100 138.0 100 138.0 72.0 135.0 96 143.0 101 143.0 101 140.0 99 141.0 96.0 136.0 97 142.0 101 142.0 101 142.0 101 140.0 120.0 142.0 96 148.0 100 146.0 99 143.0 97 148.0 144.0 137.0 98 141.0 101 139.0 99 144.0 103 140.0 312.0 129.0 91 135.0 96 139.0 99 141.0 100 141.0 480.0 124.0 91 133.0 97 136.0 99 137.0 100 137.0 648.0 121.0 89 132.0 97 136.0 100 136.0 100 136.0 TABLE X LDH 41 C. 37 C. 32 C. 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 662.0 100 662.0 100 662.0 100 662.0 100 662.0 4.0 652.0 98 655.0 99 651.0 98 651.0 98 662.0 8.0 617.0 97 623.0 98 626.0 99 633.0 100 633.0 24.0 591.0 94 603.0 96 609.0 97 622.0 99 627.0 48.0 585.0 94 604.0 97 609.0 98 608.0 98 621.0 72.0 555.0 92 556.0 93 584.0 97 590.0 98 601.0 96.0 563.0 91 585.0 95 597.0 96 612.0 99 619.0 120.0 553.0 89 572.0 92 593.0 96 601.0 97 619.0 Table XI sets forth an Arrhenius Plot for 90% life based upon the data set forth in Tables IV-X. TABLE XI ARRHENIUS PLOT FOR 90% LIFE BASED ON TEMPERATURES 41 , 37 , 32 , AND 25 C. Temperature ALP Amylase CK SGOT SGPT y-GT LDH 4 C. 9.9MÚ 6.8 Y 145.9 Y 2.4 Y 6.6 Y 206.9 Y 1.0 Y -15 C. 10.2 Yê 132.1 Y 208452.5 Y 109.8 Y 734.9 Y N/A 14.8 Y 1. H denotes months 2. Y denotes years 3. N/A denotes not available The data in Tables IV-X as well as Table XI's Arrhenius Plot based thereon demonstrate that the enzyme reference compositions within the scope of this invention are stable for periods of time far exceeding the shelf life of enzyme constituents present in prior art reference compositions. The following examples are provided for the purpose of distinguishing the instant invention over the invention set forth in Maurukas I and Maurukas II, supra. Example 4 Lots of enzyme reference compositions within the scope of the instant invention were formulated according to the procedure set forth in Example 2, supra, save that when the pH of a particular lot was other than 6.5 *0.1, that lot's pH was adjusted accordingly with either 6N HC1 or 6N NaOH. These lots were stored in plastic vials, incubated at various temperatures, and assayed at 370C at designated time intervals. The data obtained are set forth in Tables XII-XXI. TABLE XII ALP &commat; pH 6.5 Human Serum Albuminium Matrix 41 C. 37 C. 32 C, 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 107.0 100 107.0 100 107.0 100 107.0 100 107.0 4.0 110.0 103 167.0 156 109.0 102 108.0 101 107.0 8.0 113.0 97 115.0 99 115.0 99 111.0 96 116.0 24.0 103.0 96 106.0 99 109.0 102 107.0 100 107.0 48.0 101.0 89 106.0 93 107.0 94 110.0 96 114.0 72.0 98.0 89 103.0 94 108.0 98 112.0 102 110.0 96.0 94.0 84 100.0 89 104.0 93 110.0 98 112.0 120.0 95.0 81 104.0 89 105.0 90 106.0 91 117.0 144.0 88.0 78 99.0 88 102.0 90 109.0 96 113.0 312.0 78.0 68 94.0 82 100.0 88 108.0 95 114.0 480.0 70.0 64 85.0 77 94.0 85 103.0 94 110.0 648.0 66.0 60 77.0 70 89.0 81 97.0 88 110.0 TABLE XII ALP &commat; 8.5 in Human Serum Albuminium Matrix 41 C. 37 C. 32 C, -15 C. A/Dx100% B/Dx100 C C/Dx100% Hour A B C D 0.0 686.0 100 686.0 100 686.0 100 686.0 4.0 719.0 105 672.0 98 679.0 99 686.0 8.0 744.0 99 727.0 96 826.0 109 755.0 72.0 740.0 98 787.0 104 788.0 104 755.0 96.0 754.0 97 822.0 106 801.0 103 744.0 168.0 -- N/AÚ -- 744.0 99 -- N/AÚ -- 748.0 1. N/A denotes not available. TABLE XVI CK &commat; pH 6.5 in Human Serum Albumin Matrix 41 C. 37 C. 32 C, 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 340.0 100 340.0 100 340.0 100 340.0 100 340.0 4.0 330.0 97 325.0 96 335.0 99 332.0 98 340.0 8.0 309.9 93 310.0 93 335.0 101 337.0 101 333.0 24.0 249.0 75 305.0 91 322.0 96 331.0 99 334.0 48.0 184.0 55 285.0 85 316.0 94 330.0 99 335.0 72.0 130.0 39 268.0 80 312.0 94 321.0 96 333.0 96.0 -- N/AÚ -- 248.0 76 298.0 92 319.0 98 325.0 120.0 -- N/AÚ -- 245.0 72 312.0 91 324.0 95 342.0 144.0 -- N/A -- 231.0 70 303.0 91 328.0 99 332.0 1. N/A denotes not available. TABLE XV CK &commat; 8.5 in Human Serum Albuminium Matrix 41 C. 37 C. 32 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% Hour A B C D 0.0 464.0 100 464.0 100 464.0 100 464.0 4.0 440.0 95 481.0 104 451.0 97 464.0 8.0 315.0 65 425.0 88 507.0 105 485.0 24.0 62.0 13 303.0 65 415.0 89 466.0 48.0 -- N/AÚ -- 209.0 43 393.0 81 483.0 72.0 -- N/A -- -- N/A -- -- N/A -- 485.0 96.0 -- N/A -- -- N/A -- 317.0 70 450.0 168.0 -- N/A -- -- N/A -- 445.0 92 482.0 336.0 -- N/A -- -- N/A -- 201.0 43 465.0 1. N/A denotes not available. TABLE XVI SGOT &commat; pH in Human Serum Albuminium Matrix 41 C. 37 C. 32 C. 25 C. -15 C. A/Ex100% B/Ex100 C C/Ex100% D/Ex100% Hour A B C D E 0.0 194.0 100 194.0 100 194.0 100 194.0 100 194.0 24.0 187.0 96 189.0 97 191.0 98 192.0 99 194.0 48.0 179.0 92 186.0 96 191.0 98 190.0 98 194.0 168.0 153.0 79 169.0 87 178.0 92 186.0 96 194.0 TABLE XVII SGOT &commat; pH 8.5 in Human Serum Albumin Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 288.0 100 288.0 100 288.0 100 288.0 4.0 289.0 100 279.0 97 276.0 96 288.0 8.0 273.0 98 272.0 98 286.0 100 278.0 24.0 301.0 99 304.0 100 298.0 98 305.0 48.0 286.0 99 288.0 99 244.0 84 290.0 72.0 291.0 98 290.0 97 293.0 98 298.0 96.0 294.0 98 295.0 99 294.0 98 299.0 168.0 -- N/AÚ -- 300.0 99 246.0 81 304.0 336.0 -- N/A -- 271.0 93 280.0 96 292.0 ÚN/A denotes not available. TABLE XVIII SGPT &commat; pH 6.0 in Human Serum Albumin Matrix 41 C. 37 C. 32 C. 25 C. -15 C. Hour A A/Ex100% B B/Ex100% C C/Ex100% D D/Ex100% E 0.0 30.0 100 30.0 100 30.0 100 30.0 100 30.0 8.0 28.0 93 30.0 100 31.0 103 32.0 107 30.0 24.0 29.0 97 32.0 100 30.0 100 31.0 103 30.0 48.0 26.0 87 28.0 93 30.0 100 31.0 103 30.0 168.0 22.0 73 25.0 83 26.0 87 29.0 97 30.0 TABLE XIX SGPT &commat; pH 8.5 in Human Serum Albumin Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 255.0 100 255.0 100 255.0 100 255.0 4.0 250.0 98 239.0 94 246.0 96 255.0 8.0 218.0 94 216.0 94 246.0 106 231.0 24.0 191.0 79 215.0 89 222.0 92 241.0 48.0 150.0 70 193.0 91 218.0 102 213.0 72.0 140.0 56 201.0 80 227.0 91 250.0 96.0 107.0 46 175.0 75 206.0 88 234.0 168.0 -- N/AÚ -- 140.0 63 209.0 95 221.0 336.0 -- N/A -- 29.0 14 75.0 35 213.0 ÚN/A denotes not available. TABLE XX LDH &commat; pH 6.5 in Human Serum Albumin Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 416.0 100 416.0 100 416.0 100 416.0 4.0 402.0 97 407.0 98 410.0 99 416.0 8.0 407.0 104 408.0 104 410.0 105 392.0 24.0 447.0 112 492.0 123 483.0 121 400.0 48.0 378.0 92 394.0 96 394.0 96 411.0 72.0 420.0 91 432.0 94 460.0 100 461.0 96.0 346.0 85 378.0 93 396.0 98 406.0 168.0 381.0 85 -- N/AÚ -- 417.0 93 447.0 336.0 343.0 83 370.0 90 377.0 92 411.0 505.0 360.0 84 372.0 87 394.0 92 427.0 ÚN/A denotes not available. TABLE XXI LDH &commat; pH 8.5 in Human Serum Albumin Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 402.0 100 402.0 100 402.0 100 402.0 4.0 395.0 98 393.0 98 397.0 99 402.0 8.0 380.0 96 385.0 97 402.0 101 397.0 24.0 425.0 108 442.0 113 746.0 116 392.0 48.0 359.0 89 375.0 93 382.0 95 403.0 72.0 400.0 89 422.0 94 434.0 96 450.0 96.0 338.0 87 359.0 93 374.0 97 387.0 168.0 380.0 85 389.0 87 417.0 93 448.0 336.0 -- N/AÚ -- 347.0 88 360.0 91 395.0 ÚN/A denotes not available. Example 5 Lots of enzyme reference compositions of the type described by Maurukas I and Maurukas II, supra, were formulated according to the improved procedure set forth in U.S. patent 4,158,544. These lots were stored in plastic vials, incubated at various temperatures, and assayed at 370C. at designated time intervals. The data obtained are set forth in Tables XXII-XXXI. TABLE XXII ALP &commat; pH 6.5 in Human Serum Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 51.0 100 51.0 100 51.0 100 51.0 4.0 54.0 106 54.0 106 54.0 106 51.0 8.0 57.0 112 56.0 110 55.0 108 51.0 24.0 50.0 94 54.0 102 57.0 108 53.0 48.0 47.0 85 51.0 93 57.0 104 55.0 72.0 37.0 70 46.0 87 52.0 98 53.0 96.0 34.0 64 43.0 81 50.0 94 53.0 121.0 33.0 60 43.0 78 51.0 93 55.0 144.0 30.0 55 42.0 76 48.0 87 55.0 336.0 18.0 35 31.0 60 39.0 75 52.0 504.0 -- N/AÚ -- 32.0 55 43.0 74 58.0 1344.0 -- N/A -- 24.0 35 43.0 62 69.0 2016.0 -- N/A -- -- N/A -- 29.0 51 57.0 ÚN/A denotes not available. TABLE XXIII ALP &commat; pH 8.5 in Human Serum Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 295.0 100 295.0 100 295.0 100 295.0 4.0 298.0 101 295.0 100 294.0 100 295.0 8.0 288.0 99 309.0 106 309.0 106 291.0 24.0 263.0 89 -- N/AÚ -- 292.0 99 294.0 48.0 235.0 76 375.0 92 292.0 94 309.0 72.0 223.0 77 422.0 102 312.0 108 289.0 96.0 187.0 64 359.0 94 302.0 103 293.0 172.0 114.0 39 389.0 75 258.0 88 293.0 ÚN/A denotes not available. TABLE XXIV CK &commat; pH 6.5 in Human Serum Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 278.0 100 278.0 100 278.0 100 278.0 4.0 286.0 103 285.0 103 281.0 101 278.0 8.0 260.0 87 261.0 87 263.0 88 300.0 24.0 210.0 73 261.0 90 272.0 94 289.0 48.0 156.0 53 252.0 86 269.0 92 293.0 72.0 -- N/AÚ -- 238.0 83 260.0 90 288.0 96.0 -- N/A -- 213.0 73 244.0 85 292.0 121.0 -- N/A -- 195.0 66 241.0 82 294.0 144.0 -- N/A -- 183.0 62 236.0 80 294.0 336.0 -- N/A -- 101.0 40 167.0 65 255.0 504.0 -- N/A -- -- N/A -- 157.0 58 270.0 ÚN/A denotes not available. TABLE XXV CK &commat; pH 8.5 in Human Serum Matrix 41 C. 37 C. 32 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 127.0 100 127.0 100 127.0 100 127.0 4.0 115.0 91 129.0 102 124.0 98 127.0 8.0 71.0 55 119.0 93 132.0 103 128.0 24.0 2.0 2 -- N/AÚ -- 99.0 85 117.0 48.0 -- N/A -- 28.0 24 83.0 71 117.0 72.0 -- N/A -- -- N/A -- 68.0 56 122.0 96.0 -- N/A -- -- N/A -- 56.0 43 131.0 ÚN/A denotes not available. TABLE XXVI SGOT &commat; pH 6.0 in Human Serum Matrix 41 C. 25 C. 4 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 111.0 100 111.0 100 111.0 100 111.0 1.0 110.0 99 -- N/AÚ -- -- N/A -- 111.0 2.0 111.0 100 -- N/A -- -- N/A -- 111.0 3.0 109.0 98 -- N/A -- -- N/A -- 111.0 4.0 107.0 96 112.0 101 111.0 100 111.0 5.0 121.0 102 -- N/A -- -- N/A -- 118.0 6.0 114.0 96 -- N/A -- -- N/A -- 118.0 7.0 111.0 94 -- N/A -- -- N/A -- 118.0 8.0 106.0 90 -- N/A -- -- N/A -- 118.0 9.0 103.0 87 121.0 102 118.0 100 118.0 10.0 86.0 76 -- N/A -- -- N/A -- 114.0 11.0 87.0 77 -- N/A -- -- N/A -- 114.0 12.0 91.0 80 -- N/A -- -- N/A -- 114.0 15.0 79.0 70 108.0 96 112.0 99 113.0 24.0 49.0 43 108.0 95 113.0 100 114.0 30.0 43.0 38 -- N/A -- -- N/A -- 113.0 36.0 36.0 32 103.0 91 112.0 99 113.0 48.0 22.0 20 98.0 88 111.0 99 112.0 56.0 17.0 15 98.0 87 112.0 99 113.0 72.0 < 5.0 N/A 95.0 84 112.0 99 114.0 80.0 < 5.0 N/A 93.0 83 112.0 100 113.0 ÚN/A denotes not available. TABLE XXVII SGOT &commat; pH 6.0 in Human Serum Matrix 41 C. 25 C. 4 C. -15 C. Hour A A/Dx100% B B/Dx100% C C/Dx100% D 0.0 124.0 100 124.0 100 124.0 100 124.0 1.0 124.0 99 -- N/AÚ -- -- N/A -- 124.0 2.0 125.0 101 -- N/A -- -- N/A -- 124.0 3.0 123.0 99 -- N/A -- -- N/A -- 124.0 4.0 123.0 99 124.0 100 124.0 100 124.0 5.0 136.0 103 -- N/A -- -- N/A -- 132.0 6.0 135.0 102 -- N/A -- -- N/A -- 132.0 7.0 132.0 100 -- N/A -- -- N/A -- 132.0 8.0 130.0 98 -- N/A -- -- N/A -- 132.0 9.0 129.0 98 132.0 100 130.0 98 132.0 10.0 117.0 92 -- N/A -- -- N/A -- 127.0 11.0 117.0 92 -- N/A -- -- N/A -- 127.0 12.0 121.0 95 -- N/A -- -- N/A -- 127.0 15.0 122.0 96 120.0 94 125.0 98 128.0 24.0 111.0 87 126.0 99 127.0 100 127.0 30.0 107.0 85 -- N/A -- -- N/A -- 126.0 36.0 101.0 80 125.0 99 126.0 100 126.0 48.0 90.0 71 124.0 98 126.0 100 126.0 56.0 90.0 63 124.0 98 126.0 100 127.0 72.0 64.0 50 123.0 96 127.0 99 128.0 80.0 59.0 46 123.0 96 127.0 100 128.0 ÚN/A denotes not available. TABLE XXVIII SGPT &commat; pH 6.0 in Human Serum Matrix 41 C. 25 C. 4 C. -15 C. Ax100% Bx100% Cx100% D Hour A D B D C D 0.0 16.0 102 16.0 102 16.0 102 1.0 18.0 115 -- NAÚ -- -- N/A -- 16.0 2.0 19.0 121 -- NA -- -- N/A -- 16.0 3.0 20.0 128 -- NA -- -- N/A -- 16.0 4.0 20.0 100 18.0 90 20.0 100 20.0 5.0 12.0 68 -- NA -- -- N/A -- 18.0 6.0 13.0 74 -- NA -- -- N/A -- 18.0 7.0 14.0 80 -- NA -- -- N/A -- 18.0 8.0 15.0 85 -- NA -- -- N/A -- 18.0 9.0 17.0 97 18.0 102 17.0 97 18.0 10.0 22.0 90 -- NA -- -- N/A -- 24.0 11.0 7.0 29 -- NA -- -- N/A -- 24.0 12.0 29.0 119 -- NA -- -- N/A -- 24.0 15.0 15.0 88 16.0 94 17.0 100 17.0 24.0 11.0 45 29.0 119 13.0 53 24.0 30.0 12.0 72 -- NA -- -- N/A -- 17.0 36.0 10.0 60 16.0 95 17.0 101 17.0 48.0 8.0 47 16.0 94 16.0 94 17.0 56.0 8.0 42 16.0 85 17.0 89 19.0 72.0 7.0 41 16.0 95 17.0 101 17.0 80.0 < 5.0 -- N/A -- 16.0 104 17.0 111 15.0 ÚN/A denotes not available TABLE XXIX SGPT &commat; pH 8.5 in Human Serum Matrix 41 C. 25 C. 4 C. -15 C. Ax100% Bx100% Cx100% D Hour A D B D C D 0.0 20.0 100 20.0 100 20.0 100 1.0 19.0 95 -- NAÚ -- -- N/A -- 20.0 2.0 20.0 100 -- NA -- -- N/A -- 20.0 3.0 20.0 100 -- NA -- -- N/A -- 20.0 4.0 20.0 92 22.0 101 21.0 96 20.0 5.0 12.0 55 -- NA -- -- N/A -- 22.0 6.0 13.0 60 -- NA -- -- N/A -- 22.0 7.0 15.0 69 -- NA -- -- N/A -- 22.0 8.0 16.0 74 -- NA -- -- N/A -- 22.0 9.0 18.0 83 20.0 92 22.0 101 22.0 10.0 6.0 42 -- NA -- -- N/A -- 14.0 11.0 < 5.0 N/A -- NA -- -- N/A -- 14.0 12.0 13.0 91 -- NA -- -- N/A -- 14.0 15.0 5.0 50 10.0 100 11.0 110 10.0 24.0 8.0 56 15.0 104 15.0 104 14.0 30.0 9.0 50 -- NA -- -- N/A -- 18.0 36.0 7.0 39 17.0 94 18.0 100 19.0 48.0 < 5.0 N/A 15.0 80 17.0 91 19.0 56.0 < 5.0 N/A 15.0 86 18.0 103 17.0 72.0 < 5.0 N/A 18.0 93 20.0 103 19.0 80.0 < 5.0 N/A 13.0 67 19.0 98 19.0 ÚN/A denotes not available. TABLE XXX LDH &commat; pH 6.5 in Human Serum Matrix 41 C. 37 C. 32 C. -15 C. Ax100% Bx100% Cx100% D Hour A D B D C D 0.0 430.0 100 430.0 100 430.0 100 430.0 4.0 420.0 98 434.0 101 426.0 99 430.0 8.0 456.0 101 449.0 100 458.0 102 451.0 24.0 423.0 91 420.0 91 442.0 95 464.0 48.0 461.0 103 477.0 106 441.0 98 449.0 72.0 413.0 92 429.0 95 459.0 102 451.0 96.0 400.0 89 416.0 92 437.0 97 451.0 121.0 417.0 88 438.0 92 471.0 99 474.0 144.0 417.0 87 432.0 90 462.0 97 478.0 336.0 364.0 86 372.0 88 414.0 98 423.0 504.0 352.0 84 368.0 87 395.0 94 421.0 TABLE XXXI LDH &commat; pH 8.5 in Human Serum Matrix 41 C. 37 C. 32 C. -15 C. Ax100% Bx100% Cx100% D Hour A D B D C D 0.0 488.0 100 488.0 100 488.0 100 488.0 4.0 476.0 98 487.0 100 478.0 98 488.0 8.0 504.0 105 529.0 110 531.0 110 482.0 24.0 438.0 89 -- N/AÚ -- 476.0 97 491.0 48.0 436.8 88 471.0 95 482.0 97 498.0 72.0 476.0 92 505.0 97 521.0 100 520.0 96.0 428.9 81 472.0 90 504.0 96 527.0 172.0 405.0 78 451.0 86 470.0 90 522.0 316.0 353.0 69 404.0 79 449.0 87 514.0 484.0 309.0 59 406.0 78 484.0 93 521.0 1204.0 106.0 20 209.0 39 425.0 80 530.0 ÚN/A denotes not available. Table XXXII compares the shelf life for each of the constituents listed in Tables XII-XXXI at a given temperature and time. TABLE XXXII % Recovery Human Human Albumin Albumin Constituent pH Temp. ( C.)/Time (Hr.). Matrix Matrix ALP 6.5 41/144 78% 55% ALP 8.5 41/96 97% 64% CK 6.5 41/48 55% 53% CK 8.5 41/24 13% 2% SGOT 6.0 41/48 92% 20% SGOT 8.5 41/72 98% 50% SGPT 6.0 41/48 87% 47% SGPT 8.5 41/24 79% 56% LDH 6.5 41/#505 84% 84% LDH 8.5 41/96 87% 81% Table XXXII demonstrates that irrespective of pH enzymes tend to exhibit a markedly improved shelf life when stored in the human serum albumin matrix of the instant invention than when stored in the prior art human serum albumin matrix of the instant invention than when stored in the prior art human serum matrix of the composition of Maurukas I and Maurukas II, supra. Based on this disclosure, many other modifications and ramifications will naturally suggest themselves to those skilled in the art. These are intended o be comprehended as within the scope of this invention.

Claims: CLAIMS 1. An enzyme reference composition compris ing: (a) at least one enzyme constituent of known value; (b) from about 20 to about 40 weight percent of at least one alkylene polyol having from 2 to 5 carbon atoms; (c) from about 3 to about 8 grams per deciliter total protein present in a human serum albumin matrix; and (d) from about 60 to about 80 weight percent water. 2. The composition of claim 1 having a pH of frm about 5.8 to about 8.7. 3. The composition of claim 1 having a pH of from about 6 to about 8+5. 4. The composition of claim 1 having a pH of from about 6 to about 7. 5. The composition of claim 1 having a pH of from about 6.4 to about 6.6. 6. The composition of any of claims 1-4 or 5 comprising from about 30 to about 34 weight percent of said alkylene polyol, from about 4 to about 5 grams per deciliter of total protein, and from about 66 to about 70 weight percent water. 7. The composition of any of claims 1-4 or 5 wherein at least one of said enzymes is selected from a group consisting of acid phosatase, aldolase, alkaline phosphatase, amylase, cholinesterase, creatine kinase, a -glytamyl transpeptidase, 0 -hydroxybutyric dehydrogenase, isocitric dehydrogenase, lactic dehydrogenase, leucine aminopeptidase, lipase, alanine amino transferase, and aspartate amino transferase. S. The composition of any of claims 1-4 or 5 wherein said enzyme moiety comprises acid phosphatese, alkaline phosphatase, amylase, creatine kinase, aspartate amino transferase, alanine amino transferase, y-glutamyl transpeptidase, and lactic dehydrogenase. 9. The composition of any cf claims 1-4 or 5 comprising: (a) from about 0 to about 350 International Units per liter alkaline phosphatase; (b) from about 0 to about 20 International Units per liter acid phosphatase; (c) from about 0 to about 400 International Units per liter amylase; (d) from about 0 to about 600 International Units per liter creatine kinase; (e) from about 0 to about 200 International Units per liter aspartate amino transferase; (f) from about 0 to about 200 International Units per liter alanine amino transferase; (g) iror about 0 to about 300 International Units per liter-&gamma;-glutamyl transpeptidase; and (h) from about 0 to about 500 International Units per liter lactic dehydrogenase wherein the enzyme activities are stated for 370C 10. The composition of any of claims 1-4 or 5 further comprising sodium; potassium, calcium, phosphorous, and magnesium. 11. The composition of any of claims 1-4 or 5 comprising: (a) from about 0 to about 350 International Units per liter alkaline phosphatase; (b) from about O to about 20 International Units per liter acid phosphatase; (c) from about 0 to about 400 International Units per liter amylase; (d) from about 0 to about 600 International Units per liter creatine kinase; (e) from about 0 to about 2Gf nterna- tional Units per liter aspartate amino transfa ase; (f) from about 0 to about 200 International Units per liter alkaline amino transferase; (g) from about 0 to about 300 International Units per liter &gamma;-glutamyl transpeptidase; ; (h) from about 0 to about 500 International Units per liter lactic dehydrogenase; (i) from about 125 to about 157 milliequivalents per liter sodium; (j) from about 3.5 to about 4.5 millequivalents per liter potassium; (k) from about S.4 to about 10.6 milligrams per deciliter calcium; (1) from about 1.6 to about 2.4 milligrams per deciliter phosphorous; (m) from about 1.6 to about 2.4 milligrams per deciliter magnesium; and (n) from about SS to about 112 milliequivalents per liter chloride; wherein the enzyme activities are stated for 370C. 12. The composition of any of claims 1-4 or 5 comprising: (a) from about 0 to about 350 International Units per liter alkaline phosphatase; (b) from about 0 to about 20 International Units per liter acid phosphatase; (c) from about 0 to about 400 International Units per liter amylase; (d) from about 0 to about 600 International Units per liter creatine kinase; (e) from about 0 to about 200 International Units per liter aspartate amino transferase; (f) from about 0 to about 200 International Units per liter alanine amino transferase; (g) from about 0 to about 300 International Units per liter y-glytamyl transpeptidase; (h) from about 0 to about 500 International Units per liter lactic dehydrogenase; (i) from about 125 to about 157 milliequivalents per liter sodium; ; (j) from about 3.5 to about 4.5 milliequivalents per liter potassium; (k) from about 8.4 to about 10.6 milligrams per deciliter calcium; (1) from about 1.6 to about 2.4 milligrams per deciliter phosphorous; (m) from about 1.6 to about 2.4 milligrams per deciliter magnesium; and (n) from about 88 to about 112 milliequivalents per liter chloride; wherein the enzyme activities are stated for 37 C; and wherein said acid phosphatase is derived from human seminal fluid, said alkaline phosphatase is derived from calf intestine; said amylase is derived from pig pancreas; said creatine kinase is derived from monkey muscle; said aspartate amino transferase and alanine amino transferase are derived from pig heart; said y-glutamyl transpeptidase Is derived from pig intestine; and said lactic dehydrogenase is derived from chicken heart.

Inventors: LOUDERBACK, ALLAN L.
Applicants: BECKMAN INSTRUMENTS, INC.
Application year: 1980
Main classification: C12Q1
IPC codes: C12N9, C12Q1