GB2046902A - Reagents and method for detection of cancer - Google Patents

Abstract

A specific type of protein referred to as Bucovaz-protein or B- protein is generally present in the blood serum of individuals having cancer, and a method for determining the amount of the B-protein and thereby detecting cancer when present comprises adding to the serum sample a labelled coenzyme A or labelled 3-dephosphocoenzyme A, adenosine triphosphate or a salt thereof, and a coenzyme A-synthesizing protein complex thereby reacting the labelled coenzyme with serum protein, partially denaturing the resulting B-protein-labelled coenzyme complex and determining the amount of the partially denatured complex. The label may be <3>H, <14>C, <35>S, fluorescein or coumarin.

Description

SPECIFICATION Method and reagent for detecting cancer This invention relates to an improved method for detecting the presence of cancer and to reagent therefor.

Bucovaz et al reported in U.S. Patent 4,160,817 that a specific protein referred to as Bucovaz protein or B-protein is inherently present in blood serum of a person who has cancer and, therefore, cancer can be diagnosed by detecting the B-protein.

One object of this invention is to provide an improved method for detecting cancer of humans, particularly a method more convenient than the prior art method.

Another object of this invention is to provide a reagent for detecting cancer.

These and other objects of this invention will become clear hereunder.

The method of this invention for detecting the presence of cancer comprises adding either at the same time or in any order a labeled coenzyme A or labeled 3'-dephosphocoenzyme A (The labeled coenzyme A and the labeled 3'-dephosphocoenzyme A can each be referred to as "CoAX" hereunder to simplify the description.); adenosine triphosphate (referred to as ATP hereunder) or a salt thereof; and a coenzyme A-synthesizing protein complex (referred to as CoA SPC hereunder) to a predetermined amount of human blood serum (test sample) thereby reacting the serum protein with the labeled CoAX, partially denaturing thermally and by use of a chemical denaturant, the resulting B-protein-labeled CoAX complex, and determining the amount of the partially denatured complex.

The labeled coenzyme A which is useful in this invention includes a commercially available 3Hcoenzyme A, or those which are prepared by labeling in a conventional manner a commercially available coenzyme A with radioisotope such as 3H, 14C, 35S or the like or with a fluorescent dye such as fluorescein, coumarin or the like. Similarly, the labeled 3'-dephosphocoenzyme A which is useful in this invention includes those which are prepared by enzymatically dephosphating a commercially available 3H-coenzyme A at 3'-position, or those which are prepared by labeling in a conventional manner a commercially available 3'-dephosphocoenzyme A with a radio-isotope such as 3H, 14C, 35S or the like or with a fluorescent dye such as flurescein, coumarin or the like.

Commercially available ATP can be used in the method of this invention. CoA-SPC which is useful in the method of this invention includes those disclosed in the above mentioned U.S.

Patent 4,160,81 7. This U.S. Patent is incorporated in this specification by reference.

According to this invention, the reaction of this method is generally carried out by adding to 50 yl of the test sample, (1)0.01-0.1 ml, preferably, 0.04-0.06 ml of CoA-SPC, (2) ATP or its salt in an amount such that the concentration is 0.01-10 mM, preferably 1.5-5 mM in a reaction mixture of the test sample and the reactants (1), (2) and (3) one ml of a buffer containing 0.025-2,5001lM, preferably 0.25-51lM of the labeled CoAX, and allowing the mixture to stand for a period sufficient to complete the reaction with or without heating. The reaction time depends on the temperature, which ranges from 0 to 40"C. As the reaction temperature becomes higher (lower), the reaction time becomes shorter (longer).A reaction condition at 30-38"C for 3-60 minutes is particularly preferred.

In this reaction, buffers which can be used include those capable of maintaining in a pH of 6-8, typically, tris acetate buffer, phosphate buffer, and 2 N-morpholino ethansulphonic acid buffer.

A suitable formulation of the tris acetate buffer comprises 0.001-250 mM of Tris-acetate, 0.01-50 mM of magnesium acetate, and 0.001-125 mM of KCI. A suitable formulation of the phosphate buffer comprises 0.001-250 mM of KH2PO4 and 0.01-50 mM of magnesium acetate. In these buffers, magnesium acetate is added to satisfy the metal requirement (Mg+ +) of CoA-SPC. However, the addition is not critical because Mg+ + present in the test sample will suffice to meet the requirement. Of course, since the addition of magnesium acetate invariably assures sufficient results, it is safe and convenient to use.

The labeled CoAX which is mixed with the test sample is bonded with the aid of the CoA-SPC with serum protein in the case of a non-cancer patient to form a complex. While in the case of a cancer patient, CoAX is bonded with both normal serum protein and the B-protein to form two different complexes, namely, the normal serum protein complex and the B-protein complex.

Although these two complexes are very close to each other in characteristics, the water solubility of these complexes is altered differentially when the complexes are partially denatured by heat treatment and then by treating with a chemical denaturant. By utilizing the difference in water solubility, the amount of the B-protein-labeled CoAX complex can be determined.

The determination of the amount of the B-protein complex in the reaction mixture is effected by first denaturing the complex partially by heating and with a chemical denaturant. In general, the reaction mixture, as it is, is usually heated at a temperature of from 65 to 1 00'C for 1-10 minutes, preferably, at a temperature of from 68 to 70"C for 3-6 minutes. During this heat treatment, the mode of denature varies differentially between the normal serum protein-labeled CoAX complex, and the B-protein-labeled CoAX complex. Then, the partially denatured complexes are treated with a chemical denaturant, such as trichloroacetic acid, methanol, ethanol or the like whereby the B-protein-labeled CoAX complex is converted to a water insoluble form.The water insoluble material is collected by filtration or by centrifugation and the amount of the complex is determined by a scintillation counter when the CoAX is labeled with radioisotope, or by a fluorophotometer when the CoAX used is labeled with a fluorescent dye.

In this assay, the amount of the insolubilized complex of a cancer patient is 1.5 to 5 times that of a non-cancer patient and, therefore, it is possible to easily detect the presence of cancer or to diagnose cancer without a complicated operation. According to another aspect of the invention, a diagnostic reagent for multi-assay is prepared by conveniently carrying out the method described herein. The reagent can be a kit of elements, each of which contains at least one of the labeled CoAX, namely the labeled coenzyme A or labeled 3'-dephosphocoenzyme A, ATP or its salt, and CoA-SPC or a composition of all the reactants. The kit may further contain a chemical denaturant and/or a buffer as separate element.

In the kit which cintains the reactants as individual elements, each reactant may be in the lyophilized form and is used by dissolving in a buffer, or may be in the form of solution. On the other hand, in the kit at least one element of which contains two or more elements, it is preferable that one element contain the labeled CoAX, and ATP or its salt, and the other element contain CoA-SPC in the form of lyophilized form or as a buffer solution.

This invention is further illustrated by the following Examples.

Example 1 Preparation of Diagnostic Reagent (For 1,000 assays) a) Labeled coenzyme A solution: 3H-coenzyme A 1 mg (50 IlCi) 2-mercaptoethanol 4 mg distilled water to make 50 ml b) Buffer solution containing adenosine triphosphate: tris(hydroxymethyl)aminomethane 6.05 g KCI 1.85 g MgC12 6H2O 2.03 g ATP2Na3H,O 2.42 9 distilled water to make 900 ml The pH of the solution formulated above was adjusted to 8.0 with acetic acid, and then distilled water was added to make the total volume one liter.

c) CoA-SPC solution: Baker's yeast was frozen in a dry ice/ether mixture, kept in frozen state for 3 hours and then allowed to thaw to remove ether. To the treated baker's yeast, was added potassium chloride (5 g/pound) and the resulting mixture was stirred at 4"C for 30-35 hours and centrifuged at 10,000 r.p.m. (9,600 G) for 30 minutes. The supernatant was collected and centrifuged again at 35,000 r.p.m. (95,000 G) for 30 minutes. The supernatant was collected, poured into vials in a volume of 1 ml per vial and lyophilized. For use, the dried substance is reconstituted with 1 ml of distilled water.

d) 15% Trichloroacetic acid solution: Trichloroacetic acid (300 g) was dissolved in distilled water (1.5 I) and then distilled water was added to adjust the volume of the solution to 2.0 I.

Example 2 Preparation of Diagnostic Reagent (for 1 ,000 assays) a) Labeled 3'dephosphocoenzyme A solution: 3H-3'dephosphocoenzyme A 1 mg (50 iuCi) 2-mercaptoethanol 4 mg distilled water to make 50 ml b) Buffer solution containing adenosine triphosphate: Prepared as in Example 1 b).

c) CoA-SPC Prepared as in Example 1 c).

d) 15% Trichloroacetic acid aqueous solution: Prepared as in Example 1 d).

Example 3 Detection of Rabbit Transplantable Tumor (VX-2) a) Test Samples: Transplantable tumor VX-2 was exsected from a cancer-bearing rabbit, washed three times with Hanks solution and cut into small pieces. The pieces of tumor were suspended in Hanks solution and, after being filtered through a metal screen, the concentration of the pieces in the suspension was adjusted to 20% by volume. A rabbit weighing about 2.5 kg was inoculated intramuscularly with 0.5 ml of the suspension in the femur region. Just after the inoculation (0) and 3, 6, 8, 14, 20, 28 and 37 days after the inoculation, a 2.0 ml blood sample was taken from the ear vein. The blood serum was separated from each blood samples just after sampling, frozen and stored at - 20"C. Prior to assay, the serum is allowed to thaw.

b) Assay Procedure: The test sample (50 jul) was placed in a test tube, and then a buffer solution (1.0 ml) and the 3H-coenzyme A solution (50 1ll) were added to the test tube and thoroughly mixed. After allowing the mixture to stand for 5 minutes under warm condition, the CoA-SPC solution (50 yI) was added, mixed thoroughly and allowed to stand under the warm condition for 10 minutes.

Then, the mixture was heated in a water bath at 68"C for 5 minutes to terminate the reaction.

After restoring to room temperature, the reaction mixture was centrifuged at 3,000 r.p.m.

(2,000 G) for 5 minutes and the supernatant was collected in a separate test tube by decantation. A 15% trichloroacetic acid aqueous solution (2.0 ml) was added and the resulting precipitates were recovered by filtration using a Millipore filtering apparatus and Whatman No.

3 paper discs.

The precipitates collected on the discs were washed 3 times with 2 ml of distilled water per wash. Each disc was burned in a sample oxidizer and the radioactivity level was determined with a scintillation counter.

c) Results: The results obtained are shown in Table 1 below.

Table 1 Time after Inoculation (days) Measurements (cpm) 0 666 3 882 6 788 8 821 14 1575 20 2104 28 2699 37 3075 As is clear from Table 1, the amount of the B-protein-lebeled coenzyme A complex was increased time-dependently after transplantation of the tumor.

Example 4 Detection of Rabbit Transplantable Tumor (VX-2) a) Test Samples: Transplantable tumor VX-2 was exsected from a cancer-bearing rabbit, washed three times with Hanks solution and cut into small pieces. The pieces were suspended in Hanks solution and, after being filtered through a metal screen, the concentration of the pieces in the suspension was adjusted to 20% by volume. A rabbit weighing about 2.5 kg was inoculated intramuscularly with about 0.5 ml of the suspension at the femur region. Just after the inoculation (0), and 5, 9, 12, 16, 20 and 24 days after the inoculation, a 2 ml blood sample was taken from the ear vein. The blood serum was separated from each blood sample just after sampling, frozen and stored at - 20"C.

Prior to assay the serum is allowed to thaw.

b) Test Procedure: The reconstituted test sample (50 1ll) was placed in a test tube, and buffer solution (1.0 ml) and 3H-3'-dephosphocoenzyme A solution (50 ILl) were added to the test tube and mixed thoroughly. The mixture was treated as in Example 3 b) and the level of radioactivity was measured.

c) Results: The results are shown in Table 2 below.

Table 2 Period after Inoculation (days) Measurements (cpm) 0 525 5 600 9 823 12 1572 16 2713 20 3010 24 4375 As is clear from Table 2, the amount of the B-protein-labeled 3'-dephosphocoenzyme A complex was increased time-dependently after the inoculation of the tumor.

Example 5 Assays of Example 2 b) were repeated with the blood serum samples taken from 30 persons; 10 persons were diagnosed as having cancer, 10 persons were diagnosed as having a nonmalignant disease and 10 person were diagnosed as having no disease.

The results are shown in Table 3 below.

3900 Table 3 Type of persons Persons having Assay no disease non-cancer patients Cancer patient No. (cpm) (cpm: name of disease) (cpm) 1 395 316 (internal hemorrhoids) 640 (stomach) 2 540 551 (hernia) 904 (stomach) 3 400 71 9 (gallstone disease) 982 (esophagus) 4 485 575 (gastric ulcer) 2107 (breast) 5 431 532 (pancreatitis) 1699 (rectum) 6 520 565 (gallstone disease) 1 546 (sigmoid) 7 450 626 (appendicitis) 736 (stomach) 8 600 410 (mastopathy) 1 389 (large intestine) 9 460 543 (liver cirrhosis) 2593 (stomach) 10 445 386 (goiter) 590 (stomach) As is clear from Table 3, the measurements in terms of CPM on the persons having no disease and having a non-malignant disease feli in the range from 400 and 600 at a probability higher than 80%.Assuming that persons showing a measurement in cpm less than 400 have no cancer either, and that persons showing a measurement is cpm larger than 700 are cancer patients, it is possible to distinguish persons having cancer from those not having cancer with a probability higher than 80%.

Example 6 Assays of Example 4 b) were repeated except that the blood serum samples were taken from 30 persons. Ten persons were diagnosed as having cancer, ten other persons were diagnosed as having a non-malignant disease and the remaining ten persons were disagnosed as having no disease.

The results are shown in Table 4 below.

Table 4 Type of persons Persons having Assay no disease Non-cancer patients Cancer patient No. (cpm) (cpm: name of disease) (cpm) 1 395 590 (gastric ulcer) 904 (stomach) 2 547 491 (gastric ulcer) 467 (stomach) 3 437 350 (gallstone disease) 1471 (stomach) 4 485 719 (gallstone disease) 561 (stomach) 5 460 316 (internal hemorrhoids) 2320 (stomach) 6 587 410 (mastopathy) 810 (large intestine) 7 678 492 (intestinal obstruction) 1443 (rectum) 8 447 626 (appendicitis) 982 (esphagus) 9 436 532 (pancreatitis) 1477 (breast) 10 534 376 (hernia) 1140 (uterus) As is clear from Table 4, the measurements in terms of cpm on the persons having no disease and having a non-malignant disease fell in the range from 400 to 600 will a probability higher than 80%. Assuming that persons showing a measurement in cpm larger than 790 have cancer and that persons showing a measurement in cpm less than 400 do not have cancer either, the method of this invention can distinguish persons having cancer from those not having cancer with a probability higher than 80%.

Claims (16)

1. A method for detecting cancers in human which comprises adding a labeled coenzyme A or labeled 3'-dephosphocoenzyme A; adenosine triphosphate or its salt; and a coenzyme Asynthesizing protein complex to a predetermined amount of human blood serum to react the blood serum with the labeled coenzyme A or the labeled 3'-dephosphocoenzyme A, partially denaturing thermally and by use of a chemical denaturant the resulting B-protein-labeled coenzyme A complex or B-protein-labeled 3'-dephosphocoenzyme A, and determining the amount of the partially denatured complex.

2. A method according to Claim 1 wherein one ml of buffer containing 0.025-2,500 IsM of the labeled coenzyme A or the labeled 3'-dephosphocoenzyme A; adenosine triphosphate or its salt in an amount such that the concentration is 0.01-10 mM in a reaction mixture of the test sample and the reactants; and 0.01-0.1 ml of the coenzyme A-synthesizing protein complex; are added per 50 /ll of the blood serum.

3. A method according to Claim 2 wherein the concentration of the labelled coenzyme A or the labeled 3'-dephosphocoenzyme A ranges from 0.25-5 ILM; the concentration of adenosine triphosphate or its salt ranges from 1.5-5 mM; and the amount of the coenzyme A-synthesizing protein complex ranges from 0.04-0.06 ml are added per 50 yl of the blood serum.

4. A method according to Claim 1 wherein the reaction between the blood serum and the labeled coenzyme A or the labeled 3'-dephosphocoenzyme A is effected by allowing the mixture to stand at 30-38"C for 3-60 minutes.

5. A method according to Claim 4 wherein the reaction is effected at a pH of from 6 to 8.

6. A method according to Claim 1 wherein the partial denaturation is effected by first heating the reaction mixture at 65-100"C for 1-10 minutes and by treating with a chemical denaturant.

7. A method according to Claim 6 wherein the heating is effected at 68-70"C for 3-6 minutes.

8. A method according to Claim 6 wherein the chemical denaturant is selected from the group consisting of trichloroacetic acid, methanol, and ethanol.

9. A method according to Claim 1 wherein the labeled coenzyme A or the labeled 3' dephosphocoenzyme A is in the radioactive form with 3H, 14C or 35S.

1 0. A method according to Claim 1 wherein the labeled coenzyme A or the labeled 3' dephosphocoenzyme A is in the fluorescent form with fluorescein or coumarin.

11. A method according to Claim 1 wherein the determination of the amount of the B protein-labeled coenzyme A or the B-protein-labeled 3'-dephosphocoenzyme A is effected by measuring the radioactivity level or the fluorescence intensity.

1 2. A reagent for detecting cancer in humans which comprises a labeled coenzyme A or labeled 3'-dephosphocoenzyme A; adenosine triphosphate or a salt thereof; and a coenzyme Asynthesizing protein complex, said reagent being a mixture of all said compounds or being a kit of elements each of which contains at least one compound.

1 3. A reagent in the form of a kit according to Claim 12, wherein said kit comprises two ~ elements, one of which contains the labeled coenzyme A or the labeled 3'-dephosphocoenzyme A, and adenosine triphosphate or its salt, and the other of which contains the coenzyme Asynthesizing protein complex.

1 4. A reagent in the form of a kit according to Claim 12, wherein said kit contains three elements which contain three different compounds, the labeled coenzyme A or the labeled 3'dephosphocoenzyme A, adenosine triphosphate oar its salt, and the coenzyme A-synthesizing protein complex, respectively.

1 5. A reagent according to Claim 1 3 or 14 wherein said kit further contains an element containing buffer solution or a chemical denaturant.

16. A reagent according to Claim 13 or 14, wherein said compounds contained in the elements are in the lyophilized form.

1 7. A reagent according to Claim 1 3 or 14 wherein said compounds contained in the elements are in the form of solution.

1 8. A method according to claim 1, substantially as described in the foregoing Examples.

1 9. A reagent according to claim 12, substantially as described in the foregoing Examples.