EP0243448A1

EP0243448A1 - Test for intestinal polyps

Info

Publication number: EP0243448A1
Application number: EP86906566A
Authority: EP
Inventors: James X. Hartmann; Henry C. Lyko
Original assignee: Florida Atlantic University; University of Florida
Current assignee: Florida Atlantic University; University of Florida
Priority date: 1985-10-09
Filing date: 1986-10-07
Publication date: 1987-11-04
Also published as: AU6521686A; EP0243448A4; WO1987002463A1

Abstract

Une protéinase antigènique pouvant être isolée du plasma humain est utilisée comme test pour detecter la présence de polypes. Des anticorps à l'antigène sont produits et utilisés dans des tests physiologiques.An antigenic proteinase that can be isolated from human plasma is used as a test to detect the presence of polyps. Antibodies to the antigen are produced and used in physiological tests.

Description

TEST FOR INTESTINAL POLYPS

RELATED APPLICATIONS

This application is a continuation in part of copending application serial number 785,913 filed October 9, 1985.

FIELD OF THE INVENTION

This invention relates to an antigenic proteinase present in human plasma and indicative of the presence of polyps which may be benign, precancerous or approaching carcinoma in situ. It relates also to the use of the proteinase as a test for the presence of polyps which may signal the presence of adenomatosis of the colon and rectum (ACR) or a predisposition to colorectal cancer (CRC) in humans.

BACKGROUND OF. THE INVENTION

Cancer of the colon, or large bowel, is one of the leading deadly cancers among Americans. The American Cancer Society estimated that in 1985, a total of 96,000 new cases would be diagnosed and that 51,600 people would die of the disease.

CRC is highly curable when detected early, and recent studies suggest it may even be preventable by adjustments in diet. It is apparent, therefore, that a test which would both be easy to apply and give early warning of the presence of CRC or a predisposition to CRC in humans would be a valuable addition to the physician's armamentarium. CRC typically arises in an intestinal polyp, a type of benign tumor that may ay flat against the wall of the large intestine or may extend from a stalk like a mushroom. It is advisable, particularly with people with a family history of

CRC to submit to regular examinations for the presence of the polyps or active cancer.

In one type of examination for the presence of polyps and, therefore, the possibility of cancer, an endoscope is inserted rectally to reveal part or all of the colon. The endoscope is a long, flexible, narrow tubing carrying a light. It allows the entire colon to be viewed and any polyps located. It is equipped with forceps which can remove a sample for biopsy or with a wire snare which can encircle the base of the polyp to remove it by the application of fulgurating current.

This type of invasive diagnosis and treatment is obviously not suitable for screening large numbers of potential CRC victims.

A simpler procedure is based upon tests .of the stools of potential victims for the presence of blood. The principal difficulty with this procedure is that the cancer may already be well advanced before blood appears in the stool. Accordingly, the information, derived from the test may be too late to be useful.

ACR is a known, human hereditary disorder, clinically known as familial polyposis coli and Gardner's syndrome. It is an autosomal dominant disorder that invariably progresses to cancer by midlife. The phenotype is expressed in stages. ACR gene carriers are born with a mucosa void of neoplasia. The mucosa is progressively replaced by hundreds of adenomas until malignant transformation of one or more of the polyps occurs. This is the ultimate and invariable fate of the affected individuals.

It is apparent, then, that a non-invasive screening-test

*5 which would positively identify ACR victims and could be used to determine the presence of polyps in the general population would be invaluable as ^"an early warning of potential cancer since it would identify individuals with a predisposition to cancer who should be examined 10 endoscopically. Such a test has now been found.

We have earlier detected in the plasma of ACR victims with neoplasia in the form of benign polyps a factor which will transform the normal morphology of normal human colon fibroblasts in culture. See Lyko and Hartmann, Eucaryotic 15 Cell Cultures, Plenum Publishing Corp., 1984, pages 471 to 488.

We have now isolated and identified that factor. We .have been able to utilize the factor as a diagnostic test for polyps in humans and to .identify humans with ACR who are

20 therefore susceptible to CRC. It should be mentioned that, probably due to differences in dietary habits, ACR individuals develop cancer at widely different ages. It presently appears that only those who develop the polyps will also eventually develop colon cancer as an invariable

25 sequelae. It is important, therefore to detect the presence of polyps at the earliest possible stagen.

Polyps also precede the development of cancer in cancer not associated with ACR. The stages of development of such non-hereditary cancer are similar to the development in ACR -,3 individuals. Many fewer polyps are present in the colon of patients who present with non-hereditary cancer. THE INVENTION

An antigenic proteinase has now been discovered in the plasma of humans afflicted with ACR. It has been isolated and found to have the following characteristics:

1. isolatable from the plasma of a human carrying the ACR gene.

2. a proteinase with an optimum activity temperature of about 5° to 10°.

3. an optimum pH of about 4.2.

4. an isoelectric point of 6.2 to 6.4 as determined by isoelectric focusing in a pH gradient of 3 to 10.

5. . a molecular weight of from 10,000 to 30,000 as determined by fractional filtration.

6. stainable by Coomassie Blue.

7. proteolytically active in the presence of phenylmethyl sulfonyl fluoride, benzamidine and epsilon aminocaproic acid.

8. induces a transformation associated morphology in normal cells.

9. causes retraction of the cell processes, cell rescinding and eventual detachment from a smooth surface such as glass or plastic. It has also been determined that the proteinase is labile to alternate thawing and freezing. It should be frozen at -70°C immediately after isolation and should be used promptly after the first thawing.

-⁵ This isolated proteinase is useful because its presence in human plasma indicates the presence of polyps. It is useful also because it is antigenic and can be employed to produce antibodies.

For convenience the balance of this application will be 1₀ divided in,to the following sections:

1. Isolation and characterization.

2. Preparation of antibodies.

3. Diagnostic use.

4. Diagnostic kits.

₁5 5. Removal of cells from surfaces.

ISOLATION AND CHARACTERIZATION

I. Sample Preparation

A. Separation of Plasma

Blood samples were centrifuged in a Drucker Model L708 20 centrifuge at 15 rpm for ten minutes. Plasma was then pipetted into a separate sterile tube and frozen at -70 C until just prior to its use.

B. Detection of Protease Activity

Bio-Rad casein plates (Bio-Rad, Inc. California) were ^'"25 prepared by dissolving two agar-casein tablets in an 18x150 mm test tube by addition of 10 ml of distilled water. The tablets were alloed to hydrate and dissolve at room temperature for 15 minutes. The solution was vortexed and then placed in a boiling water bath for four minutes. The melted agar-casein mixture was poured into a plastic plate in a leveling table, while avoiding air bubbles. The agar- casein plate was allowed to cool and_.then six 9 mm diameter sample wells were formed. 50 μl of plasma were placed in each well. Protease activity was usually observed within 20 minutes to two hours. The plates were kept at room temperature in a humidity chamber.

C. Desalting the Plasma Samples

Using Pharmacia Columns PD-10 (Pharmacia, Piscataway, New Jersey) containing Sephadex G-25M, 2 ml- of a plasma sample was allowed to flow through the equilibrated column followed by 6.5 ml of H-O. 0.5 ml fractions were collected and 50 ul of each fraction was analyzed on an agar-casein plate. The protease of the invention was found in fractions 4 and 5. Sephadex G-25M retards the passage of salts and materials with a molecular weight of 25,000 or less. Since the protein was retarded by the column, the molecular weight by this method is probably less than 25,000.

D. Analytical Isoelectric Focusing of Plasma Samples

The desalted plasma samples and standards with known isoelectric points were separated on LKB Ampholine polyacrylamide gel (PAGE) plates with a pH gradient of 3 to 10. The electrode solution used was 0.4M HEPES (N-2- hydroxyethylpiperazine-N'-2-ethane sulfonic acid) and the cathode electrode solution used was 0.1 M NaOH. The cooling plate was maintained at 5 C. and coated with kerosene. A template supplied with the PAGE plates was laid on the cooling plate, avoiding air bubbles. The template was also provided with a coat of kerosene and the PAGE plate was placed on top with the thin plastic sheet downward. The anode and cathode electrode strips were appropriately saturated and were properly positioned as indicated on the

5 template. The plate was allowed to cool down for 15 minutes before applying the samples. Samples were applied on one half of the gel by dipping small pieces of sample application paper into the vials containing the plasma and laying these application pieces on the gel in several

10 different positions but avoiding the edges of the gel. Each sample application piece hald approximately 15_jul of sample. One half of the gel is a direct duplicate of the other half. The electrocusing apparatus was then assembled and the power supply turned on. The power was 30 watts, the voltage was

I⁵ 1500 volts, and the current was the maximum. The samples were focused for a total of 2.5 hours. However, after 30 minutes of running time, the apparatus was turned off and the sample application pieces removed. Isoelectric focusing was them resumed.

20 E. Ph of the Gel

The pH gradient of the gel ws checked immediately after the isoelectric focusing run. The pH meter was calibrated using standards kept at 5 C. After all voltage settings were turned off, a pH surface electrode was applied to the 5 surface of the gel. The pH was determined at every centimeter along the top and bottom of the gel, starting with the cathode side. A series of pH readings were also taken accross the middle of the gel close to where the active samples were applied. The isoelectric focusing unit 30 was turned on again for an additional 15 minutes to refocus any proteins that may have become mobile. F. Zymogram

After the plasma samples were separated by isoelectric focusing, one half of the gel (duplicate half) was removed and placed in a tris-barbituate buffer of pH 8;6, -for twelve minutes to elute the ampholytes. Before this time, an agar-casein gel using Bio-Rad casein tablets was poured to measure 12.5x12.7 centimeters. The gel was removed from the buffer and placed top side down onto the casein plate. The zymogram was kept moist by placing it in a humidity chamber. The zymogram was checked at 24 hours to detect the bank of hydrolysis. The isoelectric point by this procedure was found to be 6.2 to 6.4. No hydrolysis band was detected when control plasma samples were separated by isoelectric focussing.

G. Staining the Gel

The other half of the gel was immediately placed in a fixing and staining solution that consisted of 1.0 g of Coomasie Blue G-250 in 1.0 L of destaining solution 1. Destaining solution 1 consisted of 1.0 L methanol with 200 ml of glacial acetic acid and 800 ml of distilled water. The gel was fixed and stained for two hours. - The gel was then placed in destaining solution 1 for 1.5 hours. The gel was finally placed in destaining solution 2 which consisted of 50 ml of methanol with 70 ml of glacial acetic acid. 0.05 g of Coomassie Blue G-250 was added and the solution was diluted to 1.0 L with distilled water. The gel was left in this solution until an appropriate background clarity was achieved, usually 24 hours. By this means, an area corresponding to the zone of hydrolysis on the unstained plate can be localized on a stained pattern. The protease may be isolated from the gel by elution into tris-barbiturate buffer at pH 7.2, or other appropriate buffer, dialysis against distilled de-mineralized water and lyophilization.

A pure, lyophilized proteinase is convenient to obtain for structural or analytical studies. However, for most purposes, the proteinase can be employed in the gel, in the buffer or in the dialysate.

H. Fractionation by filtration

Diaflo untrafiltration membranes (Amicon Corporation,

Lexington, Mass.) were used in a graded series to determine the molecular weight of the protease. The filters were placed in an M-3 ultrafiltration cell (Amicon). After

2 addition of plasma, 35 lbs/in of nitrogen gas was applied. Both the filtrate and retentate were tested for activity by placing 50 ul of each onto an agar-casein plate. The following filters were used: XM300 (3000,000 m.w.), SM100A (100,000), XM30 (30,000) and XM10 (10,000). The proteinase activity passed through a 30,000 m.w. filter but was retained on the 10,000 m.w. filter. From this it can be concluded that the molecular weight is between 10,000 to 30,000. The determination of molecular weight by SDS polyacrylamide gel electrophoresis was not possible due to loss of enzyme activity in the presence of detergent, urea and reducing agent required for this process.

I. Induction of transformation associated morphology of normal cells

When normal human colon or epidermal skin fibroblasts were treated with ACR plasma containing active proteinase (10-100 CTA units/ml as compared to standard plasma), they assumed a transformation associated morphology. Both 24 hours and 7 day old monolayer cultures responded similarly to ACR plasma with proteinase activity.

A total of 3 x 10 cells/0.5 of culture medium were seeded into 16 mm sterile wells in a 24 multiwell plate

(Corning No. 35425). The cells were cultivated in Minimum Essential Medium (MEM) containing 10% fetal bovine serum, penicillin, streptomycin and fungizone. To test the response to ACR and control plasma, 80 μl of plasma were added directly to the well containing cells and medium. When ACR plasma containing proteinase was added to the cells, cellular proesses began retracting within one minute. Within seven minutes the cells became spherical, began piling up on the vessel durface and agglutinated. Control plasma from healthy subjects did not induce these changes.

With continued cultivation of treated cells for 24 hours the moprphological effects were reversed.

By the above procedures the presence of the proteinase in the plasma has been established as an indication of the presence of polyps in the intestinal tract and a predisposition to CRC. Any individual testing positive for the presence of the proteinase should be examined endoscopically.

J. Determination of optimum temperature for proteolytic activity

The optimum temperature for proteolytic activity of the newly discovered proteinase in the whole plasma of polyposis patients, and of normal patients, for comparison, was determined. This was achieved by applying the plasma samples under test to punches measuring 2.2 mm diameter in

casein agar plates prepared as described above and measuring the area of lysis after selected periods from 0.5 to 2 hours incubation at the selected temperature.

All samples were incubated under identical conditions. Each sample was tested in quadruplicate. The average value of the three closest diameters of the lysis zones as measured in mm by a σaliper was taken as the measure of proteolytic activity.

It was found over a temperature range of about 5 to 60 C that the plasma of normal patients had little or no proteolytic activity and that the optimum temperature for proteolytic activity for the plasma of polyposis patients containing the proteinase of this invention was 5 to 10 C.

K. Determination of optimum pH for proteolytic activity

The optimum pH Of the proteolytic activity of the newly discovered proteinase in the whole plasma of polyposis patients and normal patients, for comparison, was determined. The determination was made at room temperature for convenience since the value would be expected to be substantially the same at the optimum lower temmperature.

Each plasma sample was aliquoted in 50 μl per Eppendorf microcentrifuge tube. Each aliquot was diluted 1:12 with distilled water and the desired pH for each diluted aliquot was adjusted by adding 0.001N NaOH or 0.0025N HCl as needed. All aliquots were dried by a speed vacuum centrifugation

(Savant, Farmingdale, New York) attached to a lyophilizer. Each dry sample pellet thus produced was resuspended in 50 μl of distilled water having a pH of 7.0. The pH of each sample was retested by surface electrode and the samples centrifuged at 12,000 rpm in the Eppendorf apparatus. The supernatants were tested in triplicate on casein agar plates prepared as described in the optimum temperature determination. The lysis zone diameters were similarly measured.

It was found that the optimum pH was about 4.2.

L. Determination of proteolytic activity in the presence of inhibitors.

Proteinase activity and inhibition were tested using casein agar plates. The casein plates were made by dissolving 1 casein substrate tablet in 5 ml of water and hydrating for 10 minutes. The tubes containing the dissolved casein were then placed in boiling water for 4 minutes. The hot ^"casein agar was poured into a small petri plate and allowed to cool.

The inhibitors tested were phenylmethyl sulfonyl flouride (PMSF), benzamidine, and epsilon amino-n caproic acid. The materials are known to inhibit the proteolytic activity of many proteinases.

Each inhibitor was added while the casein was cooling in the test tube. The inhibitor was added when the temperature of the casein reached 45 C. Then the test tubes were vortexed and poured into their respective petri dishes. One petri dish was used per inhibitor. The following amounts of inhibitor were added: 20 mg caproic acid, 20 mg benzamidine to achieve a 4 mg/ml inhibitor concentration throughout the casein agar. The PMSF was diluted- with tris. buffer, to_. obtain a .2mM concentration. Then 5 microliters of this stock was placed in the 5ml casein to obtain a 2 uM concentration PMSF in the casein. 5 jul of serum from ACR patients was added to wells in each petri dish. It was found that the proteinase of this invention retained its proteoytic activity in the presence of these inhibitors.

PREPARATION OF ANTIBODIES

A. POLYCLONAL ANTIBODIES

Polyclonal antibodies to the proteinase of this invention can be prepared by at least two methods.

In one such method, the area in the polyacrylamide gel that contains the isoelectric focused proteinase is cut out with a scalpel. This area is them further sliced into 2 mm wide segments using a Bio-Rad model 195 electric gel slicer (Biorad laboratories, Richmond, CA. ) The gel containing the proteinase as immunizing antigen (1-2 mg. protein per gel segment) is injected subcutaneously .in a mouse or a rabbit (25 mg. ) using a 20 gauge needle and a 1cc. syringe. The injection is repeated every fourth day for a total of four injections. Blood is drawn via cardiac puncture from the anesthetized animal. The blood is allowed to clot for 1-2 hr at room temperature and the serum removed, centrifuged at 20,000 rpm for clarification, and stored at -70 C until use. This serum contains the anti-proteinase antibody of the invention together with other antibodies which may be produced or naturally present due to previous exposure of the animal to other antigens. The procedure affords a quicker means to produce antibody, but it lacks the specificity afforded by monoclonal antibodies. In a second method, a sufficient amount of whole plasma was mixed in 0.14N NaCl at a concentration of 2 mg/ml as the aqueous phase and emulsified with an equal volume of

Complete Freund's Adjuvant. The final antigen concentration was 2 mg/ml of adjuvant emulsion.

Antibodies against each antigen preparation (normal individual and patient plasma) were raised in four large female New Zealand White (NZW) rabbits (two rabbits per antigen preparation) . Rabbits were bled two days before immunization and the sera were collected, aliquoted and stored at -70 C to be used as controls. Antigen-adjuvant emulsion (1 ml) was initially administered intramuscularly (I.M.) deep in the quadriceps muscle of the animal with a 21-G needle for -a total dose of 2 mg of plasma proteins. At the end of the 3rd week a similar injection was administered. At the end of the 6th week and .weekly thereafter, animals were bled from the central ear artery. Immediately after bleeding, rabbits received an intravenous (I.V. marginal ear vein) injection of 350 ug of plasma proteins in 0.1 ml of saline. Blood was allowed to clot at 37°C for 1 hr. (the clot was loosened from the tube to aid retraction,) then stored at 4 C overnight, or until the serum is expressed. The antisera were stoired at -70 C in aliquots of 0.1 ml.

The evaluation of the animal response was assessed by the agerose gel immunodiffusion of Ouchterlony plates.

Gels were prepared by dissolving 2% agarose-EF (LKB Instruments, Inc.) in 2-fold dilution of the barbiton buffer, pH 8.2. The gels were cast on 8 x 10 cm plates and allowed to solidify at room temperature before cutting the appropriate wells with a punch. Samples of the whole plasma were tested against the collected rabbit anti-human plasma sera. The maximum number of precipition arcs in immunodiffusion plates was observed after 8-10 weeks of immunization. All rabbits showed consistency in antibody production. No immunoprecipition arcs were observed when normal rabbit sera were tested against human plasma.

B. MONOCLONAL ANTIBODIES

The proteinase of this invention can be employed to produce monoclonal antibodies to itself utilizing the procedure described by Fazekus et al: J. Immunol. Methods 35:1, 1980. The essentials of the procedure are as follows:

1. Immunize an animal, preferably a rodent such as a rat or mouse with the proteinase.

2. Isolate B-lymphocytes, suitable spleen lymphocytes, from the immunized animal.

3. Fuse the isolated B-lymphodyctes with myeloma cells from an animal, preferably a rodent such as a rat or mouse.

4. Select from the fused cells those hybridoma cell lines which react positively with the proteinase.

5. Clone the hybridoma cells to produce additional monoclonal antibody.

Procedures for performing each of these steps are well known to those skilled in the art who will also know the necessary reagents and how to prepare or obtain them.

To produce a much greater concentration of less pure antibody, the selected hybridoma may be injected into mice, preferably syngenic or semi-syngenic mice. The hybridoma will cause formation of antibody producing tumors in the mice after a suitable incubation time, which will result in high concentratiopn of the desired antibody (about 5-20 mg/ml) in the blood stream and peritoneal exudates (ascites) of the host mouse. Although the host mice also have normal antibodies in their blood and ascites, the concentration of these normal antibodies is only about 5% of the monoclonal antibody concentration. Moreover, since these normal antibodies are not anti-human B-cell -in their specificity, the monoclonal antibody obtained from the harvested ascites or from the serum is essentially free of contaminating antibodies.

The antibodies of this invention are characterized as antibodies to a proteinase which is identifiable by the following characteristics:

1. isolatable from the plasma of a human carrying the ACR gene. ^■

2. a proteinase with an optimum activity temperature of about 5° to 10°.

3. an optimum pH of. about 4.2.

5. a molecular weight of from 10,000 to 30,000 as determined by fractional filtration. 6. stainable by Coomassie Blue.

8. induces a transformation associated morphology in normal cells.

9. causes retraction of the cell processes, cell rescinding and eventual detachment from a smooth surface such as glass or plastic.

DIAGNOSTIC USE

As will be apparent to those skilled in the art, one of the most important medical uses for the proteinase of this invention is for the production of antibodies to itself. These, in turn can be employed to detect polyps in those individuals who are at risk for the occurrence of CRC. For these diagnostic purposes, the polyvalent or monoclonal antibody will react with the proteinase factor from the plasma of the individual under test to produce, in the case of positive individuals, a detectable product. An antibody composition used in any test designed to determine the presence of the proteinase, which for this purpose may be considered an antigen, must contain sufficient antibody to react with the proteinase, to produce a detectable product. Such diagnostically effective amounts of antibody will vary appreciably with a number of factors well knwon to those skilled in the art. These include, for example, the sensitivity and specificity of the test employed, the instrumentation available and the amount of sample under test. Any of a large number of clinical tests may be employed utilizing the antibodies of this invention. Typical tests include radioimmunoassay, enzyme linked immunoassay, precipitation, agglutination, direct and indirect immunoflourescence, and complement fixation. These tests . may employ competitive and sandwich type assays.

The tests may employ detectable labels. The proteinase (antigen), the antibody, or an antibody such as goat anti- rabbit serum which may be labeled. Useful labels include flourescent labels such as fluorescein, rhodamine or auramme. Rr.ad_i-o-i•osot-opes suchv, as 14C-, 131_τI, 125_τI and^, 35S_ may also .be employed. Enzyme labels which may be utilized include, for example, horse radish peroxidase, - -O- glucosidase, galactosidase, urease, glucose oxidase plus perixodase and acid phosphatase.

Methods for labeling biological products such as cells, antibodies, antigens and antisera are well known and need not be described.

There are several currently available procedures for detecting these labels including, for example, colorimetric, spectrophotometric, fluorospectrophotometric and gasometric techniques, as well as various instrumental methods of detecting isotopes.

All of the tests which may be usefully employed in accordance with this invention involve the formation of a detectable reaction product which includes an antibody of the invention and the proteinase. Of course there may be other components such as an anti-antibody in the detectable reaction product.

DIAGNOSTIC KITS

A wide variety of test kits are possible to take advantage of the advances in the diagnostic arts made possible by this invention. Some will be described here. Others can be devised by those skilled in.the art.

A typical test kit for use with Enzume Linked Immunoabsorbent Assay (ELISA) or Radioimmunoassay (RIA) tests will contain:

1. Plate with absorbed antibody.

2. Labeled Protein A, or labeled goat or sheep anti- rabbit proteinase.

The kit may also contain appropriate buffers such as phosphate buffered saline (PBS) containing 1% to 3% bovine serum albumin (BSA) at pH 7.2 and appropriate proteinase positive and negative controls. These materials may be provided with the kit or may be separately provided or prepared..

The term "plate" is used in the broad sense to include any flat surface such as glass or polymer.

In practice, such a kit would be employed as follows:

1. Incubate the plate with the plasma of the patient under test for an appropriate time and temperature, e.g. from 2 to 4 hours at 37 C. 2. Wash with PBS^*BSA.

3. Incubate with anti-proteinase antibody.

4. - Incubate with Protein A or sheep or goat anti-rabbit proteinase which has been tagged with a detectable label and wash with the same buffer.

- 5. Detect the formation of a reaction product in the case of a positive test by detecting the label by any of the procedures described above.

REMOVAL OF CELLS FROM A SURFACE

in the growth of cells it is customary to form a monolayer of the cells on a surface, such as the surface of a glass plate. The surface is bathed with nutrient media and the cells multiply. To obtain most efficient growth it is desirable to remove samples of the cells from the original surface and to utilize the samples as seed cells on new surfaces. It has proved difficult to remove cells from a surface without injuring them, despite extensive investigations. The present practice is to utilize trypsin. This enzyme diminishes the attraction between the cell and the surface so that the cells can be washed off.

It has been found that the proteinase of this invention can be used in place of the trypsin. The proteinase is advantageous in that it fuctions in the presence of bovine serum and escess calcium wherease trypsin does not. Normally, cell monolayers must be washed at least three times in a buffer devoid of calcium ions and bovine serum in order for trypsin to be effective in detaching the cells. The ACR proteinase can be added directly to an established momolayer and the cells will detach within a short period of time, typically 5 to 10 minutes without any additional treatment.

Claims

WHAT IS CLAIMED IS

1. An antigenic proteinase useful for the detection of intestinal polyps in humans and identifiable by the following characteristics:

1. isolatable from the plasma of a human carrying the ACR gene.

2. a proteinase with an optimum activity temperature of about 5° to 10°.

3. an optimum pH of about 4.2.

5. a molecular weight of from 10,000 to 30,000 as determined by fractional filtration.

6. stainable by Coomassie Blue.

8. induces a transformation associated morphology in normal cells.

9. causes retraction of the cell processes, cell rescinding and eventual detachment from a smooth surface such as glass or plastic. 2. An antibody which is useful for the detection of intestinal polyps in humans and is an antibody to an antigenic proteinase, said proteinase being identifiable by the following characteristics:

1. isolatable from the plasma of a human carrying the ACR gene.

2. a proteinase with an optimum activity temperature of about 5° to 10°.

3. an optimum pH of about 4.2.

6. stainable by Coomassie Blue.

8. induces a transformation associated morphology in normal cells.

9. causes retraction of the cell processes, cell rescinding and eventual detachment from a smooth surface such as glass or plastic. 3. A composition containing a diagnostically .effective amount of an antibody useful for the detection of intestinal polyps in humans and is an antibody to an antigenic proteinase, said proteinase being identifiable by the following characteristics:

1. isolatable from the plasma of a human carrying the ACR gene.

2. a proteinase with an optimum activity temperature of about 5° to 10°.

3. an optimum pH of about 4.2.

6. stainable by Coomassie Blue.

8. induces a transformation associated morphology in normal cells.

9. causes retraction of the cell processes, cell rescinding and eventual detachment from a smooth surface .. such as glass or plastic. 4. A method of detecting intestinal polyps in humans which comprises incubating plasma from the patient under test with a diagnostically effective amount of an antibody at a time and temperature sufficient to effect reaction between an antigenic proteinase in the plasma and an antibody to the proteinase and, in the case of a positive test detecting the presence of a reaction product between the proteinase and the antibody; said proteinase being identifiable by the following characteristics:

1. isolatable from the plasma of a human carrying the ACR gene.

2. a proteinase with an optimum activity temperature of about 5° to 10°.

3. an optimum pH of about 4.2.

_ 4. an isoelectric point of 6.2 to 6.4 as determined by isoelectric focusing in a pH gradient of 3 to 10.

6. stainable by Coomassie Blue.

0 7. proteolytically active in the presence of phenylmethyl sulfonyl fluoride, benzamidine and epsilon aminocaproic acid.

8. induces -transformation associated morphology in normal cells. 9. causes retraction of the cell processes, cell rescinding and eventual detachment from a smooth surface such as glass or plastic.

5. A test kit for detecting the presence of intestinal polyps in humans containing a diagnostically effective amount of an antibody to an antigenic proteinase, said proteinase being iidentifiable by the following characteristics:

1. isolatable from the plasma of a human carrying the ACR gene.

2. a proteinase with an optimum activity temperature of about 5° to 10°.

3. an optimum pH of about 4.2.

4. an isoelectric point of 6.2 to 6.4 as determined-by isoelectric focusing in a pH gradient of 3 to 10.

6. stainable by Coomassie Blue.

8. induces a transformation associated morphology in normal cells.