NL8002094A - DIAGNOSTIC METHOD. - Google Patents

Description

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Title: Diagnostic method.

The invention relates to a diagnostic test method for detecting malignant neoplasms.

There is a clear need for new methods of detecting cancer. In many cases, an early diagnosis of cancer would greatly increase the chances of a complete reduction of the disease.

Various attempts have been described in the literature to demonstrate the presence of tumor-specific components, such as hormones and anti-genes, in the blood of cancer patients. However, such attempts have been largely unsuccessful and to date, a practical non-intrusive diagnostic procedure based on the content of a tumor-specific serum component has remained an elusive target.

Recently, DM-binding proteins have been found in serum from patients with neoplasic, systemic lupus erythematosus and other inflammatory disorders (see e.g. FEBS Letters 92_ (2): 211-213 (19T8); · <·; -

Europ. J. Biochem. 71: 1-8 (1976); Amer. J. Med. 6: 5: 37- 5 (1978)).

However, this does not provide a suitable analysis procedure for such serum proteins, which procedure would be necessary for a practical diagnostic method. In addition, the previously described serum proteins do not appear to demonstrate the degree of selectivity desired in a cancer research method. .

The inhibitor serum protein, which according to the. The invention is described in The Pharmacologists (Abs.) 20 (3): 238 (1978). However, this brief summary does not indicate that the protein is present at different levels in patients with cancer than in patients without cancer.

The invention provides a generally applicable diagnostic test method for detecting malignant neoplasms in humans. More specifically, the invention provides a diagnostic method to demonstrate the existence of malignant neoplasms by analyzing human serum samples for the presence of a specific DHA binding protein. The method can be used to detect malignancies at an early stage and as indi-35. cator for progression of the condition in a cancer patient 8 0 0 2 0 04 -2- undergoing treatment.

The method of the invention is based on the finding that a particular DM-binding protein (hereinafter referred to as inhibitor protein X) is present in significantly lower concentrations in a serum of patients with cancer than that of patients without cancer. By providing a sensitive analysis for this serum level, the invention allows a rapid, non-intrusive and accurate method for detecting the presence of cancer.

The presence of a particular DNA binding protein in human serum was discovered by the inventors during a study of the blecmycin-induced degradation of IM-2 DM '- (Pseudomonas bacteriophage covalently closed circular DM). Observation of the inhibition of this degradation by human serum led to an examination of the nature of the inhibition and attempts to identify the inhibitor compound.

The inventors' hypothesis that the inhibition of the Bi-2 DM degradation reaction was caused by a serum protein was confirmed by pronase treatment leading to loss of the inhibitory activity. Treatment with DNAase I or pancreatic RNAase did not affect the inhibitory activity of the serum. Dog and calf sera were not inhibitory, nor were human albumin and immunoglobulins.

The inhibitor protein was purified under non-denaturing conditions using molecular filters, dialysis and Sephadex-25 column chromatography. Purification of the protein was significantly improved by binding the protein to DM, isolating the DNA protein complex from the unbound protein, and then separating the protein from the DM by treating with urea. Purification by SDS polyerylamine gel electrophoresis and isoelectric focusing is estimated to lead to more than 2000 fold purification. Isoelectric focusing gel electrophoresis shows that the inhibitor is a protein with a molecular weight of about 6Π.ΟΟΟ dalton and a pi of 5.9 *

This protein has been designated as the subject of research as inhibitor protein X.

The inhibitor protein X exerts its inhibitory activity by joining 800 DM with the DM. DNA binding has been demonstrated using agarose gel electrophoresis, DNA protein complex isolation, fluorescence quenching and circular dichroism studies.

When examining the nature of the inhibitor protein X, it was unexpectedly found that there was a significant difference in the content of this protein in the serum of patients with cancer and in the serum of patients without cancer. Based on this important finding, the inventors have sought a sensitive and rapid analysis of the presence of inhibitor protein X in human serum.

IM-2 DNA has previously been used in a spectrophotofluorometric analysis to determine bleomycin biochemical activity (Cancer Res. 38: 3322-3326 (1978)). The mechanism of action of blecmycin appears to be linked to its ability to degrade DNA, and the decrease in binding of ethidium branide (2,7-diamino-10-ethyl-9-15 phenyl-phenanthridinium bromide) to PM-2 DNA, induced by bleomycin, as determined by fluorescence spectrometry, can therefore be used to analyze blecmycin activity.

Inhibitor protein X, as noted above, has been shown to act as an inhibitor of blecmycin-induced degradation of IM-2 DNA.

Therefore, the fluorametric analysis method previously used to measure blecmycin activity can also be used to analyze the concentration of inhibitor protein X in human serum. The level of inhibitor protein X can then be used to predict the likelihood of cancer in the examined patient.

The invention therefore provides a method of detecting human cancer, comprising the following steps: a) taking a blood sample from a patient to be examined for cancer; b) determining the concentration of inhibitor protein X in this sample; and c) comparing this concentration, as determined in step b), with the inhibitor protein X concentration standard, which is associated with the serum of patients without cancer, significantly reducing the inhibitor protein X concentration relative to the concentration standard is an indication of the probability of cancer vomit.

The particular analysis procedure used in step b) to determine the serum inhibitor protein X concentration is not critical, and the invention is broadly based on the finding that the inhibitor protein X-5 concentration (however determined) can be used as a diagnostic tool for a wide variety of human cancers.

An advantageous analysis procedure is the EM-2 DM fluorescence analysis described in Cancer Res. J30: 3322-3326 (1976). The use of this analysis is based on the inhibitory effect of inhi-10 bitoreivit X on the known blecmycin-induced degradation of the PM-2 DM reaction. When this assay is used, the inhibo protein X ^ concentration is preferably expressed as an IC ^ value where IC ^ Q is defined as the concentration of the inhibitor protein X, which is required to obtain 50% inhibition of blecmycin. induced degradation of PM-2 DM. This IC ^ Q value can then be compared to the nominated IC ^ value, which is associated with the serum of patients who do not have cancer, and a significant increase in the IC ^ value from the nominated value will indicate the likelihood of cancer.

20 The details of the PM-2 DM fluorescence analysis on inhibitor egg X are as follows:

A mixture of a serum sample (eg, a 50 microliter sample) from a patient to be examined is first prepared with a solution of blecmycin, B4-2 DM and 2-mercaptoethanol in a buffer solution with a pH of 9 * 5 · A 50 microliter of serum sample is typically mixed with

k50 microliters of a solution containing 35 mM blecmycin, 8.3 µM EM-2 DNA

and 25 mM 2-mercaptoethanol in a sodium borate buffer with a pH of 9.5 (0.015 M NaCl: 0.05 M sodium borate).

The above mixture is then incubated at 37 ° C for 30 minutes.

A solution of ethidium brcmide in a denaturation buffer containing 3. .

a pH of 12.1 is then prepared by adding 0.1 cm of an ethidium

O

brcmide solution (22 µg ethidium brcmide per cm of the denaturation

"Oh

buffer with pH 12.1) to 0.9 cm of the denaturation buffer with a pH of 12.1. A suitable denaturing buffer contains 800 2 0 94 -5- 0.09 Μ Na-PO 2: 0.01 Μ EDTA: 0.01 Μ NaCl, adjusted to a pH of 12.1 3 ....

with 0.15 M NaOI. A portion (e.g. 100 microliters) of the incubated serum mixture mentioned above is added to 1 cm of this ethyl solution.

The fluorescence of the ethidium bramide: MM-2 DNA mixture is determined with a spectrophotofluorimeter at a 530 nm excitation and a 590 nm emission. Fluorescence emerging from the substrate is caused by ethidium bond binding to ΓΜ-2 DNA, allowing a change in fluorescence compared to a comparison reaction (identical to the sample under investigation except that it does not contain bleomycin) to quantify the extent to which bleomycin induced degradation of Bi-2 DNA occurs. From the fluorescence values, the percentage inhibition of the FM-2 DNA degradation caused by the presence of inhibitor protein X in the serum can easily be determined.

The concentration of inhibitor protein X in the serum sample is then determined by the known Lowry (folin / phenol reagent) method described in J. Biol. Chem. 193: 265-275 (1951) »determined.

By plotting the percentage inhibition of the IM-2 DNA degradation against the logarithm of the concentration of inhibitor protein X, the IC 2 value for the particular serum sample can be easily determined.

Once the IC ^ 0 value has been calculated for a particular patient, this value can be used directly to predict the likelihood of cancer in this patient. Extensive studies in healthy (cancer-free) patients and patients with a wide variety of malignant necplasmas have shown that inhibitor protein X is found in significantly smaller amounts in the serum of cancer patients. The IC ^ -30 value for cancer patients is therefore considerably greater than the normalized value for healthy individuals. This clear distinction in IC, n values provides a simple and accurate basis for determining the presence of cancer and is at the core of the invention.

The exact ICQ value obtained from blood samples from cancer-free 80 0 2 0 94-6 patients varies slightly from individual to individual.

In patients studied to date, the mean IC, Q value for cancer-free patients has been found to be 90.2-11.0 µg / cm3 (significant at the 0.001 level). The highest IC ^ Q value recorded for a healthy patient 5 is 120 µg / cm ^.

In cancer patients, the IC values obtained varied with the type of cancer, but in all cases to date the rate was significantly higher than that obtained in cancer-free individuals. The lowest IC ^ value for a cancer patient to date vas 270 µg / cm ^. In general, however, an ICG value of about 200 indicates a high probability that the patient has some type of malignant neoplasm.

The method of the invention has been successfully used to detect a wide variety of cancer types and is believed to be generally applicable to cancer diagnosis. As specific examples of detected tumor types, there may be mentioned testicular carcinoma, lymphoma, leukemia, adenocarcinoma of the breast, small cell adenocarcinoma of the lung, large cell adenocarcinoma of the lung, adenocarcinoma of the colon and melanoma, to name just a few. The method also appears to be specific for cancer diagnosis because cancer-free patients with different types of non-neoplastic conditions did not show the elevated IC ^ Q values associated with the presence of cancer.

The fluorescence analysis for inhibitor-protein X according to the invention described above is based on an intrinsic property of this material: inhibition of blecmycin-induced degradation of FM-2 DM. Serum levels of the protein determined by this method are correlated with the presence or absence of malignancies. The availability of a purified inhibitor-30X allows several other known methods of biochemistry and clinical chemistry to be used for its measurement. Prominent methods of this are immunological methods such as radio-immunoanalysis, fluoro-immunoanalysis, and zymlmmuno analysis (> eg ELISA), spin immunoanalysis, chemiluminescent immunoanalysis, fluorescence-polarization immunoanalysis, nephelometric analysis, money diffusion methods 800 2 0 94 -7- and classical methods ^ hemagglutination and component fixation.

Furthermore, other functional properties of inhibitor protein X can be "utilized for its measurement in" biological fluids. Catalytic phenomena associated with the molecule can be measured in spectrophotometric or spectrofluorometric kinetic or endpoint analyzes. Separation techniques can also be applied to the fluid in question, and various detection methods can be used to quantitate the protein, including spectral or densitometric and immunological methods. Since specific measurement of the inhibitor protein X concentration is obtained by the foregoing methods, it follows that the serum levels of the protein determined by such methods correlate with the presence or absence of malignancies.

The inhibitor protein X was purified according to the following procedures.

Partial purification - Procedure A

Inhibitoreivit X was partially purified by Sephadex-

O

column chromatography. 1 cmJ human serum was applied to a Sephadex G-75 kolan (88 x 2 cm) then eluted in sodium phosphate.

O

Buffer (0.07 M, pH 6.8) at a flow rate of 6-8 cm / h at room temperature. The inhibitor was in the void volume that was lost. . .3 was collected, lyophilized, resuspended in 1 cm HgO, dialyzed against 20 volumes of HgO for 2 h at U ° C, then on a Sephadex G-200 column. that was equilibrated in the same buf-25 fer,

Elution of the Sephadex G-200 column gave three peaks absorbing at 280 nm. Peak III, which belonged to 7.7% of the total amount of protein eluted, contained inhibitor protein X. This was treated as described above and then replenished to a Sephadex G-200 column. Elution was processed with the above-described phosph-. 3 fate buffer. 3 cm fractions from each column were collected with a fraction collector (Gilson Medical Electronics, Inc., Middleton, Wis) and the elution patterns were obtained by measuring the absorbance at 280 nm with a spectrophotometer. Protein concentrations were determined using the Lowry method.

80 0 2094 8-

Purification - Procedure B

The purification of the inhibitor protein was performed by molecular filtration with Amicon flashes, dialysis and Sephadex gel column-3.

chromatography. Human sera of 1-5 cm m 5 conical Amicon filter tubes (Amicon, Lexington, Mass.) Were centrifuged for 10 minutes at 5000 g at U ° C. The retentate, which remained in the filter cone after centrifugation, was resuspended in delonized water to the original volume of the sera. Equal amounts of the retentate and the filtrate were tested for their inhibitory effect. The active retentate was dialyzed in a cellulose tube against 20 volumes of deionized water at 1 ° C for 2 hours. The retentate and dialysate were lyophilized and resuspended in deionized water. The results of analyzes of the two fractions showed that the inhibitory activity in the retentate was exerted. 1 cm retentate was applied to a 60 x 5 cm Sephadex G-50 column and eluted with 0.07 M sodium phosphate buffer pH 6.8 at a flow rate of 30 cm / h at U ° C. The column eluate (5 cm per tube) was collected with a Buchler Fractcmette Alpha 200 (Searle, Fort Lee, H.J.) fraction collector. The 280 nm absorbance of each fraction was measured with a spectrophotometer and the tubes were pooled according to the absorbance peaks. The pooled fractions were lyophilized, dialyzed and tested for inhibitory activity as described above. The "active" material, which was in the empty volume fraction, was applied to Se-25 phadex G-75 and G-100 columns and eluted to the G-50 column in the same manner. The inhibitory activity was found in the empty volume fractions. of both columns The "active" material collected in the empty volume fraction of the Sephadex G-200 'column was eluted with 0.07 M sodium phosphate buffer with a pH of 6.8 at room temperature at a flow rate of 6-8 cm / hour.

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The fractions were collected (3 cm / tube) with a Gilson microfraction collector (Gilson, Middleton, Wis.) The column eluate was fractionated based on the 280 nm absorbance profile by pooling the tubes under each absorbance peak. Each fraction was then lyophilized, dialyzed and tested for inhibitory activity as described above. The inhibitory inhibition was found in fraction III. The material in fraction III was then added to the Sephadex G-20 column and eluted under the same conditions. Fraction III of the Sephadex G-200 column, containing each inhibitor protein X, represented more than 1000 fold purification.

The EM-2 DNA fluorescence analysis procedure for inhibitor level X is explained in more detail below:

Determination of IC ^ Q for inhibitor level X.

The determination of the IC ^ q (the egg protein concentration required to obtain a 50% inhibition of the PM-2 DNA degradation) for inhibitor protein X in human sera is performed in two steps: 1. The egg protein concentration in the serum determined using the Lovry method.

2. Using the FM-2 DNA fluorescence analysis for bleomycin 15 cm, obtain a dose response response for the serum protein inhibitor. The IC ^ Q values were derived from graphs plotting the percentage inhibition of DNA degradation against the

N

egg protein concentrations on log probxt graphs.

Procedures: 20 I. Lowry reaction.

A. Preparation of stock solutions:

Solution A. 2% NagCO 2 in 0.1 N NaOH.

Solution B. 1% CuS0 ^ .5H2 O in H ^ O.

Solution C. 2% Na + K + tartrate in HgO.

Solution D. 2 N Folin-Ciocalteau Phenol Reagent.

B. Preparation of reagent solutions.

Reagent solution 1: Mix 1 volume of stock solution B with 1 volume of stock solution C.

Reagent solution 2: Mèmg 50 volumes of stock solution A with 30 1 volume of reagent solution 1.

Reagent solution 3: Mix 1 volume of stock solution D with. 1 volume HgO.

C. Reactions.

1. Dilute the sera 1:10 with 100%.

2. Transfer 10 microliters of the diluted sera into a 13 x 100mm glass test tube and add 290 microliters of HgO.

800 2 0 94 3 -10- 3. Add 1 cm of reagent solution 2, mix well, and mix for 10 minutes at room temperature.

h. Add 100 microliters of reagent solution 3, mix well, and incubate at room temperature for 30 minutes.

5 5 · Use a spectrophotometer to measure the absorbance at 750 nm.

6. Determine the protein concentration from the BSA standard curve.

D. Bovine Serum Albumin (BSA) Standard Staple.

1. Prepare a series of dilutions of BSA (i.e. 25, 50, 75s 100, 150 3 3 10 Mg / cm) in a final volume of 0.3 cm.

2. Carry out the reactions, given under C, starting at step C (addition of reagent solution 2).

3. Plot the absorbance at 750 nm against BSA concentrations.

II. The protein inhibitor analysis.

15 A. Perform series of dilutions of the serum protein based on the serum egg white concentrations determined by the Lowry reaction. Make the dilutions in a constant volume of a sodium borate buffer (0.05 M, pH 9S5) · B. Add the samples of serum protein (50 µl volume) to 10ul 20 bleomycin (3 ^ 5 nanamol) present in a glass test tube '' (13 x 100 mm).

C. Add 390 µl of a sodium borate buffer (0.05 M, pH 9-5) containing 25 mM 2-mercaptoethanol.

D. Add 50 µl PM-2 DM (8.3 x 10 ~ 6M in 0.15 M HaCl).

25 E. Incubate at 37 ° C for 30 minutes.

F. Transfer triplicates (100 yul) into glass test tubes (13 x 100 mm) containing 0.9 cm ^ lia ^ PO ^ (0.09 M, pH 12.1), 0.01 M EDEA and 0.01 .MiCla; stir well.

G. Add 100 µl of ethidium bremide (55 7 × 10 10 µM) in the 30 PO 10 buffer, pH 12.1. Stir well.

H. Comparative Tests: 1. Sodium borate (0.5 M, pH 9.5), containing 2-mercaptoethanol, without bleomycin or proteins.

2. Protein without bleomycin or PM-2 DM.

35 3. Protein without bleomycin.

80 0 2 0 94 -11- k. sodium borate (0.05 M, pH 9.5) containing 2-mercaptoethanol as a bianco sample.

I. Spectrophotofluorametry (Amine o-Bo-wm an).

Transfer the sample to a 1 cm quartz cuvette and measure the 5 fluorescence at a 530 nm excitation and a 590 nm emission.

8 0 0 2 0 94

Claims (3)

A method for detecting cancer in humans, comprising the following steps: a) taking a blood sample from a patient to be examined for cancer; B) determining the inhibitor level X concentration in this sample; . and c) comparing this concentration determined in step b) with the inhibitor protein X concentration standard, which is associated with the serum of patients who do not have cancer, whereby a significant decrease of the inhibitor protein X concentration compared to the concentration standard indicates the likelihood of cancer. 2. A method for detecting human cancer, comprising the following steps: a) taking a blood sample from a patient to be examined for cancer; b) determining the concentration of inhibitor protein X in this sample, which is required to obtain a 50% inhibition for the bleo-mycine-induced degradation of EM-2 DNA; and c) comparing this IC value as determined in step b) with the named IC value, which is associated with the serum of patients who do not have cancer, with a significant increase in IC value, n value '.ov the numeric value indicates an indication for pu the likelihood of cancer. 3. Process according to claim 2, characterized in that the determination of the IC ^ Q value in step b) takes place according to the following steps: 1. preparing a mixture of the blood sample with a solution of bleomycin, B1-2. DM and 2-mercaptoethanol in a buffer with a pH of 9.5; 2. incubating the mixture from step 1) for 30 minutes at a temperature of 3 ° C; 3. adding a portion of the mixture from step 2) to a mixture of ethidium bramide in a denaturation buffer with a pi of 12.1; It) determining the fluorescence of the ethidium bramide: R4-2 DM 35 mixture from step 3) with a spectrophotofluorometer at a 530 nm 800 2 0 94> -13 excitation and a 950 nm emission; 5) "determining the percent inhibition of blecmycin-induced EM-2 DM degradation from the fluorescence change of the sample relative to a comparative sample containing no" blecmycin 5; 6) determining the concentration of inhibitor protein X by the Lowry method; and T) determining the concentration of inhibitor protein X5 obtained in steps 5) and 6) required to obtain 50% inhibition of the blecmycin-induced EM-2 DM degradation. 800 2 0 94