JPS6054697A - Diagnostic method and apparatus - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to early diagnosis of cancer and other diseases such as heart disease, diabetes, atherosclerosis.
This is based on the fact that diseased humans or animals have relatively high concentrations of virus-like DNA polymerases in their blood.

According to one aspect of the invention, in a method for detecting illness or potential illness by analyzing blood of a patient, the proportion of viral-like DNA polymerase is quantified relative to that of a hematologically normal patient. A method is provided which is characterized in that:

Regarding the presence of virus-like polymerases in cancer cells,
Already reported. For example, Gallo et al.
tal,, Co1d Spring Harbor
Symposium Quant Biol, 39
: 933 (1974)) and Baxt et al.
et al., Nature New Biol, 2
40 Near 2-7.5 (1972)).

The presence of virus-like DNA polymerase can be detected by various methods. In a highly preferred embodiment of the invention, platelets or platelet particles, which are nuclear-free cell fragments with a reduced range of polymerases, are used. It has been discovered that platelet abnormalities often predict the onset of metastasis. The use of platelets rather than white blood cells or cancer cells allowed for a correlation between enzyme activity and disease status.

Platelets can be obtained by platelet electrophoresis or directly from whole blood. Separation of platelets from other blood components is carried out by differential centrifugation or by depositing the individual blood components separately on a solid surface, such as a microscope slide. To prepare platelet particles, 99.99% pure platelets are disrupted by means such as homogenization, sonication, or mixing. Preferably, the plate is tightly packed with 5 to 50 strokes.
Divided by a mating Dounce homogenizer. Large organelles and cell debris are approximately 40'O, OOO×N
can be removed by centrifugation for - minutes.

The small virus-like particles are then broken down by centrifugation of the particles into a density gradient, usually sucrose-containing particles due to their buoyant density. Known amount of platelets usually &'4 equivalent to 14gK
The 00,000 x 9-min supernatant is layered onto the density gradient and centrifuged to equilibration. This is 100,0
00x9 was found to require 2 to 24 hours. Both fractions from the gradient were collected for DNA polymerase activity. This is accomplished in a preferred embodiment of the invention through the ability of this substance to catalyze DNA synthesis in response to a synthetic RNA template.

In a preferred feature, a small volume of the density gradient fraction is added to a solution containing buffer, detergent, K(J, NaCJ, reducing agent, divalent cation, radioactive nucleoside triphosphate, synthetic RNA template, and complementary primers). 10
A Na concentration of ˜100 mM was found to be optimal. Generally Mn is used as the divalent cation, Triton X-100 is used as the detergent, and dithiothreitol is used as the reducing agent. However, using Ic M9 etc. instead of Mn, Tr
It was also possible to use NP4Q instead of iton and fcB-mercaptoethanol instead of dithiothreitol. Divalent cations in the range of 0.5-10 mM are required.

“Standard measurement conditions” are 50 mM, Tris-HCJ
,P)('7.s, 68mMKcL, 0.03%Tr
iton, 05ml/mlBSA, 5m
MNacJ! ,,32μ! q/ml! template primer,
15mM dithiothreitol, 0.5mM MnCf2
, 0.05 mC/ml"H nucleoside tribosphate.

DNA synthesis is preferably carried out at 37°C for 20 minutes. Different degrees or shorter times are suboptimal. DNA formation is tracked by acid insolubility, DBAE-binding, radioactivity, etc. accumulation. The total DNA radioactivity synthesized by platelet particles of high buoyant density (e.g. greater than 1.1697 ml in sucrose) is determined by the amount of platelet material, the volume of the density gradient fraction analyzed, and the specific radioactivity of the nucleoside IJ phosphate. and after correction of deviation from standard measurement conditions -
This is a measure of platelet DNA polymerase activity.

The present invention further provides a test kit for identifying a disease from platelets obtained from a blood sample, comprising: a) a template primer; and b) a measurement solution of known composition for permitting and enhancing DNA polymerase activity; This allows for maximum DNA synthesis (dT) for biological specimens of virus-like particles prepared from platelets.
)m(rA)n in vitro using primer template
ro.

In order to facilitate understanding of the present invention, the present invention will be explained below using examples. All measurements are by weight unless otherwise noted.

Platelet electrophoresis or essential thrombocythemia (ET)
, 20 untreated patients with polycythemia vera (PV) and myelofibrosis (MF) with myeloid metaplasia,
Nine normal blood donors and eight myeloid leukemia (CML)
) was obtained from a blood unit from a patient with The number of patients and their clinical conditions are summarized in Table 1 below.

Platelets were collected at 25°C using the method of Plotsky et al. (B
rodsky et al., J., Nat., Canc., I.
n5t.

55 1069-. 1074 (1975)]. One unit of whole blood was collected in a triple pack;
Centrifuged for 6 minutes at 3300 rpm. The platelet-rich plasma is separated into one bag, and the batufi coat is a second bag.
The blood cells were separated into a third bag, and the blood red cells were separated into a third bag. Place Batuficoat in a 50ml centrifuge tube at 11,000 rpm.
The cells were recentrifuged for 12 minutes at VcM and the supernatant was added to platelet-rich plasma. The bag is then rotated to 3300 rpm.
Centrifugation was performed for 30 minutes at . platelets 30-4'
Suspend in Oml supernatant, transfer to 50ml tube,
Centrifuged for 30 minutes at rpm.

The platelet electrophoresis sample was placed in a 50ml tube and heated at 1000 r.
Centrifuged for 12 minutes at pm. Platelets from supernatant, 3
It was pelleted by centrifugation for 30 minutes at 300 rpm. There was less than one white cell per IO' platelets as determined by light microscopy.

All operations were performed at 4°C unless otherwise noted. Platelet concentrate was added to an equal volume of buffer C (50mM Tris-H
The cells were resuspended in pH 7.9, 1 mM dithiothreitol, 20% chrycerol, and 1 mM halamethylsulfonyl fluoride) and incubated for 20 minutes. Platelets are driven by a motor with 30 strokes, tight-fitting Do
Disintegrated using an un Ce homogenizer. Large particles (
Membranes, mitochondria, granules) were removed by centrifugation at 20,0 OOX, F for 20 minutes.゛゛\ Small particles in the supernatant liquid according to buoyant density? (i.e. according to biochemical composition)
The particles were fractionated into a sucrose gradient by centrifugation.

20.000 in a volume corresponding to 4 g of platelet starting material
The supernatant of 9X was added to TNE buffer (100mM NaCj
! , 1mM EDTA, 10,mM tr? s
-H(J, 30~b% in PI(7,5) 100.000X in 5W270-tar,
! ; centrifuged in NC for 16 hours until equilibration. Fractions of 1 ml were collected from the bottom of the tube and analyzed for DNA polymerase activity.

Add 5 μl of sucrose fraction to 10 μl of premix (4mMt
ris-HcL% pH 7,5,680mM K (J.

0.3%'riton X -100,5TQ/
ml B8A.

40mM NaCj! and 320μ97ml of template primer, poly(rA), oligo(dT) 12-1
8 or poly(dA), oligo(dT)12-8)
and incubated on ice for 15 minutes. 85μ
l cocktail mix (47rnM tris-HC
f,, p) I7.8.17 mM dithiothreitol,
0.58mMnCl! 2.0.07 mCi/m
l3HTTP,5Q75Ci/mM)
After incubation, acetic acid was added to 10% and the precipitate was collected on a nitrocellulose or cellulose acetate membrane. Scintillation counting was performed on each dry filter in 9-10 ml Econoflour.

To detect any terminal T. fulanisis activity that may be present, 10 μl of the sucrose gradient fraction was mixed with 10 μl of the premix (
320μ97ml of oligo(d', )12-18 (same as the DNA polymerase measurement premix except that the template primer was replaced) was added and incubated on ice for 15 minutes. 80μl cocktail mix (70mMt
ris-HCJ, pH 8, 3% 1.25 mM' dithiothreitol, 12.5 mM MIC, 12.0,
42mCj, 7fn13HdGTP, 5”-15C
i 7mM) and incubation at 37°C.
This continued for 30 minutes. Add 10% trichloroacetic acid to each reaction.
The calcined solution was added until the solution was stopped, and the precipitate was collected and counted.

The results are shown in Table I under the heading 1 "Extrinsic Activity". These results indicate that sick patients had higher exogenous activity values than healthy patients. This was true even when the presence of disease could not be diagnosed clinically. Ta.

The platelet DNA polymerase assay of Example I uses a synthetic template utilized by platelet DNA polymerase solubilized from platelet particles. Template and primer molecules are also present in platelet particles (oiles
pie.

Ga5pari and 5trayer, Bioche
m, BiophysRes, Comm, 99:
1191-1198.1981). Retained platelet RNA
The conditions for measuring DNA synthesis by platelet DNA polymerase are as follows.

Platelets obtained by platelet electrophoresis were purified in the same manner as in Example I. Platelet division, particle fractionation and DNA polymerase activity were determined by Brodsky et al.
'1. J, Nat, Cane, In5t.

55.1069-1074 (1975) and 59.6
1-67 (1977)] and using the technique described above.

Fractions from the sucrose gradient containing DNA polymerase activity were combined in one equal volume of EP buffer (50 mM Tris-HCJ).
, pH 8.0, 1mM dithiothreitol, 0.5
mM EDTA, 5 mM MyCj! 2 and 30
mMNaCJ! ) TE dilution L TA. 100.
(Collected as a pellet after 1 hour centrifugation at 100Xg. Particles from 2 g of platelets were mixed with 1.25 μl of R buffer (50 mM Na(J, 20 mM dithiothreitol, 50 mM tris-'HCJ, pH
7, 8, 10mM MgCj! 20.5mM MnC
J! 2.52 μg/me actinomycin D, 0.3
3mM unlabeled nucleotide triphosphate (dC
TP%dGTP, TTP), 3.2 mci/mA'
32 p-aATp (400-600 Ci/
mmol) and 0.01% Triton x −
100) and incubated at 37°C for 30 minutes.

375 μl after incubation (7) LETS
Soaking solution (100mM LiCL, 1mM EDTA,
10mM tris-H (J, pH 7,2,0,0
5% 8DS), 100 μl diethylpyrocarbonate, 25 μl 20% SD8 and 100 μl 0.
Add IM EDTA/2.0M NaCJ and mix the suspension on a portex mixer. LETS buffer (Q, 8+++l) and 8-hydroxyquinoline (3
, 7g7100ml) of phenol-cresol (7:1.50mM tris-H
Saturated with CJ, pH 7,6) was added, and the emulsion was
Shake at 5°C for 5 minutes. This emulsion is 10,0
Disrupted by centrifugation at 00xg for 10 minutes.

The cDNA-containing upper layer was removed, re-extracted with phenol-cursol, and recentrifuged.

The extraction was repeated until no interface remained between the top and bottom layers. After adding 25 μl of yeast t RNA and Na(J to 0.3 M), nucleic acids were collected by three ethanol precipitations.

The third ethanol precipitate is Q, 3mA! TE(0,
1M tris-HCL, 0.1M EDTA%p
H8). To a polyaromer centrifuge tube were added 7.4 ml water, 0.5 ml 'rE buffer, 0.2 ml 5tisalkosyl and 6.635 g solid Cs 2804. After fully dissolving 082SO4, the sample was added and the refractive index was adjusted to 1.375 at 25°.

This system was placed in a T150 rotor (36,00 Orpm) at 85. Centrifugation was performed at 20°C for 3 days in OOOXg.

The total radioactivity that migrated with the RNA was taken as a measure of platelet DNA polymerase activity and the results are shown in Table I as 1-intrinsic activity. It is observed that diseased patients have higher values than normal patients.

Using the technique of Example 2, platelet DNA polymerase was quantified in patients whose symptoms were insufficient for current clinical diagnosis. The results were compared with the diagnosis based on subsequently obtained clinical data. The results are shown in Table H. Patients included one patient with polycythemia vera without elevated platelet count and two patients with essential thrombocythemia suspected of having secondary thrombocythemia. All three patients had elevated platelet DNA polymerase levels consistent with a subsequent clinical diagnosis of myeloproliferative disease; the first patient (patient no. 14) had an elevated hematocrit and normal Platelet counts were shown. When one unit of this patient's blood was analyzed for DNA polymerase activity using a microassay, it was positive (Table ■).

This patient is described in Example 2 and 1. cDNA was synthesized in an HRNA-templated reaction. Subsequent measurements of the erythrocytosis and spleen diameter confirmed true erythropoiesis.

A second patient (patient no. 10) had an elevated platelet count after surgery. Platelets were positive for particulate DNA polymerase (Table ■). Platelet counts were greater than 106 in dogs on several measurements. The patient's records showed a history of elevated platelet levels that had previously been ignored. Her platelet function was normal. Subsequent studies did not reveal the morbidity normally associated with secondary thrombocythemia. Therefore, a diagnosis of instinctive thrombocythemia was made.

A third patient (patient number 4) had recurrent symptoms of abdominal pain that was diagnosed as pneumonitis, but her pancreas did not show any abnormalities. This patient was then noted to have an elevated platelet count. Her platelets showed high particulate DNA polymerase (Table I).

Her records showed a history of high platelet counts, and a diagnosis of essential thrombocythemia was made. This patient's elevated platelet count responded to low-dose busulfan treatment and she is currently asymptomatic.

As in Example I, platelet DNA polymerase was repeatedly quantified in thrombocythemic myeloproliferative disease patients.

Platelets were collected pre-chemotherapy, post-chemotherapy, before and after decline in bratallet counts if a decline occurred.

All patients who responded to chemotherapy with a decrease in thrombocytopenia showed a pre-decrease in platelet DNA polymerase.

The results are shown in Table ■.

These examples can use DNA polymerases to diagnose pre-leukemic conditions and predict a patient's response to chemotherapy. This provides a means of monitoring and/or treating certain high-risk cancer patients.

The use of in vitro enzymatic activity is merely an example of quantifying platelet DNA polymerase in human platelet particles. Other assays can also be used, such as immunoassays using anti-polymerase antibodies on whole platelet preparations.

Table 1 Patient Platelet count Exogenous Intrinsic] CML 1.0 265 4.4 2 CML 1.0 240 3 CML, 2.5 226 34 4 ET 0.8 191' 19 5 pv 0.7 175 6 ET 1. 5 174 23 7 gT O,814315 8pv O1313626 9cMr, 2.5 122 6.3 10 ET 1.2 115 16 1i pv O,69937 12CML O,683 13CML 2. i 70 14 pv O,263 15pv 1.0 55 4.5 16 ET 2.0 51 4.2 17 ET O,8497,3 18MP O,8454,5 19MP O,94112 2Q pv O,441 21CML 1.5 40 22 pv O,63924 23CML O,537 24MF 1.0 34 1.4 Ogami Wataru' Patient Platelet count Exogenous Intrinsic 25 MF 1.0 29 1.3 26 Normal 0.3- 29 27 Normal 0 .2 28*3.3 28 Normal Q,4 27 29 pv O,s 24 5.1 30 pv O,5232,9 31MFo,8 21 32 Normal 0.4 18 33 Normal 0.4 14 34 Normal 0.3 12 2.9 35 Normal 0.2 9 1.0 36 Normal 0.4 7*1.2 37 Normal 0.33 *No peak Table 1 14 63 PV 10 115 ET 4 191 ET Table l A PV 25 0.8 11 1.0 0.2BET
53 2.0 1 2.0 0.2cET230 1
．． 6 25 1.0 0.2D IE'I' 154
1.0 73 1.0 0.5E PV 175 0,
7 109 0.8 1. OF'MF 40' 1.
0 3B 1.0 1. Oo ET 174 1,0
1B5 1.0 1.0 Agent Patent Attorney Akira Kono Continued from page 1 [Partner] Inventor David Gillispie America.

Candy 0 shots bright person David Strayer Amelie.

More, 1, Hensylvania, United States 19343% Glenmooy Pulflower Road, Box 138, Pennsylvania, United States 19010. Brinerock Creek
Road, 918 Procedural amendment amendment voluntary) iOctober 12, 1998 To the Commissioner of the Japan Patent Office1, Indication of matter 1'1 Patent application No. 163107 of 19882, Diagnosis method of the name of the invention and aA@3, Amendment Relationship with the patent applicant's name: Isadora Protoskyi (and 2 others) 4. Agent: 107 (1 location: 2-2-21-6 Akasaka, Minato-ku, Tokyo) Typed engraving (in the contents) No changes have been made) and the letter of appointment and its translation (7), contents of Supplement I.

Claims (1)

[Claims] (11) A method for detecting a disease or a potential disease by analyzing the blood of a patient, which includes quantifying the proportion of virus-like DNA polymerase relative to that of a hematologically normal patient. Characteristic method: (2) isolating platelets and/or platelet particles from blood;
The method according to claim 1, wherein virus-like DNA polymerase is detected therein. (3) The method according to claim 1 or 2, which comprises reacting Tavirus-like DNA polymerase obtained from platelets or platelet particles with a synthetic RNA template primer and measuring the synthesized DNA. (4) The method according to claim 3, wherein the template is a (dT)(rA) primer template m n , a virus-like DNA polymerase is incubated with a premix on the template, and DNA is precipitated and measured. (5) The method according to claim 1 or 2, wherein DNA synthesis produced by platelet DNA polymerase is measured using platelet RNA retained in platelet particles. (6) The method according to any one of claims 2 to 5, wherein the primer template is platelet RNA. (The method according to claim 1 or 6, wherein the force measurement is immunological using antibodies produced against platelet DNA polymerase or cross-reactive DNA polymerase. (8) The method according to any of claims 1 to 7, wherein the test blood is that of a human or non-human animal. (9) The blood is first subjected to: a) separation of platelets from other blood components; b) preparing small particles from a known amount of platelets, split bodies using differential centrifugation, and C) separating virus-like particles from other small particles using isobaric centrifugation in a gradient of known volume. 9. A method according to any one of claims 1 to 8, characterized in that the method comprises: (10) A test kit for identifying a disease from platelets obtained from a blood sample, comprising a) a template primer, and b) a measurement solution of a known composition for allowing and enhancing DNA polymerase activity; For maximum DNA synthesis for biological specimens of virus-like particles prepared from platelets ('
dT)(rA), pram, n imer template for enzymatic determination of DNA polymerase activity in vitro.