AU616897B2 - Detection method of abnormally-responding lymphocytes as well as detection reagent and kit therefor - Google Patents

Description

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AUSTRALIA

Patents Act COHPLET SPECIFICATION

(ORIGINAL)

Class 616897 Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: e S 000 I c. t- 0 0e *0sS PPLICANT'S REFERENCE: FP -OP-6/P :Name(s) of Applicant(s): Japan Immuno Research Laboratories Co., Ltd Address(es) of Applicant(s) ii l 5 0 5.

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17-5, Sakae-cho, Takasaki-shi, Gunma-ken,

JAPAN.

Address for Service is: PHILLIPS RW0DE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Complete Specification for the invention entitled: DETCICN MGTH0D OF ABNORMALLY-RESPONCDING LYPHOCYTES AS WELL AS DETECICI REAG=h AND KIT 'ITFEREOF Our Ref 101533 POF Code; 1544/88523 The following statement is a full description of this invention, including the best method of performing it known to applicant(s) 6003q/1 I i ir I I 1~ lA- TITLE OF TH2 INVENTION: DETECTION METHOD OF ABNORMALLY-RESPONDING LYMPHOCYTES AS WELL AS DETECTION REAGENT AND KIT THEREFOR BACKGROUND C2T THE INVENTION 1) Field of the Invention: This invention relates to a method for the detection of lymphocytes which have responded abnormally to to transformed cells contained in an 10 organism, infusion or the like as well as detection reagent and detection kit useful for the method.

2) Description of the Related Art: It has been known that a serious disease is S induced when intravital cells are transformed by a 15 certain cause. For example, histocytic cells transformed by oncogenes become cancer cells and develop a cancer while T 4 cells transformed by an HIV virus may cause acquired immune deficiency syndrome (AIDS). In addition, hepatitis is said to be attributed to transformation of liver cells by a virus.

It is hence extremely important for the detection of the above-described diseases and the judgement of the degrees of their progress to determine the existence of transformed cells in an organism and their population.

2 However, the diagnosis of a disease developed by the existence of these transformed cells have heretofore been effected by detecting biochemically, hematologically or immunologically a specific change caused by the disease. These diagnoses are therefore accompanied by drawbacks that they cannot be performed with ease in an early stage of a disease in many instances and they have difficulties in determining the degree of its progress. They involve a further 10 drawback that with respect to each disease, a reagent suited for the diagnosis of the disease must be provided.

0 6 SUMMARY OF THE INVENTION The present inventors have carried out an 9.

1 15 extensive investigation with a view toward developing a method for detecting and diagnosing, in an early stage, cancers and various diseases caused due to infection with viruses. It has now been found that there are certain specific common characteristics on lymphocytes which have responded abnormally in the presence of transformed cells.

Namely, the investigation of the present inventors has revealed that one of saccharide chains expressed in cancer cells is expressed on T cells infected with HIV and the saccharide chain is also

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3 expressed on lymphocytes responded abnormally to these transformed cells. It has also been found that the expression of the saccharide chain on the abnormallyresponding lymphocytes is stronger than that on the transformed cells and detection of the saccharide chain on the lymphocytes permits easy judgement of the development of the abnormal response by the lymphocytes to the transformed cells.

Accordingly, this invention provides a method S 10 for the detection of abnormally-responding lymphocytes, HIV-infected T cells by transformed cells, which comprises causing an antibody, which can specifically recognize a saccharide chain represented by the following formula 15 Fucal--2Gal1l-->4GlcNAc3l->3Gall-94Glcl-91Cer, 3

(I)

Fucal to act on a sample containing lymphocytes and then detecting lymphocytes conjugated with the antibody, and also a reagent and a kit useful for the practice of the above method.

any abnormal rpote to transformed cells can ibe rectly detected by the present inven f n, this invention can conduct not, only the d. gno s of various diseaes but also the direct- \j'r 5 5 'I I @0 S S 0 0 i LYna~ -3a- As used herein in the claims and description, Fuc, Gal, GlcNAc, Gal, Glc and Cer respectively represent fuc&se, galactose, N-acetylglucosamine, galactose, glucose and ceramide respectively. An arrow represents a glycosidic linkage between the numbered carbon atom of one sugar Asd the numbered carbon atom of an adjacent sugar by the presented configuration, a or B, and the central number 3 is the carbon number of GlcNAc.

Since any abnormal response of lymphocytes to transformed cells can be directly detected by the prsent invention, this invention can conduct not only the diagnoses of various diseases but also the direct e* o See o i r, DMW/3210U

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S- 4 determination of the degrees of progress of such diseases. The present invention does not detect transformed cells themselves but does detect lymphocytes responded abnormally to them. Accordingly, body fluids such as blood can be used and the testing has been facilitated.

BRIEF DESCRIPTION OF THE DRAWINGS The above and other objects, features and advan- 90 tages of the present invention will become apparent to 0 310 from the following description and the appended claims, *taken in conjuction with the accompanying drawings, in 0 Swhich: FIG. 1 illustrates the relationship between *incubation time and percent positive conversion for HIV-infected T-cells; FIG. 2 illustrates results of cytofluorometric analyses conducted respectively on various refined antibodies to detect HIV-infected and uninfected .I T-cells; FIG. 3 depicts the reactivity between T-cells and various antibodies; and FIGS. 4(A) through 4(D) show results of immunofluorometric staining of peripheral human lymphocytes.

1 i DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS The saccharide chain which the antibody useful in the practice of this invention can recognize, has already been known as "Ley aintigen" (Hakom ri, 1rK ibi 663v o. )o rV o S .3S N.lmn. Lvory, atd-Ka"na", af 1? 1rmR-1v C11 3) Biol. Chem., 25, 4672-4680- 1984. It is hence possible to use, as an antibody useful in the practice of this invention, a monoclonal antibody produced, for

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10 example, by immunizing an animal with Le y antigen in a manner known per se in the art, obtaining hybridomas I from antibody-producing cells of the animal by a cell

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S" fusion technique and then culturing the hybridomas.

The antibody useful in the practice of this invention S* 15 may be used as a crude antibody solution, namely, in the form of a culture supernatant of the anti-Le 7 producing hybridomas or a mouse ascitic fluid without any further treatment or processing. It may also be used after purification by ammonium sulfate fractio- 20 nation, ion-exchange chromatography, or affinity chromatography on a protein A or antigen column.

As one example of antibodies available readily, may be mentioned the antibody already known as "BM-1" (Abe, McKibbin, and Hakomori, J. Biol.

Chem., 258, 11793-11797, 1983).

c -6- As samples containing lymphocytes, blood, lymph, ascitic fluid, amniotic fluid, cerebrospinal fluid and the like may be used by way of example. When blood is used, it is preferable to collect 0.1-10 ml of blood and generally, to use it as serum, plasma or lymph.

In the method of the present invention, the above-mentioned antibody-conjugated lymphocytes can be detected by a known method. This detection may be performed preferably by radioimmunoassay (RIA), enzyme 10 immunoassay (EIA) or fluoroscent antibody technique (FAT), which is a usual immunological assay and makes use of the competitive method. Operational procedures of these techniques may be carried out in a usual manner. More specifically, T cells are isolated from a o 15 sample by a method known per se in the art. A slide glass is coated with the T cells and the antibody useful in the practice of this invention is added dropwise onto the thus-coated T cells. After washing the T-cells, they are stained with a secondary antibody 20 by fluoroscent antibody technique or enzyme immunoassay.

As a detection method of a virus or the like which is present in serum or plasma and may transform vital cells, cultured cell line T cells TALL-1) are exposed to a serum or plasma to be tested so as to infect the cells by a virus or the like contained in 7the serum or plasma, and the virus or the like present in the serum or plasma can then be detected as described above.

Labelling of the antibody, which is useful in the practice of this invention, with an enzyme as a marker may be carried out by the method described in Tanpakushitsu.Kakusan.Koso (Proteins-Nucleric Acids.Enzymes) 20(11), 1007-1013 (1975). Labelling of the antibody with"a fluorescent marker may be 10 conducted following the method described in Kiso Seikagaku Jikkenho (Basic Biochemical Experimental see* Procedures) Vol. 6 (Biochemical Assays), page 167. Any antibody may be used here so long as it has binding ability for the aforementioned antibody.

15 For example, the secondary antibody may be obtained by immunizing an animal other than human being, such as a rabbit, goat, mouse or the like, with an antibody employed as a primary antibody in a kit and then collecting same from serum or ascitic fluid or by S. 20 purchasing an antibody, which can specifically bind the aforerient'oned antibody, for each isotype. The enzymelabelled or fluorescence-labelled secondary antibody may be prepared in the above-described manner or a commercial product may be purchased. A suitable enzyme substrate solution may be chosen in accordance with the I t -8 kind of the enzyme carried on the antibody. That is, a 3',3'-diaminobenzidine solution, 9-amino-9-ethylcarbamizole solution or the like may be used where the enzyme is horse raddish peroxidase, whereas a p-toluidine 5-bromo-4-chloro-3-indolylphosphate solution or the like may be employed where the enzyme is an alkali phosphatase.

A suitable staining agent may be chosen depending on the enzyme. 5-aminosalicylic acid, o-phenylene- G OD" o0 diamine or the like may be used where the enzyme is .4 S horse raddish peroxidase, while p-nitrophenyl phosphate S or the like may be employed where the enzyme is an alkali phosphatase.

In a direct method, an antibody itself is 15 fluoroscence- or enzyme-labelled and the thus-labelled antibody is used. Although the direct method therefore requires fewer staining steps compared with an indirect method making use of a secondary antibody, it is accompanied by a drawback that the background is high.

20 The fluorescent antibody technique making use of a fluoroscent marker requires fewer steps and is thus simple and convenient.

The method of this invention can be easily and conveniently practised by using a reagent which c;ontains an antibody capable of specifically recognizr 9 ing the saccharide chain of the formula It may be practised more easily and conveniently by additionally using a kit for the direct fluorescent antibody technique, said kit containing a fluorescence-labelled antibody useful in the practice of this invention, (2) a kit for the indirect fluorescent antibody technique, said kit containing the antibody useful in the practice of this invention and a secondary antibody carrying thereon a fluorescent marker capable of binding the 10 antibody, a kit for the direct enzyme antibody technique, said kit containing an enzyme-labelled antibody useful in the practice of this invention, (4) a kit for the indirect enzyme antibody technique, said kit containing an antibody useful in the practice of 15 this invention and a secondary antibody carrying thereon an enzymatic marker capable of binding the antibody, or the like.

The kit is supposed to contain an antibody useful in the practice of this invention and a 20 secondary antibody. The kit may also contain, for example, a stabilizer such as glycerol or bovine serum protein and/or a preservative. The antibody reagent may be lyophilized and the kit may contain a watersoluble or water-miscible solvent. The antibody reagent may also be added with a buffer for maintaining the thus-reconstituted reagent at a constant pH and/or 10 a preservative and/or a stabilizer for preventing deterioration of a sample. Although the buffer is not an essential component of the kit reagent, it is preferable to use a buffer capable of controlling the pH of the kit reagent within a range of from about to about 9.0 upon practise of the assay of this invention. Although the reconstituting agent may preferably be that containing water, a portion or the whole portion of the water may be replaced by a solvent e 10 miscible with water. As illustrative examples of the water-miscible solvent, may be mentioned glycerin, alcohols, glycol ethers, etc., all of which are well known in the art.

Lymphocytes which have abnormally responded and are detectable by the method of the present invention reflect the existence of transformed cells. The term "transformed cells" as used herein means those occurred due to infection of cells with a virus, canceration of cells and the like and causing, for example, various Cancers, AIDS, hepatitis, certain mental diseases, autoimmune diseases, etc.

It is however evident from subsequent examples that lymphocytes detectable in accordance with this invention are not transformed cells themselves.

Namely, It han been demonstrated that lymphocytes, which may be infected with HIV, are not T 8 cells but T 4 11 cells but those reactive most strongly to Ley antigen are T 8 cells which are not infected with HIV.

The present invention will hereinafter be described in further detail by the following examples.

Example 1: Peripheral blood samples drawn reas,,.ively from healthy subjects and patients of various diseases a. caused by the existence of transformed cells or see probably attributed to their existence were subjected 10 to two-color flow cytometry to check for the existence of Le y antigens expressed on T-cells. The tested I subjects consisted of 27 healthy subjects, 10 cancer patients, 80 HIV patients, 31 schizopherenia patients, 13 hepatitis patients, 1 Guillain-Barre syndrome patient, and 1 Sezary syndrome patient. Results are summarized in Table 1.

For the two-color flow cytometry, 10 mi of heparinized blood was drawn from each subject's vein, added to 1 ml of "KAC-2" (trade name; 5% silica suspension; product of Japan Immunoresearch Laboratories), and, to eliminate phagocytotic cells, incubated for one hour at 37°C, after which the blood was subjected to FICOLL-HYPAQUE gradient centrifugation to yield a mononuclear fraction. Next, the lymphocytes (5-10 x 105 cells/100 pA) were mixed with 100 lp of BM-1 antibody at a concentration of 5 pg/ml, 12 incubated for one hour, and washed once in a phosphate buffer saline solution (PBS). The mixture was then added with 100 pi of FITC conjugated anti-mouse IgM (product of Tago Inc.) at a concentration of 15 pg/ml, incubated for 30 minutes, and again washed once with PBS. Subsequently 10 pi of T-cll markers-CD3 (Pan-T marker)~ CD4 (helper/inducer T marker), or CD8 (suppressor/killer T marker) all from Becton bo Dickinson and Co. was added, and the resultant mixture was incubated for 30 minutes, washed once with PBS. .fter the washing, the resulting cells were added with 1 ml of 1.5% formalin in PBS, fixed for

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minutes in ice water, and then washed once with PBS.

bo Finally, 300 pL of PBS was added to prepare a cell suspension and then subjected to an analysi5. To detect the FITC-conjugated BM-1 antibody, the suspension was analyzed at an excitation wavelength of 488 nm and a fluorescence wavelength of 520 nm.

Measurement of T-cell markers directly marked with phycoerythin were, however, conducted by measuring the fluorescence strength at an excitation wavelength uf j 488 nm and a fluorescence wavelength of 580 nm.

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[Results] Subjects. Ley antibody level in T-3 cells in T-4 cells in T-8 cells Healthy 27 9.3 0.77 10.0 0.66 11.4 1.25 cancer 3,4 5 -14.7 0.57 14.9 1.42 22.6 I.7 7 0,1,2 5 16.2 5.09 ]17.7 4.54 j17.1 5.11 HIV CDC** IV T31 15.5 2.18 24.8 3.22 22.1 2.44 CDC** 111149_ G9J 0.74 7.j 0.83 J14,8 1.56 Schizo- Active phase 26 19.1 1.97 120.3 2.03 24.0 2.11 phrenia Inactive phase. 5~ 12.6 2.09 13.6 2.54 18.5 2.41 Hiepatitis Acuce 4 15.6 4.08 J 9.2 2.36 25.0 3.75 Chnro mec 9 10.1 1.24 10.7 1.32 7.7 1.32 Gili-aesnrm1 18619. 146.6 Sezary syndrome 1 11.6E 17.5 -27.1 Mk_ i r 'r r

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14 P.S. (Performance Status)

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55o 06 S 0 S. S 0 P.S. Patient's status Patient is entirely without symptoms, can S take part in social activities, and is able to move without restriction as before the onset of the illness.

Has slight symptoms which limit his ability 1 to do physical work, but can walk and perform light labor such as house or office work.

Sometimes requires assistance in walking or 2 in performing minor tasks. Cannot perform light work, but is out of bed at least half of the day.

Frequently requires assistance in performing 3 minor tasks, and spends more than half of the day in bed.

4 Normally requires assistance even in minor tasks, and spends the entire day in bed.

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The Center for Disease Control's CDC classification system for symptoms of HIV infection P.S. Patient's status Tests positive to anti-HIV antibodies, and I has transient symptoms peculiar to thr early stage of HIV infection.

Has no symptoms of infection, but tests positive for anti-HIV antibodies.

Has continuous, body-wide enlargement of lymphnodes, has lymphnode swelling of III at least 1 cm for at least 3 months in at least two areas outside the groin, but has no other symptoms.

Has continuous, body-wide Subgroup A symptoms (fever, weight loss, diarrhea, etc.).

Has neurotic symptoms Subgroup B' dementia, myelopathy, peripheral neuropathy).

IV Subgroup C Has a cellular immune deficiency caused by HIV infection.

S bo Has secondary malignant tumors Subgroup D caused by HIV infection.

ubgroup E ass IV patients who do not fall under categories A-D.

-I 16 With combinations of BM-1 and various T-cell markers, tests were conducted on the 10 advanced cancer patients whose cancers were clearly observable on X-ray or CT scan pictures (5 patients at P.S.'s 3 and 4; patients at P.S.'s 0, 1 and As a result, expression of Ley was observed on T-cell subsets, CD4 (helper/inducer T-cells) and CD8 (supperssor/killer T- 6* o cells). In particular, strong expression of Ley was a** observed on CD8 cells of patients with high P.S.

numbers.

Strong expression of Ley was also observed on CDC IV patients (those with apparent clinical i symptoms), rather than in CDC III patients (those with lasting, body-wide lymphnode enlargement) or CDC II patients considered to be HIV carriers.

With the schizopherenia patients, analysis of peripheral lymphocytes drawn from the patients both in active and inactive phases revealed an unusual staining pattern in the lymphocytes of the active phase patients, and showed a high level of Ley expression on T-cells. Although the patients in the inactive phase had a similar unusual pattern in their peripheral lymphocytes, their levels of Ley expression were not significantly different from those of healthy subjects.

The lymphocytes of the single Guillain-Barre syndrome patient showed an usual staining pattern, and ri 'mE f 17 a high level of Le y expression was observed on T-cells. The Sezary syndrome patient, who was probably infected with an unknown virus and whose helper T-cells were reported hyperactive, had been classified as carrying a type of leukemia. That patient showed an unusual Le y staining pattern in marker CD4-positive cells of helper T-cells.

From these results, it is indicated that Le y antibody is expressed by lymphocytes which respond abnormally due to the presence of virus-infected or cancerous cells (transformed cells), and that the detection of the antibody makes it possible both to s predict the onset of symptoms and to diagnose already apparent symptoms for patients with a variety of illness.

1* Example 2: Expression of Antibodies over Time by HIV-Infected Cells TALL-1 cells were cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum containing 50 g/mi of gentamyciA\. HIV having reverse transcriptase activity of 58000 cpm/ml containing 1 pg/mi of "Polybrene" (trade mark; product of Sigma Chemical Company, St. Louis, Mo) was added to a 3 x 10 5 /mi suspension of the TALL-1 cells on the 1st, 3rd, and 6th days as shown by arrows in _1 i i I V 18 FIG. 1, and HIV antigens expressed on the cell surfaces were analyzed.

The analysis was carried out by cytofluorometry, using anti-p24 monoclonal antibodies, blood serum from AIDS patients, and BM-1 antibodies, whereby the ratio of positive cells was determined.

"In FIG. 1, stands for the reactivity with BM-1 antibody, for the reactivity with the patient's blood serum, and for the reactivity 1 0 with anti-p24.

As a result of the above experiment, it has Sbecome evident that from the 18th day after infection o with HIV, TALL-1 cells begin to change to positive 00 o cells which react strongly with BM-1 antibody.

Example 3: Cytofluorometric Analysis of Membrane Antigen 5 x 105 cell portions of HIV-infected or uninfected H-9 cells or TALL-1 cells were mixed respectively with 100 pC of various refined antibodies, and after incubation at 4 C for minutes, were separately washed twice with RPMI-1640 medium. The resultant solutions were each added with 100 V1 of 35-times diluted FITC-conjugated goat F(ab) 2 fragment directed to anti-mouse IgM or IgG (Tago Inc.; Code No. 4352). After incubation at 4 0

C

for 30 minutes, the cells were washed twice with RPMI 19 medium and fixed with 1.5% formalin-PBS. Purified mouse IgM (Coulter Corporation) was used as a primary antibody for a control. Cytofluorometry was performed using an EPICS-C (Coulter Electronics), and the results are shown in FIG. 2.

The graphs in FIG. 2 are assigned as follows: Ta: HIV-uninfected H-9 cells treated with .O mouse IgM.

e* Ib: HIV-infected H-9 cells treated with mouse IgM.

IIa: HIV-uninfected H-9 cells treated with BM-1 monoclonal antibody.

IIb: HIV-infected H-9 cells treated with BM-1 S monoclonal antibody.

IIIa: HIV-uninfected TALL-1 cells treated with S" mouse IgM.

IIIb: HIV-infected TALL-1 cells treated with mouse IgM.

IVa: HIV-uninfected TALL-1 cells treated with monoclonal antibody BM-l.

IVb: HIV-infected TALL-1 cells treated with monoclonal antibody BM-1.

As shown in FIG. 2, Groups lib and IVb both expressed strong fluorescence. Namely, both the HIVinfected H-9 and TALL-1 cells, after treatment with BM-1, showed an increase in fluorescence intensity.

S- Example 4: For each type of cells, the difference between their reactivity with each antibody both before and after infection with HIV was determined. Results are shown in in FIG. 3. As illustrated there, it is understood that the established cell lines, T-cells TALL-1), react strongly with BM-1 antibody only after infection with HIV.

o*"o The antibodies used in this experiment were prepared by the procedures described respectively in the following articles: FH-2: Fukushi, et al., J. Biol. Chem., 259, 4681-4685, 1984.

ACFH-18: Fukushi, et al., J. Biol. Chem., 259, 4681-4685, 1984.

SFH-6: Fukushi, et al., J. Biol. Chem., 259, 10501-10517, 1984.

KH-1: Kaizu, et al., J. Biol. Chem., 261, 11254-11258, 1986.

Example Separation and Immunofluorescent Staining of Human Peripheral Lymphocytes Heparinized blood samples (10 mi) drawn respectively from AIDS and ARC patients' veins were each mixed with 2 ml of "KAC-2" (trade name; 5% silica suspension; product of Japan Immunoresearch

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21 Laboratories) and after elimination of phagocytotic cells, maintained at 37 0 C for one hour. The KAC-2 treated blood samples were then subjected separately to FICOLL-HYPAQUE gradient centrifugation to obtain mononuclear cell fractions. Alliquots of the lymphocytes were stained by immunofluorescence with various antibodies. Results are depicted in FIGS. 4(A) through 4(D).

FIG. 4(A) is an optical micrograph of T-cells of an AIDS patient, and FIG. 4(B) a fluorescent micrograph of same. Further, FIG. 4(C) is an optical micrograph #0 of T-cells of an ARC patient, and FIG. 4(D) a fluorescent micrograph of same.

As is shown in FIG. the lymphocytes of the AIDS patie't reacted strongly with BM-1 antibody.

However, the ARC patient's lymphocytes showed much weaker fluorescence staining than those of the AIDS patient as shown in FIG. 4(D).

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j Example 6: BM-l Antibody based differential diagnoses of cancers: The counts of positive cells to BM-1 antibody were taken with 19 cancer patients and a study conducted on a possible relationship between BM-1 reactive lymphocytes and the life expectancy of each of the cancer patients.

FIG. 5 diagrammatically shows the relationship between the percentage of BM-1 positive cells in CD4 (helper-inducer/suppressor-inducer) and that in CD8 (killer-suppressor) of each patient. In the drawing, indicates cases in which the patients died within 3 months of the measurements of BM-1 positive cells (11 cases); 0 represents cases in which although the average periods of their life continuation were expected to be about 10 months, treatments were effective and the patients remained alive 1 year or more under absolute non-curative conditions, with no recrudescense observable in diagnostic imaging, exploratory excision, etc. (8 cases).

From these results, it has been found that among cancer patients those with a high CD8 level (said CD8 being reactive to BM-1) have a low apothanasia rate, and those with a high CD4 level have a high apothanasia rate. As has been demonstrated above, measurement of !t 9.* 'II .9 f* 4 9 *4 4 2-3 BM-l positive cells in a patient is believed to become an important means for grasping the prognosis of the patient and also for determining suitable treatments to the patient.

BM-l Antibody based differential diagnoses of hepatitides: Of 106 cancer patients 9 hepatitis A patients, hepatitis B patients, 32 non-A, non-B hepatitis patients and other hepatitis patients positive cells to BM-1 antibody were counted and a study was conducted on a possible relationship between BM-1 reactive lymphocytes and the conditions of the patients.

Identification of lymphocytes in each patient was conducted by a simple stain analysis based on the combinations of CD3, CD4, CD8, CD19 and NKH-1 (marker i for CD16) and BM-l; and also by flow cytometry of a two color analysis based on the combinations of CD3, CD4, CD 8, CD19 and NKH-1 (marker for CD16) and BM-l.

i) Out of the 106 cases, 34 cases showing an acute pattern typical to acute viral hepatitis were selected and classified into the foll(' 'ng groups: 4 Acute stage 13 cases (indicated by 0 in FIG. 6).

Descending stage 11 cases (indicated by A in FIG. 6).

Convalescent stage 10 cases (indicated by j in FIG. 6).

ic r 1 'i i r ~1 I r r c: c -91C FIG. 6 diagrammatically illustrates the result of plotting the relationships between the percentages of BM-1 positive cells in CD4 cells and those in CD8 cells. In each stage acute, descending, and convalescent the ranges of means S.D. (standard deviations) are indicatd by surrounding them with a rectangular frame.

As FIG. 6 indicates, the development of a symptom characteristic to acute viral hepatitis results in the percentages of CD8 positive cells, increasing from the corresponding normal levels of the healthy subject (ranges in which the percentages of BM-1 positive cells in both CD4 and CD8 are not higher than It has also been found that this phenomenon peaks in the acute stage; and the levels of BM-1 positive cells then return to the normal ranges respectively during convalescence.

As has been described above, greater variations were observed in the percentage of positive cells in CD8 rather than CD4 in acute viral hepatitis and the percentage of positive cells in CD8 decreased toward the recovery. As has been demonstrated above, selective counting of BM-1 positive cells is useful for the determination of the degree of convalescence from acute viral hepatitis. it is hence possible to r

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0 5i UF I i S I j S. S determine suitable treatments on the basis of the results of the counting.

ii) Of those with chronic viral hepatitis among the above-mentioned 106 cases, 19 cases marked by continuity, persistency and intractableness were classified in accordance with a standard to be described below, and the results of plotting of the relationships between the percentages of BM-l positive cells in CD4 cells and those in CD8 cells in these cases are diagrammatically depicted in FIG. 7.

Chronic hepatitis B namely, cases found to be chronic aggressive hepatitis by a histological tissue diagnosis in exploratory liver excision and in which abnorrality in the transaminase (s-GPT) level continued over 6 months 8 cases.

Among non-A, non-B hepatitides a) Cases diagnosed as chronic aggressive hepatitis by a histological tissue diagnosis in exploratory liver excision and in which the transaminase level remained 100 units or higher over 6 months 7 cases.

b) Cases diagnosed as acute hepatitis by a histological tissue diagnosis in exploratory liver excision but in which abnormal I variations were observed in the transaminase Slevel over 6 months 4 cases.

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4: In the ,'rawing, the 19 chronic viral hepatitis cases are indicated by O. In addition, out of the acute viral hepatitides i) above, the 13 acute-stage cases and the 11 descending-stage cases are also indicated by and A respectively.

From the above results, it has been found that in acute hepatitides the percentages of BM-1 positive cells in CD8 are distributed in a high region in the acute stage but in a relatively low region in the convalescent stage; whereas in chronic hepatitides the percentage of BM-1 positive cells are distributed in a region where the percentage of positive cells in CD8 and that in CD4 are comparable and also in a region where the percentage of positive cells in CD4 is high.

Namely, measurement of BM-l positive cells has made it possible to determine any variation on the side of effector cells, said variation being undetectable when s-GPT values alone are relied upon alth(ugh such s-GPT values are said to reflect the degree of damage of liver cells. Thi.s indicates that the history of convalescence from acute hepatitis can be traced by measuring BM-1 positive cells along with s-GPT upon diagnosis of hepatitis and the counting of BM-1 positive cells also permits differential diagnosis of a patient suffering from continuous, persistent and intractable chronic hepatitis and monitoring of curative effects.

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Claims (2)

1. 4. A method as claimed in claim 1 substantially as hereinbefore defined with reference to any one of the examples. DATED: 19 August 1991 PHILLIPS ORMONDE FITZPATRICK Patent Attorneys for: JAPAN IMMUNO RESEARCH LABORATORIES CO LTD to .00. 9 0,
2. 660. 000.. DMW/3210U I- ;i