JPH06242116A - Immunologic measuring method for pgl-1 similar material or antibody to the same - Google Patents

Abstract

PURPOSE:To measure PGL-1 similar material or an antibody to the material existing in a biosample quickly and with high accuracy by using an antibody to phenol glycolipid (PGL-1). CONSTITUTION:An antibody used may be any antibody to PGL-1. For example, both a polyclonal antibody made by giving PGL-1 to a horse or a cow as immunity and a monoclonal antibody produced by a hybridoma obtained by fusion of the spleen cell of a mouse previously rendered immune and a cell from a tumor line may be used. For example, in the case of measuring PGL-1 similar material, a biosample such as blood plasma diluted with physiological saline solution, urine or the like is dotted on a nitrocellulose film or the like to make anti-PGL-1 monoclonal antibody react therewith. After reaction, anti-mouse immune globulin antibody marked by a marker material such as emzyme or the like is made react therewith, and then the quantity of the marker material solidified on the film is measured. The measurement result is applied to an analytical curve showing the relationship between the already manufactured marker material and PGL-1 similar material so as to accomplish the measurement quickly and with high accuracy.

Description

Detailed Description of the Invention

[0001]

The present invention relates to, for example, serum, plasma,
A substance (hereinafter, abbreviated as PGL-1 analogue) having an antigenicity similar to that of a phenol glycolipid derived from Mycobacterium leprae (hereinafter abbreviated as PGL-1) present in biological samples such as urine and spinal fluid. Or), or a method for measuring an antibody against the substance.

[0002]

2. Description of the Related Art Special substances produced by cancer cells, such as α
-Fetoprotein (AFP), carcinoembryonic antigen (CE
A), etc., or cancer virus-related substances, oncogenes, etc., give important information for knowing the existence of cancer, its site and type, or the progression of cancer. being called.

[0003] These cancer markers include CEA which is elevated in most cancers, specific organs such as AFP which is said to be specific to liver cancer, CA19-9 which has high specificity to pancreatic cancer and the like. It is known that there is something peculiar to cancer.

Needless to say, the existence of a cancer marker that can be used for diagnosing a specific organ cancer is extremely useful when conducting drug administration, surgery, or a follow-up study of prognosis. However, the current situation is that many specific cancer markers corresponding to various cancers have not been found yet.

[0005] For example, although the incidence of cancer in the kidney is not particularly low as compared with other organs, even with sialyl SSEA-1, which is said to have an increased measurement value in kidney cancer, the positive rate of kidney cancer patients is high. It is about 15%, and it is hard to say that it is a definitive marker for kidney cancer. at present,
The cancer marker used for the follow-up investigation after removal surgery of kidney cancer is CEA which is elevated in most cancers, and the cancer marker specific to kidney cancer is not yet found.

[0006]

SUMMARY OF THE INVENTION The present invention has been made in view of the above situation, and is present in a biological sample which is considered to be highly useful as a cancer marker, in particular, a marker for renal cancer. -1 It is an object of the present invention to provide a method for rapidly and accurately immunologically measuring a similar substance or an antibody against the substance.

[0007]

The present invention is an invention of a method for immunologically measuring a PGL-1 analogue present in a biological sample, which comprises using an antibody against PGL-1. . Further, the present invention provides an immunological assay method for an antibody against a PGL-1 analogue existing in a biological sample, which comprises using a carrier on which PGL-1 or a part thereof is immobilized. is there.

[0008] That is, the present inventors have been earnestly studying the relationship between PGL-1 (or an anti-PGL-1 antibody) and infection with Leishmania, and the plasma, serum and urine of humans not infected with Leishmania. ,
In some cases, PGL-1 analogs and antibodies against them may be present in biological fluids such as spinal fluid, and it was found that these are highly useful as cancer markers, and the present invention has been completed.

More specifically, the inventors of the present invention reacted with a specimen (eg, a biological sample such as plasma, serum, urine, or cerebrospinal fluid) on gelatin particles on which PGL-1 was immobilized for the presence or absence of infection with Leishococcus. Then, the presence of anti-PGL-1 antibody in the sample was examined based on the presence or absence of the resulting aggregation. As a result, in the serum of pregnant women, particularly in the serum of pregnant women 9 months pregnant. It was also found that anti-PGL-1 antibody appears at a high rate and that the antibody titer decreases with delivery. Therefore, when the serum of these pregnant women was investigated in more detail, the positive reaction in the pregnant women was not due to the presence of the anti-PGL-1 antibody expressed by leprosy infection, but the PGL-1 expressed in the serum of the pregnant women.
It was found that the analogy substance was produced as an antigen and was caused by the presence of an antibody against it. Also, PGL
-1 analog and anti-PGL-1 analog antibody expression,
Since it was observed at a high rate in pregnant women, particularly in pregnant women in the latter half of pregnancy, it was suggested that this PGL-1 analogue has a very close relationship with the fetus, in other words, is a fetal antigen. Based on the above results, when the present inventors measured the presence or absence of the PGL-1 similar antigen in the urine of patients with renal cancer, the positive rate was as high as 70%. In addition, as a result of examining whether or not anti-PGL-1 analog antibody was present in the sera of various cancer patients, about 70% of renal cancer patients showed a positive value. From these things, this PGL-1 analogue and anti-P
The present inventors have found that the GL-1 analog antibody is highly likely to be used as a new carcinoembryonic antigen (cancer marker) different from CEA, and have completed the present invention.

The PGL-1 analogue according to the present invention is not known in detail in what part of the body it is produced, but from the results of the studies up to now, fetal cells and certain cancer cells have been obtained. It is presumed to be derived from. Also, P
Although the detailed physical properties of the GL-1 analogue are unknown, in addition to causing an antibody-antigen reaction with an antibody against PGL-1 derived from S. bacilli, the results of sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis, It is known that the substance has a molecular weight estimated to be about 40,000 to 50,000 (the molecular weight of PGL-1 derived from Mycobacterium leprae is about 1,000).

The antibody used in the method for measuring a PGL-1 analogue of the present invention is not particularly limited as long as it is an antibody against PGL-1. That is, a conventional method, for example, "Introduction to Immunological Experiments, Second Edition, Nao Matsuhashi,
In accordance with the method described in Academic Society Publishing Center, 1981, etc., animals such as horses, cows, sheep, rabbits, goats, rats, and mice can be treated with PGL-1 or a part thereof (even if derived from PGL-1. ,
It may be a synthesized one or any of them. ), Or PGL-1 or a part thereof according to a conventional method, that is, a cell fusion method established by Keller and Milstein (Nature, 256, 495, 1975). Splenocytes of a mouse previously immunized with (derived from PGL-1 or synthesized).
Any monoclonal antibody produced by a hybridoma obtained by fusing cells from a mouse tumor line may be used, and these may be used alone or in appropriate combination. If necessary, these antibodies may be digested with an enzyme such as pepsin or papain to obtain F (a
It goes without saying that it may be used as b ') ₂ , Fab' or Fab.

P used as an antigen for immunization
As a part of GL-1, for example, the method of Fujiwara et al. (Agric.
Biol. Chem., Vol. 51 (9), 2539-2547, 1987), which is preferably a sugar chain portion of PGL-1 and the like.
Needless to say, when actually used, it is bound to bovine serum albumin or the like by a conventional method.

The method for measuring a PGL-1 analogue of the present invention is a method known per se [eg enzyme-linked immunosorbent assay (EIA) except that the anti-PGL-1 antibody obtained as described above is used as the antibody. ), Radioimmunoassay (RIA), fluoroimmunoassay (FIA), etc.], for example, a buffer, a color former, a fluorescent substance, an enzyme, a substrate to be used at that time. Also, reagents such as radioisotopes may be appropriately selected and used from those used in the immunological assay methods known per se.

The method for measuring the PGL-1 analogue of the present invention will be specifically described below by taking the dot blot method as an example. That is, first, for example, on a film such as a nitrocellulose film, a nylon film, or a polyamide film, for example, 50 mM
Dot a biological sample such as plasma, serum, urine, spinal fluid, etc. appropriately diluted with phosphate buffer (pH 7.4), physiological saline, etc.
Then, the anti-PGL-1 monoclonal antibody (derived from mouse) obtained as described above is added thereto at 4 to 40 ° C., preferably 20 to 40 ° C. for usually 10 minutes to overnight, preferably 15 minutes to React for about 4 hours. After the reaction, an anti-mouse immunoglobulin antibody labeled with a labeling substance such as an enzyme, a fluorescent substance, or a radioactive substance is usually added thereto, usually at 4 to 40 ° C., preferably
20 to 40 ° C, usually 10 minutes to overnight, preferably 15 minutes to 4
After reacting for about a time, it is washed by a conventional method, and then, the amount of the labeling substance immobilized on the membrane is measured by a conventional method according to its property, and the result is prepared in advance. It can be easily carried out by applying it to a calibration curve showing the relationship between the amount of labeled substance and the amount of PGL-1 analogue.

The anti-PGL-1 analog substance antibody measuring method of the present invention comprises PGL-1 or a part thereof immobilized.
For example, operation is carried out in accordance with a measurement operation method of per se known immunoassay (eg, EIA, RIA, FIA, etc.), except that carriers such as latex particles, magnetic particles, gelatin particles, glass beads, plastic beads, and microtiter plates are used. Reagents such as buffers, color formers, fluorescent substances, enzymes, substrates and radioisotopes used at that time are appropriately selected from those used in the immunological assay known per se. It is sufficient to select and use it.

PG used for fixing to a solid phase
As a part of L-1, for example, the method of Fujiwara et al. (Agric.Bi
Ol. Chem., vol. 51 (9), 2539-2547, 1987), which can be used as the sugar chain portion of PGL-1.

The method for measuring the anti-PGL-1 analog antibody of the present invention will be described more specifically below. That is, first, a synthetic sugar chain having a structure peculiar to PGL-1 or the above-described method of Fujiwara et al., Which is unique to PGL-1, is bound to a protein such as bovine serum albumin by a conventional method, For example, it is adsorbed by a conventional method to a solid phase such as a microtiter plate and plastic beads which has been widely used in the conventional immunological assay such as EIA, RIA and FIA.
On this solid phase, a biological sample such as plasma, serum, urine, cerebrospinal fluid, etc., which is appropriately diluted with 50 mM phosphate buffer (pH 7.4), physiological saline or the like, is usually 4 to 40 ° C., preferably 20 to 40 The reaction is carried out at 0 ° C. for usually 10 minutes to overnight, preferably 15 minutes to 4 hours. After the reaction, the solid phase is washed by a conventional method, and then an anti-human immunoglobulin antibody labeled with a labeling substance such as an enzyme, a fluorescent substance or a radioactive substance is usually added at 4 to 40 ° C, preferably 20 to 40 ° C. The reaction is usually performed for 10 minutes to overnight, preferably 15 minutes to 4 hours. After the reaction, the solid phase is washed by a conventional method, and then the amount of the labeling substance on the solid phase is measured by a method according to its property. It can be easily carried out by applying the obtained results to a calibration curve that represents the relationship between the amount of labeling substance and the amount of anti-PGL-1 analogue substance antibody that has been prepared in advance.

Specific examples of the labeling substance used in the measuring method of the present invention include alkaline phosphatase, β-galactosidase, peroxidase, microperoxidase, glucose oxidase and glucose-6-phosphorus used in EIA. Enzymes such as acid dehydrogenase, malate dehydrogenase and luciferase, for example ^99m Tc, ¹³¹ I, ¹²⁵ I, ¹⁴ C, ³ H used in RIA
Radioisotopes such as fluorescein, dansyl, fluorescamine, coumarin, used in FIA
Fluorescent substances such as naphthylamine or derivatives thereof, such as luciferin, isoluminol, luminol, bis
Luminescent substances such as (2,4,6-trifluorophenyl) oxalate, such as phenol, naphthol, anthracene, or their derivatives, which absorb in the ultraviolet, such as
4-amino-2,2,6,6-tetramethylpiperidine-1-oxyl, 3-amino-2,2,5,5-tetramethylpyrrolidine-1-oxyl, 2,6-di-t-butyl- α- (3,5-di-t-butyl-4-oxo
-2,5-cyclohexadiene-1-ylidene) -p-tolyloxyl such as a substance having a property as a spin labeling agent represented by a compound having an oxyl group.
Needless to say, it is not limited to these.

As a method for binding the above-mentioned labeling substance to anti-mouse immunoglobulin antibody, anti-human immunoglobulin antibody, etc., there are known per se EIA, RIA or FIA.
And the like, which are generally known in the art (for example, Medical Chemistry Laboratory Course, Volume 8, supervised by Yuichi Yamamura, No. 1
Edition, Nakayama Shoten, 1971; Illustrated fluorescent antibody, Akira Kawao, No. 1.
Edition, Soft Science Co., Ltd., 1983; enzyme immunoassay,
Eiji Ishikawa, Tadashi Kawai, Kiyoshi Miyai, 2nd Edition, Medical School, 1982
Etc.) can be mentioned without exception and may be performed according to these. Further, it goes without saying that the method for binding the labeling substance to the binding substance or the substance to be measured may be a conventional method utilizing the reaction of avidin (or streptavidin) and biotin.

In the measuring method of the present invention, the amount of the labeling substance is measured according to a predetermined method depending on the kind of the labeling substance. For example, when the labeling substance is an enzyme, EI
A standard method, for example, “Enzyme Immunoassay, Protein / Nucleic Acid Enzyme Separate Volume No. 31, Edited by Tsunehiro Kitagawa, Toshio Minamihara, Akio Tsuji, Eiji Ishikawa, pages 51-63, Kyoritsu Publishing Co., Ltd., September 10, 1987. It is possible to carry out the measurement according to the method described in "Issuing daily" and the like. When the labeled substance is a radioisotope, the liquid is determined according to the type and intensity of radiation emitted by the radioisotope according to the usual method of RIA. Measurement may be performed by appropriately selecting and using a measuring device such as an immersion type GM counter, a liquid scintillation counter, a well type scintillation counter (for example, medical chemistry experiment course, Volume 8, supervised by Yuichi Yamamura, 1st edition, Nakayama). See bookstores, 1971, etc.). When the labeling substance is a fluorescent substance, a conventional FIA method using a measuring instrument such as a fluorometer, for example, “illustrated fluorescent antibody, Kawao Akira, 1st edition, Soft Science Co., Ltd., 1983”, etc. The measurement may be carried out in accordance with the method described in, when the labeling substance is a luminescent substance, a conventional method using a measuring device such as a photon counter, for example, “enzyme immunoassay, protein nucleic acid enzyme separate volume No. 31, Kitagawa Tsunehiro, Minamihara Toshio, Tsuji Akio, Eiji Ishikawa, edited, pp. 252-263, Kyoritsu Shuppan Co., Ltd., published September 10, 1987. Further, in the case where the labeling substance is a substance having absorption in the ultraviolet region, the measurement may be carried out by a conventional method using a measuring instrument such as a spectrophotometer, and when the labeling substance is a substance having a property as a spin labeling agent. Is an ordinary method using an electron spin resonance apparatus, for example, “enzyme immunoassay, protein / nucleic acid / enzyme separate volume No.31, edited by Tsunehiro Kitagawa / Toshio Minamihara / Akio Tsuji / Eiji Ishikawa, pages 262-171, Kyoritsu Shuppan Co., Ltd., The measurement may be performed in accordance with the method described in “September 10, 1987”.

It has not been known at all until now that PGL-1 analogs and anti-PGL-1 analog antibodies may be present in the serum of patients who are not infected with M. leprae.
It was a surprising fact that the present inventors discovered for the first time, and it was also very surprising that these are highly likely to be used as cancer markers. Hereinafter, the present invention will be described in more detail with reference to Reference Examples and Examples, but the present invention is not limited thereto.

[0022]

【Example】

Reference example 1. Preparation of anti-PGL-1 monoclonal antibody (1) Immunization PGL-1 bound to bovine serum albumin by a conventional method
Was performed by inoculating mice 3 times at 2-week intervals (inoculation amount per time: 50 μg / mouse). (2) Preparation of hybridoma Cell fusion of spleen cells and myeloma cells (P3 / NS1-1-Ag4-1) extracted from the mouse on the third day after the final immunization was performed by the method of Keller and Milstein (Nature, vol.256, 495, 1975) and screened by the following method. That is, a 0.2% bovine serum albumin solution prepared by dissolving it in 50 mM carbonate buffer (pH 9.6) or a 0.2% PGL-1 conjugated bovine serum albumin solution prepared by dissolving it in 50 mM carbonate buffer (pH 9.6) 50 μl of the above was dispensed into a 96-well microplate and incubated at 37 ° C. for 1 hour to immobilize. After washing with 10 mM phosphate buffered saline (pH 7.4), cell fusion was performed and the culture supernatant of various hybridomas was 50μ.
l was dispensed into the microplate and reacted at 37 ° C. for 1 hour. After washing with 10 mM phosphate buffered saline (pH 7.4),
Peroxidase-labeled anti-mouse immunoglobulin antibody (DAKO) appropriately diluted with 0 mM phosphate buffered saline (pH 7.4)
50 μl) was dispensed into the microplate and the temperature was adjusted to 1 at 37 ℃.
Reacted for hours. Then, after washing with 10 mM phosphate buffered saline (pH 7.4), with 2 mg / ml o-phenylenediamine
Citrate buffer (pH 4.9) containing 0.017% hydrogen peroxide 50
μl was dispensed into the microplate and left standing at room temperature for 10 minutes to cause a color reaction, and then 6N sulfuric acid was dispensed into the microplate to stop the color reaction. Based on the results obtained above, a hybridoma producing a culture supernatant that does not react with bovine serum albumin but reacts only with PGL-1-bound bovine serum albumin was selected and used as a parent hybridoma. This parent hybridoma was cloned by the limiting dilution method to produce an anti-PGL-1 monoclonal antibody-producing hybridoma GT.
-1 was established. (3) Preparation of Monoclonal Antibody 0.5 ml of pristane (2,6,10,14-tetramethylpentadecane, manufactured by Wako Pure Chemical Industries, Ltd.) was intraperitoneally injected into the abdominal cavity of a mouse 3 days after the injection. Hybridoma G obtained in 2)
1 × 10 ⁶ T-1 was inoculated. 1 after hybridoma inoculation
On the second day, ascites fluid collected in the abdominal cavity was collected, 10 ml of the obtained ascites fluid was fractionated with 40% ammonium sulfate, and then dialyzed against 10 mM phosphate buffered saline (pH 7.4) (1 liter). × 3 times),
An anti-PGL-1 monoclonal antibody solution was obtained.

Example 1. Detection of PGL-1 analogues (Procedure) Urine of patients with renal cancer serially diluted with 50 mM phosphate buffer (pH 7.4) was used as nylon membrane (Nitran 13N, Schlei).
cher and Schuell) and then 2μl each,
Air dried. The obtained nylon membrane was immersed in a 1% bovine serum albumin solution at 25 ° C for 1 hour, and then 50 mM phosphate buffer solution (pH
It was washed in 7.4). This is then treated with the anti-P obtained in Reference Example 1.
GL-1 monoclonal antibody solution (antibody titer 20 μg Ab / ml)
It was immersed in the solution at 25 ° C for 1 hour and then washed. The obtained nylon membrane was applied to an alkaline phosphatase-labeled anti-mouse immunoglobulin antibody solution (Kappel, antibody titer 10 μg Ab / ml) 25
After soaking for 1 hour at ℃, wash and then nitrotetrazolium blue (NBT) and 5-bromo-4-chloro-3-indolyl-phosphoric acid (X-phosphoric acid) (both are Boehringer Mannheim Yamanouchi Co., Ltd.) (Manufactured by K.K.) was used for color development. Instead of patient urine, 50 mM phosphate buffer solution (pH 7.4) was used to perform the same procedure as a negative control, and the dilution factor of urine giving a positive spot was calculated.
-1 It was determined that the analogue was positive. (Results) As a result of the above measurement, 7 out of 10 urine samples of kidney cancer patients were determined to be positive (positive rate: 70%). In addition, healthy person 8
The same operation as described above was performed on the urine of the first name, and the PGL-1 analog was detected.

Example 2. Detection of Anti-PGL-1 Analogue Antibody (Procedure) Fujiwara's method (Agric.Biol.Chem., Vol.51
(9), 2539-2547, 1987), and the sugar chain portion of PGL-1 synthesized by (9), 2539-2547, 1987) was bound to bovine serum albumin by a conventional method.
50 μl of this 0.1 mg / ml solution was dispensed into each well of a 96-well microtiter plate and incubated at 37 ° C. for 1 hour. Next, this microtiter plate was washed with 50 mM phosphate buffer (pH 7.4), and then 1% bovine serum albumin solution 100
Dispense μl into each well, incubate at 37 ° C for 1 hour for masking, and then use 50 mM phosphate buffer (pH 7.
Washed in 4). To each well of the obtained microtiter plate, 100 μl of renal cancer patient serum serially diluted with 50 mM phosphate buffer (pH 7.4) was dispensed and incubated at 37 ° C. for 1 hour, and then 50 mM phosphate buffer ( It was washed with pH 7.4). Then, a peroxidase-labeled anti-human immunoglobulin antibody solution (manufactured by Kappel, antibody titer 24 μg Ab /
ml) 50 μl was dispensed, incubated at 37 ° C for 1 hour, washed with 50 mM phosphate buffer (pH 7.4), and then 3,3 '
A 5,5'-tetramethylbenzidine (TMB, manufactured by Kappel) reagent was dispensed to cause a color reaction. Instead of patient serum
The same procedure was performed using 50 mM phosphate buffer (pH 7.4) as a negative control, and the serum dilution factor at which the OD 595 nm value was 2 times or more that of the negative control was obtained.
500 times or more was determined to be positive for anti-PGL-1 analogue substance antibody titer. (Results) When the anti-PGL-1 analogue substance antibody titer was measured for 40 sera of renal cancer patients, 29 of 40 sera were determined to be positive (positive rate: about 70%).

[0025]

INDUSTRIAL APPLICABILITY As described above, the present invention can be expected to be used as a cancer marker, especially as a renal cancer marker.
The present invention provides a rapid and highly accurate method for measuring GL-1 analog and anti-PGL-1 analog antibody, and is a great invention that contributes to the industry.

Claims (5)

[Claims] 1. A phenolic glycolipid derived from Mycobacterium leprae (hereinafter referred to as P
It is abbreviated as GL-1. An immunological assay method for a substance having an antigenicity similar to PGL-1 (hereinafter abbreviated as PGL-1 analogue) present in a biological sample, characterized by using an antibody against (1). 2. The measuring method according to claim 1, wherein the biological sample is plasma, serum, urine or spinal fluid. 3. PGL-1 present in a biological sample, characterized by using a carrier having PGL-1 or a part thereof immobilized thereon.
An immunological assay method for an antibody against a GL-1 analogue. 4. The measuring method according to claim 3, wherein the biological sample is plasma, serum, urine or spinal fluid. 5. The measuring method according to claim 3, wherein the carrier is latex particles, magnetic particles, gelatin particles, glass beads, plastic beads or a microtiter plate.