JP2915530B2 - Laminin fragment - Google Patents

Description

The present invention relates to a novel human laminin fragment, a method for detecting a disease using the amount of the fragment as an indicator, and an immunoassay kit for the fragment.

[Conventional technology]

The basement membrane is composed of components of collagen and non-collagen, and is an extracellular matrix that is universally present in a living body. In recent years, attention has been paid to the relationship between various diseases that progress irreversibly, such as liver disease, kidney disease and cancer, and the basement membrane. Laminin (hereinafter abbreviated as LN) is a high-molecular protein that exists specifically in the basement membrane, and is unfragmented laminin (hereinafter abbreviated as nLN) or fragmented laminin when the basement membrane is degraded. Hereinafter, abbreviated as fLN) is considered to be released into body fluids.
Measuring nLN or fLN is important for clinical tests in diagnosing the progression of degradation of basement membrane due to disease, and establishment of a simple and rapid measurement method is desired.

[Problems to be solved by the invention]

Recently, a radioimmunoassay using eel antiserum against fLN by pepsin degradation has been developed [DG Brocks, Clinical Chemistry 32, 787-791 (198
6)].

In this assay, a radioactively labeled pepsin-degraded fLN and a sample are competitively reacted with an anti-pepsin-degraded fLN antibody. By measuring the activity, LN in the sample
It measures the amount. The radioimmunoassay uses rabbit antiserum to fLN by pepsin degradation,
There is a problem in terms of specificity by not using fLN in body fluid as an antigen, and when using an antiserum as an antibody to the target antigen, the binding force of the antibody to the antigen thus fragmented is reduced, It was thought that accurate measurements were not possible, and this also involved a major problem.

In addition, urinary LN measurement is present at a low concentration of about 1/10 or less of blood LN, although it has important clinical significance in the analysis of pathological conditions such as diabetic nephropathy. For this reason, it was not possible to quantify with sufficient sensitivity using conventional measurement methods.

Although the above problem can be solved by using a monoclonal antibody that can also recognize fLN in body fluid instead of antiserum, the structure of fLN molecules in body fluid that can be a target antigen has not been identified, and To measure fLN in
Identification of the fLN was required.

An object of the present invention is to identify fLN in a human body fluid,
To provide a method for specifically and highly sensitively measuring, a kit for measurement, and a method for detecting a disease by measuring the amount of fLN.

[Means for solving the problem]

In general, the present invention relates to human fLN, which is obtained from a human body fluid and has a molecular weight of about 42,000 by SDS-polyacrylamide gel electrophoresis, and has the following formula I: (Wherein X represents an unidentified amino acid residue)
It has a terminal amino acid sequence.

The human fLN of the present invention can be purified from, for example, urine of a healthy subject using, for example, an anti-human nLN polyclonal antibody column, an ion exchange resin column, or the like. Purified human fL
N was detected as a band having a molecular weight of about 42,000 by, for example, 10% SDS-polyacrylamide gel electrophoresis.
Compared with LN (molecular weight about 800,000), it is extremely low molecular weight. The human fLN has an amino acid sequence of the above formula (I) at its N-terminus, which is the amino acid sequence of human nLN [T. Pikkarainen et al., Journal of Biological Chemistry (J. Biol. Chem.), No.
263, p. 6751 (1988)], from the N-terminus of the B2 chain.
Matches the amino acid sequence at the 18th position. That is, the fLN
Is identified as being composed of the domains V and VI of the B2 chain of human nLN, and is the first human isolated and identified in human body fluid
fLN.

The method for measuring human fLN of the present invention includes, for example, a method using HPLC, an immunological method, and the like.An immunological method using a specific antibody against the human fLN enables easy and high sensitivity in a sample. Human fLN can be measured. In the present invention, a sample refers to a human body fluid, for example, serum or urine.

The specific antibody against human fLN of the present invention refers to the human fLN
However, it is preferable to use a pair of monoclonal antibodies that recognize the human fLN and have different antigen recognition sites from the viewpoints of sensitivity, operability, and the like. Examples of the monoclonal antibody include monoclonal antibodies HLN5 and HLN82 prepared by the present inventors,
Using the pair of antibodies, human fLN in a sample can be measured with high sensitivity, for example, by an enzyme immunoassay.

For example, if fLN in urine of a patient such as a malignant tumor, liver disease, kidney disease, or arteriosclerosis is measured, the urinary fLN value is higher than that in a healthy person, and diagnosis of these diseases is performed using a urine sample. It can be easily performed.

In addition, even if a serum sample is used, these diseases can be detected with high sensitivity.

Specific antibodies against human fLN of the present invention include, for example, human nLN
Can be prepared as an antigen, and human nLN for obtaining the antibody.
For example, from the human placenta, by a method known per se,
For example, it can be obtained by separation and purification using methods such as salt precipitation, ion exchange chromatography, and affinity chromatography alone or in combination [Journal of Biological Chemistry, vol. 258, pp. 12654-12660 ( 1983)]. Further, human fLN is, for example, human nLN obtained by the above method,
According to a method known per se, for example, proteases such as pepsin, trypsin, thrombin, plasmin, and chymotrypsin are subjected to limited or complete degradation using alone or in combination, and various separation methods such as gel filtration, ion exchange chromatography, and affinity It can be obtained by separation and purification using a method such as chromatography, and used as an antigen for antibody preparation.

Further, human fLN of the present invention, for example, lung cancer, stomach cancer, colon cancer, breast cancer, malignant tumor patients such as pancreatic cancer, kidney disease patients, patients with arteriosclerosis or liver disease purified from serum or urine as a raw material, It is also possible to collect. Furthermore, in recent years humans
The synthetic peptide having the amino acid sequence of human fLN of the present invention based on the determination of the partial amino acid sequence of LN can also be used.

Specific antibodies against human fLN of the present invention include, for example, human fLN of the present invention purified from urine of the above-mentioned patients as an antigen, and non-human mammals such as guinea pigs, rabbits, rats, mice, goats and the like. After immunization using an animal capable of producing an antibody according to a conventional method, blood is collected to obtain an antiserum, and the antibody is further separated. To obtain the antibody, for example, 0.1 to 1 mg of the human fLN of the present invention is dissolved in 0.1 to 5 ml of physiological saline, and the same amount of complete Freund's adjuvant is added thereto. For example, it is injected subcutaneously or intradermally into rabbits or mice, and injected several times every 1 to 3 weeks for immunization. Thereafter, after a certain period from the day of the final immunization, blood is collected to obtain an antiserum containing an antibody against human fLN of the present invention.

As the animal used in this case, any animal may be used as long as it has an antibody-producing ability, and it is preferable to use a large animal in order to obtain a large amount of antibody.
Goats are used, but are not limited in any way. Furthermore, in order to obtain an anti-human fLN antibody from an anti-serum containing an anti-human fLN antibody obtained from these animals, a method commonly used for antibody purification can be used, for example, by fractionating antisera with ammonium sulfate, Then ion exchange chromatography,
Alternatively, it may be purified and collected by gel filtration. For further purification to high purity, it may be obtained by adsorption by affinity chromatography using an insolubilized carrier on which human fLN is immobilized as a substrate, followed by elution. As a further alternative, spleen cells of a non-human mammal immunized with the human fLN of the present invention as an antigen are fused with myeloma cells, and from the fused cells, a monoclonal antibody-producing cell against human fLN of the present invention is produced. There is a method for producing an anti-human fLN monoclonal antibody using the fused cells, and a method using a mouse as a mammal is often used [Nature, vol. 256, p. 495 (1975) )]. As described above, a domain-specific antibody against human LN is obtained.

On the other hand, the monoclonal antibody against human fLN of the present invention can be obtained using the above human nLN as an antigen. That is, spleen cells of a non-human mammal immunized with human nLN as an antigen are fused with myeloma cells, and a clone producing the monoclonal antibody against human fLN of the present invention is isolated from the fused cells. The monoclonal antibody thus obtained is a monoclonal antibody that reacts with the human fLN of the present invention. Mice are often used as animals.

The thus obtained monoclonal antibody against human fLN of the present invention can effectively detect the human fLN of the present invention by using a combination of antibodies having different antigen recognition sites.

The fLN-specific antibody used in the measurement of human fLN of the present invention may be either a polyclonal antibody such as an antiserum or a monoclonal antibody, as described above, but has high specificity and high affinity. In this regard, it is particularly desirable to use a monoclonal antibody.

As the assay using the monoclonal antibody against human fLN of the present invention, immunoassays well known in the art, namely, enzyme immunoassay, radioimmunoassay immunoturbidimetry, latex agglutination, hemagglutination, SRID Method (immune diffusion method) or the like is used. Among them, the enzyme immunoassay is most practical in terms of sensitivity, simplicity and the like. That is, an anti-human fLN monoclonal antibody is treated with an insoluble carrier such as polystyrene beads, glass beads, or a polystyrene microtiter plate, and covalently or physically adsorbed to these carriers to form an insoluble antibody bound with the anti-human fLN monoclonal antibody. obtain. On the other hand, the anti-human fLN monoclonal antibody is labeled with an enzyme using a conventionally known method. For example, a compound (eg, β
-M-maleimide ester for galactosidase, periodate for peroxidase) and then the antibody are coupled to the reaction to obtain an enzyme-labeled anti-human fLN monoclonal antibody.

Using the thus obtained anti-human fLN monoclonal antibody-binding carrier (insoluble antibody) and enzyme-labeled anti-human fLN monoclonal antibody (labeled antibody), the amount of human fLN in blood and urine was measured. Compared with malignant tumor patients, patients with renal disease, patients with arteriosclerosis, in the blood and urine of patients with liver disease, it was found that the concentration of the human fLN of the present invention increased, It was shown to be useful for the diagnosis of the disease.

By using the anti-human fLN antibody used for measuring the amount of human fLN of the present invention as a kit, the amount of the human fLN of the present invention in a sample can be easily measured. The reagent used in the kit may be in the form of a solution or a lyophilized product.

〔Example〕

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

Reference Example 1 (1) Isolation of human nLN antigen For extraction of human nLN antigen, human placenta was extracted with 0.5M NaCl
After homogenizing in 0.05M Tris-HCl buffer (pH 7.2) containing, and removing the insolubilized substance by centrifugation, NaCl was added to a final concentration of 4M, and the resulting precipitate fraction was collected. Collect. Collect collected precipitate to 0.05M with 0.5M NaCl
The fraction solubilized in Tris-HCl buffer (pH 7.2) is fractionated on a CL-6B Sepharose column equilibrated with the same buffer, and the fraction eluted in the first peak is a human nLN sample Collected as.

(2) Preparation of polyclonal antibody against human nLN antigen 0.5 mg of the purified human nLN antigen obtained in the above (1) was dissolved in 0.5 ml of physiological saline, and an equal amount of complete Freund's adjuvant was added thereto to emulsify. The rabbit was then injected subcutaneously. Every two weeks, the same amount of antigen was emulsified with incomplete Freund's adjuvant and injected subcutaneously four times. The whole blood was collected 10 days after the final immunization, left at room temperature for 60 minutes, and centrifuged. By separating, an antiserum containing an anti-human nLN antibody was obtained. Protein A is obtained from this antiserum by a standard method.
The anti-human nLN polyclonal antibody was purified by the column.

Example 1 (1) Purification and identification of human fLN in urine The anti-human nLN polyclonal antibody obtained in Reference Example 1- (2) was adsorbed to CNBr-activated Sepharose (Pharmacia) by a conventional method. Normal human urine 5 was passed through the column, and the adsorbed fraction was eluted with PBS containing 8 M urea. The crude human urine fLN fraction eluted from the column was subjected to Mono-Q
Fractionation was performed using an ion exchange column (Pharmacia) to obtain purified human urine fLN. When purified human urine fLN was separated by 10% SDS-polyacrimamide gel electrophoresis, a single band was observed at a molecular weight of about 42,000, and the target human urine fLN was significantly smaller than human nLN (molecular weight 800,000). It was confirmed that the fragment was formed into a low molecular fragment.

When the N-terminal amino acid sequence analysis of this human urinary fLN was performed, the sequence of the above formula (I) was detected.
LNB 2 chain amino acid sequence (T. Pickalainen et al.,
Journal of Biological Chemistry, Chapter
263, p. 6751 (1988)].
Since it completely matched the second sequence, it was confirmed that human urinary fLN was composed of a polypeptide having a molecular weight of 42,000 (domains V and VI) derived from the human LNB2 chain N-terminus.

Example 2 (1) Preparation of Monoclonal Antibody Against Human fLN 50 μg of human nLN obtained in Reference Example 1- (1) was dissolved in 1.0 ml of physiological saline, emulsified by adding an equal amount of Complete Freund's adjuvant, and emulsified by Balb / c. The mice were injected intraperitoneally. 4
One week later, only 50 μg of the antigen was injected intraperitoneally into the mouse. Three days later, spleen cells were obtained from the spleen removed from the mouse, mixed with mouse myeloma cells (P3-X63-Ag8-U1) at a cell ratio of 10: 1, and mixed with 50% polyethylene glycol and 20% dimethyl sulfoxide. The cells were allowed to stand in the presence for 1 minute to perform cell fusion. After adding and diluting a serum-free DMEM medium, the supernatant is removed by centrifugation, and the cells are suspended in a DMEM medium containing 10% fetal bovine serum, to give 2 × 10 ⁴ cells per well in a 96-well microtiter plate. Was dispensed as follows. Thereafter, half of the medium was replaced with a HAT medium every 1 to 3 days, and after 10 to 20 days, the culture supernatant of the well in which the fused cells (hybridomas) had grown was collected, and the presence or absence of antibody production was determined.
It was examined by ELISA or the like, and 5 hybridomas producing an antibody against human nLN were selected.

Of the above five hybridomas, Example 1- (1)
Three strains producing antibodies reactive with human urinary fLN obtained in the above were selected.

These hybridomas were subjected to limiting dilution by 2
Cloning was performed twice, and clones HLN5 and HLN5 were used as hybridoma clones producing the highest titer antibodies.
Acquired two strains of HLN82.

The clone strains are designated as Hybridoma HLN5 and designated as Microtechnological Laboratory No. 11727 (FERMP-11727), and designated as Hybridoma HLN82 and designated as Microtechnological Laboratory Microorganism No. 10799 (FERM P-10799). Deposited at the Industrial Technology Research Institute.

In order to confirm that the monoclonal antibodies produced by these two hybridomas have the same fragment specificity, Example 1 was performed by Western blotting.
It was confirmed that both reacted with the human urinary fLN obtained in (1) and had different antigen recognition sites. In order to obtain these monoclonal antibodies in large quantities, about 2 × 10 ⁷ hybridomas were injected intraperitoneally into Balb / c mice, ascites tumors were formed, and ascites was collected 10 days later, and the anti-human fLN monoclonal antibody HLN5 And HLN82.

(2) Preparation of anti-human fLN monoclonal antibody-bound beads Anti-human fLN monoclonal antibody HLN82 obtained in (1) above
20 ml of 0.1 M phosphate buffer (pH 8.0) containing 1 mg of
Polystyrene balls (Sekisui Chemical Co., particle size 6.35mm) 10
0 grains were added and reacted at 5 ° C. for 16 hours and at 37 ° C. for 1 hour to immobilize the antibody on the beads. After washing the beads sufficiently with physiological saline, the beads are immersed in 10 mM phosphate buffer (pH 7.4) containing 1% bovine serum albumin (BSA), 0.05% sodium azide, and 0.9% NaCl, and left at 5 ° C overnight. Thus, beads binding to an anti-human fLN monoclonal antibody were obtained.

(3) Preparation of anti-human fLN monoclonal antibody enzyme-labeled product Anti-human fLN monoclonal antibody H obtained in (1) above
Peroxidase (Boehringer-Mannheim) was added to LN5 according to the method of Nakane et al. [Journal of
Histochemistry and cytochemistry (J.Hist
ochem. Cytochem.), Vol. 22, p. 1084 (1974)] to obtain a labeled antibody. That is, 10 mg of peroxidase is dissolved in 2 ml of purified water, and 0.2 ml of 0.1 M potassium periodate is added. After reacting at room temperature for 20 minutes, the mixture is dialyzed overnight at 4 ° C. against 1 mM acetate buffer (pH 4.0). To this was added 0.2M carbonate buffer (pH 9.5) to adjust the pH to 9-9.5.
Adjust to On the other hand, 2 mg of anti-human fLN monoclonal antibody
Dissolve in 1.5 ml phosphate buffered saline (pH 7.4) and add 10 mM
It was dialyzed overnight at 4 ° C. against a carbonate buffer (pH 9.5), mixed with the above periodate-treated peroxidase, allowed to react at room temperature for 2 hours, and then sodium borohydride (4 mg / d). 0.1 ml), reacted at 4 ° C. for 2 hours, and fractionated by gel filtration using Ultrogel AcA22 (manufactured by LKB) equilibrated with phosphate buffered saline (pH 7.4). Fractions having the same peroxidase activity and antibody activity were collected, added to a final concentration of 0.01% sodium merthiolate, and stored at 4 ° C.

Example 3 (1) Measurement of human fLN The EIA method was performed as follows. 200 μl of the sample is placed in a tube, and the insolubilized antibody beads are placed one by one in the tube, and the first incubation is performed at 37 ° C. for 1 hour. Next, the beads are washed three times with 3 ml of physiological saline, 200 μ of the labeled antibody solution (300-fold dilution) is placed in the tube containing the beads, and a second incubation is performed at 37 ° C. for 1 hour. The beads were then washed three times with 3 ml of physiological saline, and the beads were transferred to another tube, to which 300 μl of a coloring reagent (o-phenylenediamine 1 mg / ml, 0.01% of H ₂ O ₂ , 0.1 M citrate buffer pH 5) was added. .0) and reacted at 37 ° C. for 30 minutes. The reaction was stopped by adding 1 ml of 1N H ₂ SO ₄ . Wavelength 4
The absorbance at 92 nm was measured.

In such a procedure, 19 cases of malignant tumor, 27 cases of liver disease, 10 cases of kidney disease, 9 cases of arteriosclerosis, and serum of 18 cases of healthy persons, and 32 malignant tumors, 16 cases of liver disease, 14 cases of kidney disease For example,
The amount of human fLN in urine of 13 cases of arteriosclerosis and 21 healthy subjects was measured. In urine, the amount of creatinine is simultaneously measured using a commercially available kit (creatinine test Wako: manufactured by Wako Pure Chemical Industries, Ltd.), and the ratio of the amount of fLN to the amount of creatinine (fLNμg / g · Cr ).

FIG. 1 is a graph comparing the distribution of human fLN in serum between various patients and healthy individuals, and FIG. 2 is a graph comparing the distribution of urinary human fLN amounts between various patients and healthy individuals.

As a result, in the combination of monoclonal antibodies HLN5 and HLN82, human fLN in urine and serum can be measured, and the amount of human fLN in serum and urine of various patients is clearly higher than that in healthy subjects, and this measurement was performed in disease diagnosis. Has been shown to be useful.

Example 4 (1) Preparation of Kit The composition of the EIA kit (one dose) is shown below.

1. One antibody-immobilized bead [Hybridoma micro-technical research bacterium No. 10799 (FERM P-10799) deposited at the Research Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology)
Beads with immobilized monoclonal antibody HLN82 produced by E. coli. 2. Peroxidase-labeled antibody 0.3 ml [Hybridoma Microtechnical Laboratory No. 11727 deposited with the Institute of Microbial Engineering, National Institute of Advanced Industrial Science and Technology (FERM P-11727)
Was labeled with peroxidase.) 3. Coloring reagent 0.3 ml (10 mg / ml of o-phenylenediamine HCl, hydrogen peroxide 0.
Citrate buffer solution containing 01% pH5.0) 4. Reaction stop solution (1N sulfuric acid) 1.0 ml 5. Standard (4, 2, 1, 0.5 μg / ml of human fLN of the present invention)
1% BSA / TBS containing) [Effect of the Invention] As described in detail above, the present invention provides
An LN measurement reagent was provided. The measurement reagent of the present invention has made it possible to diagnose novel malignant tumors, liver, kidney, and arteriosclerosis.

[Brief description of the drawings]

FIG. 1 is a graph showing the results of measurement of serum human fLN levels, and FIG. 2 is a result of measurement of urinary human fLN levels for various patients and healthy persons, respectively, using the present measurement reagent.

──────────────────────────────────────────────────続 き Continuing on the front page (72) Inventor Ikunoyuki Kato 3-4-1, Seta, Otsu City, Shiga Prefecture Takara Shuzo Co., Ltd. Central Research Laboratory (56) References JP-A-3-42572 (JP, A JP-A-3-27296 (JP, A) Biol. Chem. , Vol. 14 (1988) p. 6751-8 (58) Field surveyed (Int.Cl. ⁶ , DB name) C07K 14/78 REGISTRY (STN) CA (STN) WPI (DIALOG) BIOSIS (DIALOG) JICST file (JOIS)

Claims (1)

(57) [Claims] (1) a molecular weight of about 42,000 as determined by SDS-polyacrylamide gel electrophoresis obtained from a human body fluid; (Wherein X represents an unidentified amino acid residue)
A human laminin fragment having a terminal amino acid sequence.