JP4505800B2 - Anti-renal type IV monoclonal antibody - Google Patents

Description

  The present invention relates to a nephritis detection method and detection reagent using renal type IV or anti-renal type IV monoclonal antibody. In addition, it includes therapeutic tools and medicines.

Conventionally, urinary protein, albumin, and type IV collagen have been measured as main indicators for nephritis detection. In particular, in the case of diabetic nephropathy, which is secondary glomerulonephritis, the measurement of this was noted for a while because type IV collagen appears in urine earlier than albumin. That is, when glomerulonephritis occurs, the glomerular basement membrane with filtration function breaks, the filtration hole expands, and serum protein and albumin flow out into the urine, but type IV collagen, which is a component of the basement membrane. Appeared in the urine at the time of the destruction of the basement membrane before the enlargement of the filter pores, so that glomerulonephritis could be detected early.
Further, a conventional method for definitive diagnosis of nephritis has been to stain kidney sections obtained by renal biopsy and observe immunoglobulin deposition, meniscus formation, and the like. There was no way to distinguish between disease and normality by immunostaining.

Problems to be solved by the invention

These had the following problems.
Conventional measurement of type IV collagen in serum and urine uses placenta type IV as an antigen (anti-placenta type IV antibody) and standard placenta type IV. This is because the difference due to the type IV collagen-derived tissue was not taken into account, and is not suitable for the measurement of renal type IV appearing in glomerulonephritis.
In addition, the conventional definitive diagnosis requires advanced diagnostic techniques by experienced pathologists. In addition, glomerulonephritis sometimes takes decades to reach the immunoglobulin deposition stage, and gin function is also significantly reduced. Therefore, it has been desired that a definitive diagnosis can be performed very easily and accurately at an early stage in which specific pathological images such as immunoglobulin deposition and meniscus formation are not observed.

Means for solving the problem

The present invention provides a method and a reagent capable of detecting nephritis at an early stage by using a glomerulus-derived type IV collagen spiral region (kidney type IV), and further detecting serum nephritis. Is to provide.
Type IV collagen is widely present in the basement membrane of living tissue and consists of three α chains. Six types of α chains from α1 to α6 are known, and the configuration of the three α chains differs depending on the tissue. In the placenta, α3 and α4 chains are not found, rich in α1 and α2 chains, and in the glomerular basement membrane, it is rich in α3 and α4 chains. Furthermore, the homology of the amino acid sequence between each α chain was determined by DNASIS Mac ver3.5. The homology of the other α chain to the α3 chain amino acid sequence 1201-1439 is 47% for the α1 chain and 46% for the α2 chain. Similarly, the homology of the other α chain to the α4 chain amino acid sequence 1229-1459 is 49 for the α1 chain. %, Α2 chain was 45%, and although it was a partial measurement, both were 50% or less. On the other hand, whether or not kidney type IV of different animal species can be used for measurement of other animals was examined from the homology of amino acid sequences as described above. As a result, bovine α3 chain was 76% of human α3 chain and bovine α4 chain was 82% of human α4 chain. Therefore, it can be said that the same tissue of different animals has higher homology than the different tissues of the same animal. Therefore, for measurement of renal type IV and anti-renal type IV antibody, it is better to use kidney type IV, preferably kidney type IV of the same animal, rather than placenta type IV as the origin.
Therefore, the present inventor first developed an anti-renal type IV antibody detection method by ELISA using bovine kidney type IV as an antigen, and found the appearance of this antibody in glomerulonephritis urine. At the same time, when human placenta type IV and bovine placenta type IV were measured as antigens, it was found that the response of anti-placenta type IV antibodies in urine was weak against both human and bovine placenta type IV. Therefore, the measurement of serum anti-renal type IV antibodies in urine and preceding serum is a useful indicator for detecting glomerulonephritis. Next, the inventor of the present application prepared a kidney type IV measurement kit in urine and serum using an anti-renal type IV antibody obtained by immunizing an animal using kidney type IV as an antigen. The measurement kit may be a sandwich ELISA method using only polyclonal antibodies, but a sandwich ELISA method incorporating one or more monoclonal antibodies is desirable.
Specifically, kidney type IV isolated and purified from bovine kidney glomeruli is sensitized to mice to produce anti-renal type IV monoclonal antibodies, which are also suitable for Western blotting and immunostaining of kidney biopsy tissues. Obtaining “anti-renal type IV monoclonal antibody” and “labeled anti-renal type IV monoclonal antibody”, and “anti-renal type IV monoclonal antibody” incorporated “anti-kidney type IV monoclonal antibody” ELISA kit by kidney method It is.

The procedure is as follows.
1 [Separation and purification of antigen] Using bovine kidney glomeruli as a raw material, a type IV collagen triple chain region (hereinafter referred to as kidney type IV) is separated and purified by column chromatography (J. Biol. Chem., 263). , 10481-8).
2 [Preparation and Selection of Anti-Renal Type IV Monoclonal Antibody] Monoclonal antibodies are prepared using a mouse by a conventional method (Monoclonal Antibody Experiment Manual. Kodansha Publishing Co., Ltd., 1987). In screening of fused cells, many positive holes with high antibody titers are selected by ELISA, and then antibodies that react with kidney type IV are selected by Western blot. Subsequently, immunostaining is performed to select those that react with glomeruli of monkey anti-glomerular basement membrane (GBM) antibody nephritis. In addition, choose one that does not react with monkey normal kidney. In this manner, an anti-renal type IV monoclonal antibody that can be obtained by ELISA, Western blotting, or immunostaining can be obtained.
Of course, the anti-renal type IV monoclonal antibody may have only one function of ELISA, Western blotting, immunostaining, cancer cell growth inhibition, or other uses, or may have multiple functions. Anything that works is desirable. Immunostaining may react with normal kidneys, such as polyclonal antibodies prepared from rats, goats, rabbits, etc., but cannot be used for discrimination from nephritis. It can be used to confirm the existence of an organization.

The “anti-renal type IV monoclonal antibody” and “fluorescently labeled kidney type IV monoclonal antibody” of the present invention are the anti-glomerular basement membrane (GBM) antibody nephritis in the former by the indirect staining method and the latter by the direct staining method. Alternatively, the glomerular basement membrane of the kidney is stained with a pathological section of human IgA nephropathy. Of course, rats, mice, other animal species, and various types of nephritis other than IgA nephropathy are similarly stained.

The present inventor has also established the following specific means for the “renal type IV measurement ELISA kit” for the initial detection of glomerulonephritis. That is, the method for detecting renal type IV occurring in glomerulonephritis from the urine of a patient and the measurement reagent are illustrated side by side in the case of serum, but the present invention is not limited to the measurement method and reagent described. .

1 Method and reagent for detecting kidney type IV from serum and / or urine.
As reagents, 1) a plate coated with anti-renal type IV antibody (derived from rabbit), 2) enzyme (HRP) -labeled anti-renal type IV monoclonal antibody, 3) chromogenic substrate (TMB), 4) reaction Measure using stop solution (sulfuric acid).
Here, “2)” may be unlabeled and “enzyme (HRP) labeled anti-mouse IgG (or IgM) antibody” may be added as “2) -2”. In addition, the antibody portions “1)” and “2)” may be interchanged, or both may be monoclonal antibodies or polyclonal antibodies. Any immunized animal that produces antibodies can be used, and any animal species such as goats, pigs, rabbits, rats, mice, horses, cows, chickens, and monkeys can be used.
At this time, it is most desirable that the standard is produced from human kidney when a human is a subject, and it is more desirable that the homology of the amino acid sequence with the animal to be measured is closer.

As an immune reaction, an enzyme immune reaction is typically exemplified, but is not limited thereto, and includes an AB method, an RIA method, an immunoluminescence method, a precipitation reaction, an agglutination reaction, and the like. In the enzyme immunoreaction, the enzyme-labeled antibody may be a polyclonal or monoclonal antibody. Further, it may be a radioactive substance (RIA method), a substance labeled with a luminescent substance (immunoluminescence method), or a non-labeled substance (precipitation method or aggregation method).
The reaction format is not limited to the sandwich method, and may be a competitive method or the like, but the sandwich method is particularly desirable. As a constitution of the measurement reagent, the plate for coating the anti-renal type IV antibody may be glass or a magnetic substance, and it may be omitted without using the solid phase method.
When an anti-renal type IV antibody (hereinafter referred to as an antibody) is coated on a plate, it may be an indirect coating, and the coating substance may be avidin, biotin, or a component to which these are bound.
The antigen may be not only a biological extract or a recombinant but also a constituent peptide (including a specific fraction and a synthetic product), and an antibody may be prepared from these antigens.
The animal species of the antigen used for the measurement reagent is preferably human, and it may be monkey, cow, pig, chicken, sheep, goat, rabbit, rat or other animal, and is not limited thereto. Furthermore, the antigen may be a mixture of multiple animal species.
The antigen-derived tissue is preferably the kidney, but is not limited thereto.

2 An instrument for removing anti-renal type IV antibody and / or an instrument for removing renal type IV.
When blood is passed through an affinity column prepared with an anti-renal type IV antibody, only renal type IV in the serum is removed. Subsequently, the blood is passed through an affinity column made of kidney type IV, the anti-renal type IV antibody in the serum is removed, and the blood from which the antibody and antigen have been removed is returned to the body again. Using this principle, when tested in a rat nephritis model (sensitized with “K35 NC1” (produced by Collagen Technical Training Institute)), urine after treatment contains 1/5 of the antigen and antibody before treatment. It became the following. Conventional dialysis is patient serum and no such difference is seen before and after dialysis. Of course, as long as it is a device that can remove kidney type IV and anti-renal type IV antibodies in the blood, it is not limited to the aforementioned devices. In addition, even if the kidney type IV or anti-renal type IV antibody is replaced with α3 chain, α4 chain or α5 chain, or an aggregate thereof or an antibody thereof, the α3 chain, α4 chain or α5 chain which causes nephritis, or these It may be replaced with a specific part of the aggregate and / or its antibody. The antibody used in the instrument may be either polyclonal or monoclonal, but more preferable is a monoclonal because it has a semipermanently the same performance.

The invention's effect

The present invention is useful for early detection, definitive diagnosis of nephritis, and improvement of nephritis and cancer patients.

[Separation and purification of antigen] Using bovine kidney glomeruli as a raw material, a type IV collagen triple chain region (hereinafter referred to as kidney type IV) is separated and purified by column chromatography (J. Biol. Chem., 263, 10481). -8).
[Preparation and selection of antibody] The antibody is prepared using a mouse (Monoclonal antibody experiment manual. Kodansha 1987). In screening of fused cells, many positive holes with high antibody titers are selected by ELISA using the culture supernatant. Next, after growing cells in the abdominal cavity of the mouse, ascites is collected, and one that responds to renal type IV is selected by Western blotting. Subsequently, immunostaining is performed using a normal kidney of a cynomolgus monkey and a kidney of a monkey nephritis model (anti-GBM antibody nephritis), and one that reacts with the glomeruli of monkey anti-GBM antibody nephritis but does not react with a monkey normal kidney is selected.
(The cynomolgus monkey nephritis model created here differs from the previously reported administration site in that it is made with 1 mg of the initial NC1 and 3 mg of booster immunity in the back skin. No difficulty and low chance of infection)
Since the anti-renal type IV monoclonal antibody is not labeled with fluorescence, an indirect method is used for immunostaining. At this time, a commercially available rhodamine-labeled anti-mouse IgG (H + L) antibody (Cortex Co., Ltd.) was used for the secondary reaction. CB1545, absorbed by human serum, diluted 50-fold) reacts with other types of immunoglobulins (in this case, IgG deposited on monkey kidney glomeruli), that is, a nonspecific reaction is feared. Therefore, when rhodamine-labeled anti-mouse antibody is sprinkled directly on the glomeruli of monkey anti-GBM antibody nephritis, it is stained slightly. However, staining is clearly weaker than indirect immunostaining using the product of the present invention.
Furthermore, an anti-renal type IV polyclonal antibody (rabbit derived) was prepared, and a normal monkey kidney and a kidney of a nephritis model were compared by an indirect staining method. As a result, both normal kidneys and nephritis model kidneys are stained. Conversely, the anti-renal type IV monoclonal antibody of the present invention stains better towards nephritis.
Therefore, the anti-renal type IV monoclonal antibody of the present invention is a staining reagent useful for identifying glomerulonephritis.
In fact, anti-renal type IV monoclonal antibodies selected in this way stain glomerular basement membranes and tubules in kidney frozen sections of human glomerulonephritis, eg IgA nephropathy.
Furthermore, for the confirmation of the characteristics of this antibody, immunological reaction by Western blotting using type IV collagen (derived from human placenta, treated with pepsin) and type IV collagen (derived from bovine kidney glomerulus, treated with pepsin) as antigens. , It reacts with kidney type IV but not with placenta type IV.
It is an anti-renal type IV monoclonal antibody that can be used for ELISA, Western blotting, and immunostaining.
[Fluorescent labeled anti-renal type IV monoclonal antibody]
The fluorescent substance is a condition (0.05M carbonate buffer, pH 8.0 to 9.0, room temperature × 30 minutes + 4C × 2 hours), which is more relaxed than the literature (“Basic Technology for Immunological Research”, page 25, published by Yodosha). Label with IgG: FITC = 1: 4 (molar ratio) /MW.IgG160,000 FITC400 / = 10 mg: 0.1 mg), and remove the free fluorescent substance with the column. After labeling, dissolve in PBS (pH 7.4) and compare the antibody titer with unlabeled anti-renal type IV monoclonal antibody by ELISA to confirm that no antibody degradation has occurred. The protein concentration (IgG concentration) is determined by measuring the absorbance (A280 nm).
Since F / Pratio = 0.6-3.0 has the ability as a fluorescence labeled antibody, this is used, and 6.0 or more is not used because reactivity with kidney type IV is lost. In addition, staining of a kidney section of monkey anti-GBM antibody nephritis with an anti-renal type IV monoclonal antibody (hereinafter referred to as a monoclonal antibody) is confirmed by direct immunostaining. Furthermore, the pathological section of human nephritis is confirmed.

[A device for removing anti-renal type IV antibody and / or a device for removing renal type IV]
When blood is passed through an affinity column prepared with an anti-renal type IV antibody, only renal type IV in the serum is removed. Subsequently, the blood is passed through an affinity column made of kidney type IV, the anti-renal type IV antibody in the serum is removed, and the blood from which the antibody and antigen have been removed is returned to the body again. From the cynomolgus monkey (male, body weight, estimated age) of the nephritis model, 4 ml of blood corresponding to serum is collected and returned to the blood vessel again through the two kinds of affinity columns described above. Repeat this operation three times. As a result, when the antibody titer in urine before and after the work was measured, the antibody titer decreased to 1/5.

[Anti-renal type IV antibody measurement kit using renal type IV as an antigen] and [A renal type IV measurement kit using an anti-renal type IV antibody prepared using renal type IV as an antigen]
In order to obtain kidney type IV, glomerular basement membrane is taken out from bovine kidney and extracted by pepsin degradation in a conventional manner. At this time, the NC1 fine powder that may be mixed is removed with an anti-NC1 antibody affinity column prepared separately. By performing this operation, type IV collagen derived from pure kidney can be obtained. Further, by using this as an antigen, a highly specific antibody can be obtained.
"Antibody measurement kit"
A 96-well plate was coated with antigen (1) kidney type IV, 2) human placenta-derived pepsin solubilized type IV collagen, added with 100 ul of sample, reacted at room temperature for 2 hours, and then labeled with HRP-labeled antibody ( Anti-mouse when the sample is derived from a mouse, anti-rabbit when it is a rabbit, and anti-goat when it is a goat), react for 1 hour at room temperature, add TMB solution, react for 10 minutes at room temperature, and then 1N Stop the reaction with sulfuric acid and immediately measure at 450 nm.
Specimen and result;
Specimen / anti-human placenta-derived type IV collagen monoclonal antibody (immunized animal / mouse, 3 types; 1A, 1B, 1E);
1) Both are negative (the background is subtracted, the same shall apply hereinafter)
2) 1A / 1.826, 1B / 2.188, 1E / 2.222
Specimen / anti-human placenta-derived type IV collagen polyclonal antibody (immunized animal / rabbit, YOKO); 1) 2.391 2) 2.231
Sample / marketed anti-human placenta-derived type IV collagen monoclonal antibody (immunized animal / mouse, F59); 1) 0.047 2) 2.135
Sample / anti-human placenta-derived type IV collagen polyclonal antibody (immunized animal / goat)
1) 0.450 2) 2.037
・ Sample / Human glomerulonephritis patient urine (24 cases);
1) Average absorbance 0.487 2) Average absorbance 0.124
In immunized animals, only the antibody YOKO reacted with both bovine kidney glomerular pepsin-solubilized type IV collagen and human placenta-derived pepsin solubilized type IV collagen. Only one type IV collagen reacted.
-Anti-renal type IV antibody was prominently detected in the urine of human glomerulonephritis patients.
Conclusion: When measuring an anti-type IV collagen antibody for evaluation of renal function, it is preferable to use a type IV collagen derived from the kidney as an antigen.
"Antigen measurement kit"
1) Standard (bovine kidney type IV) and human placenta type IV were added to a 96-well microplate coated with 10 ug / ml of anti-renal type IV antibody (derived from rabbit). 2) Add anti-renal type IV monoclonal antibody (derived from mouse) and wash after standing for 2 hours at room temperature. 2) -2 Add enzyme (HRP) -labeled anti-mouse IgG antibody and leave for 1 hour at room temperature and wash. ) A chromogenic substrate (TMB) was added and allowed to stand at room temperature for 10 minutes. 4) A reaction stop solution (1N sulfuric acid) was added, and the absorbance at a wavelength of 450 nm was immediately measured.
result;
Standard (bovine kidney type IV) was measured to a minimum concentration of 0.1 ng / ml, but human placenta type IV could not be detected. In this measurement, “2)” was changed to the simultaneously prepared anti-renal type IV polyclonal antibody (derived from rat), and “2) -2” was changed to the corresponding enzyme (HRP) -labeled anti-rat IgG antibody. Bovine kidney type IV) was measured to a minimum concentration of 1.0 ng / ml, but human placenta type IV was also measured to a minimum of 10 ng / ml.
Conclusion: When the measurement target is renal type IV, a measurement kit using an anti-renal type IV antibody increases the detection sensitivity.

Claims (5)

A method for detecting nephritis, in which urine and / or serum from humans and / or animals is used as a sample, and whether or not a kidney-derived type IV collagen triple chain region and / or an antibody thereof is present therein And the presence of a kidney-derived type IV collagen triple chain region and / or antibody thereof indicates that nephritis has occurred. A method according to claim 1 for detecting nephritis, comprising: a sample-derived kidney-derived type IV collagen triple-chain region and a separately added anti-renal-derived type IV collagen triple-chain region antibody. Nephritis is caused by detecting immune complex and / or immune complex of anti-kidney-derived type IV collagen triple-chain region antibody and separately added kidney-derived type IV collagen triple-chain region in the sample A method consisting of showing that. A method according to claim 2 for detecting nephritis, comprising: a sample-derived kidney-derived type IV collagen triple-chain region and a separately added anti-renal-derived type IV collagen triple-chain region antibody. Nephritis is caused by detecting immune complex and / or immune complex of anti-kidney-derived type IV collagen triple-chain region antibody and separately added kidney-derived type IV collagen triple-chain region in the sample An enzyme immunoassay method comprising: A method according to claim 3 for detecting nephritis, comprising a sample-derived kidney-derived type IV collagen triple-chain region and an anti-renal-derived type IV collagen triple-chain region antibody that specifically binds separately. A sandwich enzyme immunoassay method comprising detecting nephritis by detecting an immune complex. The method of claim 3 for detecting nephritis, comprising:
Nephritis is caused by detecting an immune complex between the anti-renal-derived type IV collagen triple-chain region antibody in the sample and the kidney-derived type IV collagen triple-chain region separately immobilized on a microplate. An enzyme immunoassay method comprising: