JPH0379995B2 - - Google Patents

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a monoclonal antibody, and more particularly to a monoclonal antibody, which is a marker for advanced cancer.
(psedouridine ψ). [Prior art and its problems] In recent years, clinical diagnostic tests using monoclonal antibodies against cancer-related antigens have been actively carried out.
Similar efforts have also been made using antibodies against carcinoembryonic antigen (CEA), α-fetoproten, and other substances that are known to increase in cancer. By the way, among substances that increase when cancer develops, that is, substances called tumor markers, euduridine ψ is known to increase in the urine of patients with advanced cancer. This substance is one of the components of transfer ribonucleic acid (t-RNA), and its increase is due to the breakdown of t-RNA in cancer tissues compared to normal tissues. It is said that this is due to an increase in the number of children, but the details of the mechanism behind this increase have not been elucidated. Currently, attempts are being made to measure the amount of pseudourisine ψ (hereinafter sometimes abbreviated as "ψ") and determine the presence of advanced cancer from this.
Quantification is performed using high performance liquid chromatography (HPLC).
Since the method is carried out in a vacuum, pretreatment of the sample is complicated, and it takes a very long time to measure a large number of samples. The present inventors previously conducted various studies in order to develop a simple method for measuring ψ, and as a result, they created a new monoclonal antibody against ψ. We discovered that it was possible to carry out the simultaneous measurement of multiple samples using the ELISA method without performing any processing operations, and that the measurement could be made faster and simpler.
No. 143484 (Unexamined Japanese Patent Publication No. 62-299765)]. However, the monoclonal antibody against ψ described in Japanese Patent Application No. 143484/1984 has uridine and 95
This monoclonal antibody was used because it has ~99% and 30-40% cross-reactivity with uracil.
When quantifying ψ by ELISA, there was a possibility that uridine and uracil were also measured in addition to ψ. Therefore, conventional HPLC has shown that there are few cases of high values of ψ even in patients with advanced cancer.
The drawback was that results contradicting the quantitative data obtained by the method were obtained. Therefore, the present inventors further conducted research to obtain a monoclonal antibody with high reaction specificity for ψ. As a result, it was found that the inhibition was strong against free ψ, and there was no inhibition or very little inhibition against uridine and uracil. We have completed the present invention by discovering that a weak monoclonal antibody has high reaction specificity for ψ, and when used in a method for measuring ψ, it agrees well with quantitative data by HPLC or the like. That is, the present invention has the following characteristics (1) Antibody class: IgG ₁ (2) Antibody titer (the highest value when performing ELISA in which the culture supernatant is diluted in two stages and each is reacted with ψ-BSA) Dilution factor that maintains absorbance): 64
The present invention provides a monoclonal antibody against ψ having cross-reactivity: 100% pseudouridine, 0% uridine, 7% uracil, 2.8% 5-fluorouracil, and a method for measuring ψ using the same. The monoclonal antibody against ψ of the present invention is prepared, for example, as follows. That is, first, an animal is immunized using ψ, and splenocytes from the animal are collected. In this process,
Since ψ cannot act as an immunogen by itself, it is used as an immunogen after being combined with an appropriate carrier protein. As ψ, for example, a known method can be used to extract urine from advanced cancer patients, such as a standard product sold by Sigma, and as a carrier protein, the hapten portion is recognized by immunocompetent cells. Proteins used for this purpose, such as keyhole limpet hemocyanin and bovine serum albumin, can be used. Also,
Examples of a method for binding this ψ to a carrier protein include oxidizing the sugar moiety of the nucleobase with periodic acid and then binding it to the carrier protein. Furthermore, examples of immunized animals include mice, rats, and the like. Next, the obtained splenocytes of the immunized animal are fused with myeloma cells to obtain hybridomas. For cell fusion, either a known method using polyethylene glycol or a method using a virus may be used, but when screening hybridomas, care must be taken to remove hybridomas that produce antibodies against carrier proteins. be. For this purpose, it is desirable to use a method such as screening as an antigen a protein derived from a different species than the carrier protein used for immunization, in which ψ is bound. The hybridoma thus obtained was further examined to see if it was inhibited by free ψ, uridine, and uracil, and those that were strongly inhibited by ψ and not or extremely weakly inhibited by uridine and uracil were selected. Then, a hybridoma producing the antibody of interest is obtained by cloning using a conventional method. The monoclonal antibody of the present invention produced by this hybridoma has the properties described above. An example of measuring ψ using the monoclonal antibody obtained as described above is as follows. That is, a conjugate of ψ and bovine serum albumin (BSA) is added at 0.2 μg/well to a 96-well plate, and then left at 4° C. for 12 to 24 hours. Next, 100 μl of phosphate buffered saline (PBS) containing 1% BSA is added to each well to prevent nonspecific adsorption of antibodies and other proteins. Next, 50μ of a sample (urine, etc.) was added to each well, and the monoclonal antibody of the present invention (20-fold dilution) was added to each well.
Add 50 μl of to each well. After stirring well, leave at 4°C for 1 hour. reaction mixture
After thorough washing with PBS, add 100 μl of a 1000-fold dilution of alkaline phosphatase-labeled goat anti-mouse IgG antibody to each well. After reacting at room temperature for 30 minutes, washing thoroughly with PBS and draining water, add 100μ of substrate solution (para-nitrophenyl phosphate 1mg/ml, PH9.8 diethanolamine buffer) and incubate at 37℃. Incubate for 30 minutes. The amount of ψ present in the sample is quantified by measuring the absorbance of each well at 405 nm using an EIA reader. [Effects of the present invention] As described above, by using the monoclonal antibody of the present invention, ψ can be measured without pre-treating the sample, and also does not require large-scale equipment such as HPLC. , the presence or absence of advanced cancer can be determined simply and quickly. Furthermore, it is possible to quantify ψ more specifically than by using the monoclonal antibody described in Japanese Patent Application No. 143484/1984. [Example] Next, the present invention will be explained with reference to Examples. Example 1 (1) Preparation of immunogen: ψ isolated from the urine of cancer patients by a conventional method,
Keyhole lympet hemocyanin, a carrier protein
hemocyanine (KLH) was combined by the method of Erlanger and Bieser (periodate oxidation method). That is, ψ
After oxidizing with periodic acid and decomposing excess periodic acid with ethylene glycol, it is combined with KLH under alkaline (PH9-9.5) conditions to form Schiff's base. Then reduced with _NaBH4 ,
Generate stable compounds. The reaction mixture was dialyzed overnight in a buffer solution (phosphate buffered saline, pH 7.4) to remove unreacted ψ, and then freeze-dried and stored in a -20°C freezer. (2) Creation of monoclonal antibody: () The ψ and KLH conjugate obtained in (1) was added to Freund's Complete Adjuvant (Freund's Complete Adjuvant).
Adjuvant) was mixed in equal amounts to form an emulsion, and then 50 μg per BALB/c mouse was administered intraperitoneally. From the second time onward, the emulsion using an incomplete adjuvant was intraperitoneally administered twice at 10-day intervals. Final immunization is ψ-KLH100μg/
ml solution was administered intravenously. () Three days after the final immunization, splenocytes (1.3 × 10 ⁸ cells) and BALB/c were excised from hyperimmunized mice.
Mouse-derived myeloma cell line SP2/0-
Ag14 (1.2 × 10 ⁷ pieces) was fused using polyethylene glycol (PEG) 4000. The cells are
100μ/well was added to a 96-well plate, half of the medium was replaced with HAT medium after 24 hours, and the medium was replaced every two days. After 7 to 10 days, growth of pigeon-resistant hybridomas is observed (288 out of 288 wells). At this time, the medium was changed to HT, and after culturing for about 10 days, the medium was changed to hybridoma growth medium. () Screening for antibody-producing cells is carried out using a two-antibody ELISA using a combination of ψ and bovine serum albumin (BSA) as an antigen.
It was done according to the law. Using this method, we succeeded in excluding hybridomas that produce antibodies against carrier proteins. Next, we selected hybridomas that produced monoclonal antibodies that specifically reacted with ψ by examining whether they were inhibited by ψ. For this hybridoma, ANTICANCER further investigated whether or not it is inhibited by uridine and uracil.
RESEARCH6:135-138 (1986), CANCER
RESEARCH46, 3164−3167 (1986),
Journal of Immunological Methods.81
(1985) 169-185, etc., and selected those that had no or very weak inhibition against these. The cell lines selected here were cloned by the limiting dilution method to establish monoclonal antibody-producing hybridoma clones (3 strains). (3) Properties of monoclonal antibody: The class (subclass) of the monoclonal antibody against ψ was IgG ₁ . The antibody titer was 64 times (the culture supernatant was diluted in two stages, and
When performing ELISA to react with the antibody, the dilution ratio at which the highest absorbance persisted was taken as the antibody titer). In addition, when we examined cross-reactivity with various purines, pyrimidines, modified and unmodified nucleosides, and bases, we found that there was no cross-reactivity with uridine, 7% with uracil, and 5-fluorouracil.
It showed 28% cross-reactivity with other compounds.
No cross-reactivity was shown. Example 2 ψ of 200, 100, 50, 20, 10, 5, 2, 1, 0.5
(μg/ml) solution in advance with ψ-BSA0.3μg/
A competitive inhibition experiment was performed by adding 50 μl each of the 20-fold dilution of the monoclonal antibody obtained in Example 1 to a 96-well plate coated with holes. As a result, as shown in FIG. 1, it was revealed that the binding between the antibody and ψ-BSA was inhibited depending on the dose of free ψ. The quantification range of ψ using this monoclonal antibody was 1 to 50 μg/ml. Example 3 A competitive inhibition test was conducted in the same manner as in Example 2, using urine from a normal person and a cancer patient as samples instead of the ψ solution in Example 2. Using the calibration curve obtained from the results of Example 2, the amount of ψ in the sample was measured. Patent application 1986-143484
When comparing both monoclonal antibodies by performing the same measurement for the monoclonal antibody against ψ described in the issue, it was found that cancer patients whose ψ value exceeded the (average + 2 x standard deviation) value was 5/6 for the new antibody and 1/6 for the old antibody. The results are shown in Table 1. 【table】

[Brief explanation of drawings]

FIG. 1 is a calibration curve for ψ of the monoclonal antibody of the present invention.

Claims (1)

[Claims] 1. The following properties: (1) Antibody class: IgG ₁ (2) Antibody titer (ELISA in which the culture supernatant is diluted in two stages and each is reacted with the conjugate of pseudouridine ψ and bovine serum albumin). Monoclonal antibody against pseudouridine ψ having the following dilution ratio: 64 times (3) to maintain the highest absorbance when tested. 2. A method for measuring pseudourisin ψ, which comprises adding a monoclonal antibody against pseudourisin ψ having the properties shown below to a specimen and measuring the amount of the resulting pseudourisin ψ-monoclonal antibody complex. (1) Antibody class: IgG ₁ (2) Antibody titer (when the culture supernatant is diluted in two stages and an ELISA is performed in which the culture supernatant is reacted with the conjugate of seuduridine ψ and bovine serum albumin, the highest absorbance is maintained) Dilution ratio): 64 times (3) Cross-reactivity: Euridine 100% Uridine 0% Uracil 7% 5-Fluorouracil 2.8%