JPS60123765A - Preparation of peculiar antibody to vanylmanderic acid - Google Patents

Abstract

PURPOSE:To prepare a peculiar antibody to vanylmanderic acid, by a method wherein bovine serum albumin is chemically bonded to vanylmanderic acid to impart immunogenicity thereto and, after an animal is innoculated with the resulting conjugate, blood is collected from said animal and the specific antibody is separated from the blood and purified. CONSTITUTION:Vanylmanderic acid is dissolved in a saline solution and 1-ethyl- 3, 3-dimethylaminopropyl hydrochloride and bovine serum albumin are added to said solution. This reaction solution is applied to column chromatography using Sephadex G-25 to separate a conjugate of vanylmanderic acid and bovine serum albumin. In the next step, a rabbit is hypodermically innoculated with the resulting conjugate as a protein concn. along with a Freund's complete adjuvant and, thereafter, blood is collected from the rabbit. Whereupon, the production of a peculiar antibody to vanylmanderic acid is discovered in the serum of the blood. This specific antibody is separated and purified by affinity chromatography prepared by coupling AH-Cephalose 4B with vanylmanderic acid.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing a specific antibody against vanylmandelic acid.

Conventionally, urinary vanylmandelic acid was determined by separation and determination using high-performance liquid lithography, but this quantitative device itself was expensive, and the useful life of consumables such as empty books was short, and it was difficult to quantify. The drawback is that it takes time to operate and cannot measure multiple samples at once.

Therefore, the present inventor developed an enzyme immunoassay method that can quantitatively measure many samples at once in a short time.
After much trial and error to see if it could be used to quantify dibric acid, the inventors conducted intensive research on the production of a specific antibody for use in the enzyme immunoassay described above, and as a result, they came up with the present invention.

Vanylmandelic acid itself does not act as an immunogen, so by chemically bonding the vanylmandelic acid with bovine serum albumin, the immunogenicity is 1. They found that this binding substance easily binds to serum albumin and is immunogenic, and furthermore, they found that specific antibodies to vanylmandelic acid were produced in animal serum by inoculating animals with this binding substance. I found it.

Example Panilmandelic acid 10 mg % 0. ．． 1-ethyl-3-3-dimethylmino-propyl hydrochloride (EDAC-1(Cl) 1 (
Add 10μ and adjust the pH to 50. 3 at room temperature
Stir for 0 minutes. Next, 1100 mg of EDAC-HC was added, and 35 mg of bovine serum albumin was added in total.
Stir all day and night. This reaction solution was mixed with Sephadex G-
The conjugate of vanylmandelic acid and bovine serum albumin is separated by column chromatography using 25.

The protein concentration of the above-mentioned conjugate was 5 mg, and the rabbit was subcutaneously inoculated with Freund's complete azygos 5 to 6 times every two weeks. When blood was collected, the serum contained specificity for vanylmandelic acid. Production of an antibody was found, and the specific antibody of O was isolated and purified by affinity chromatography in which panilmandelic acid was coupled to AH-Sepharose 4B.

Next, use the specific antibody obtained in Example An example of the quantitative determination used is shown below.

Quantification Example 1 Specific antibody appropriately diluted in a microtray 0. i n+1 is adsorbed to produce an antibody in phase. After washing, urine was diluted with phosphate buffered saline (PBS).
Add 1 mjl and react at 37°C for 3 hours. When urine is collected using a mouthpiece, a certain area of the mouthpiece is cut out, eluted with PBS, and received. After the reaction, wash and add specific anti-antibody 0.1
ml and reacted for 1 hour at 37°C. After washing, the enzyme substrate 0-phenylenediamine was added, and the colorimetric value was measured using a spectrophotometer, followed by quantitative determination using a calibration curve.

Quantification Example 2 0.2 ml of an appropriately diluted specific antibody is adsorbed onto a microtray to prepare an antibody solid phase. After washing, 0.1ml of urine diluted with PBS and 0.1ml of specific antibody.
Add mJ and react at 37°C for 3 hours. After the reaction, the mixture was washed, 0.2 nJ of enzyme substrate 0-phenylenediamine was added, and colorimetry and quantitative determination were performed using a spectrophotometer.

Quantification Example 3 Appropriately diluted urine was placed in a test tube of about I x 7 cm.
1 m11 specific antibody 0.1 ml and PBSo, 2
After reacting day and night at 4°C, 0.1 mJ each of normal rabbit serum and specific antibody were added. After reacting at room temperature for 4 hours, centrifuge, discard the supernatant, add enzyme standard 0-phenylenediamine, and perform colorimetry and quantitative determination.

Through the above quantification examples 1 to 3, it was possible to quantify urinary vanylmandelic acid in the nanogram range5.

To date, to the best of the present inventor's knowledge, no specific antibody against vanylmandelic acid has been produced at all. The present invention finally makes it possible to produce a specific antibody against vanylmandelic acid.

Moreover, by using the specific antibody according to the present invention, it is possible to quantitatively process approximately 500 samples at a time, and not only can high-sensitivity measurements be performed, but the equipment necessary for measurement is a spectrophotometer that is available everywhere. be.

Procedural amendment (method) March 7, 1980 1, Indication of case Patent application No. 58-231053 2 Title of invention Method for producing specific antibody against vanylmandelic acid 3,
Relationship with the case of the person making the amendment Patent applicant: Yamaguchi Kei Telephone 271-3751 (Representative) 5. Date of amendment order: February 8, 1980 (Shipping date: February 28, 1980) 6. Subject of amendment

Claims (1)

[Claims] Vanirmandelic acid is chemically bound to bovine serum albumin to impart immunogenicity, and the bound product is inoculated into an animal and then separated and purified from collected blood. A method for producing a specific antibody against derric acid.