Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Patent
1991-06-19
1994-03-29
Rosen, Sam
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
435 79, 436518, 436536, 436547, 436548, 5303889, C12Q 100, G01N 3353
Patent
active
052983978
DESCRIPTION:
BRIEF SUMMARY
TECHNICAL FIELD
This invention relates to a method of assaying D-vanillylmandelic acid contained in a specimen of a living organism and a reagent and a kit used for the assay.
BACKGROUND ART
As tumor producing acidic catecholamine metabolites, pheochromocytoma and neuroblastoma have been known. Neuroblastoma is generated primarily in infants and has been one of the assaying items of mass screening in Japan.
Presence of neuroblastoma in vivo has been determined by assaying vanillylmandelic acid (VMA) and homovanilic acid (HVA), which are acidic catecholamine metabolites, in urine.
As methods used for assaying VMA and HVA in urine, qualitative methods such as spot test and dip test and quantitative methods such as high performance liquid chromatography (HPLC) and immunoassay have hitherto been reported. The spot test and the dip test suffer from high appearance ratio of false positive, and thus they are not quite satisfactory. On the other hand, HPLC is an excellent method which is reliable with respect to precision, but a skilled specialist is required for maintenance and management of the device. Also it has a drawback in that a plurality of test samples cannot be treated at the same time.
In contrast, immunoassay has been expected to overcome the drawbacks as mentioned above [see Japanese Patent Laid-Open Publications Nos. 123765/1985 and 11165/1987; Biogenic Amines, 4, No. 3, 229-235 (1987); Infant Cancer, 24, 250-252 (1988); Revised Neuroblastoma Mass Screening, 148-152 (1989), published by Shakai Fukushi Hojin Onshi Zaidan Boshi Aiikukai; Journal of Immunological Methods, 118, 101-107 (1989)].
VMA, as shown by the following formula, has an asymmetric carbon in the molecule, and two optical isomers with different optical activities (D form and L form) exist. ##STR1## wherein * represents asymmetric carbon.
However, there is nothing in the articles hitherto published or reported in the art which teaches or suggests as to whether D-VMA can be specifically assayed by means of immunoassay.
DISCLOSURE OF THE INVENTION
We have conducted extensive research in order to develop a method of assaying D-VMA specifically and simply. As a result, we have successfully obtained an anti-D-VMA antibody and established a method of assaying specifically D-VMA contained in a specimen of a living organism according to an extremely simple method with the use of said antibody, whereby the present invention has been accomplished.
More specifically, the present invention is concerned with a method of assaying D-VMA contained in a specimen of a living organism through the following steps (A) to (D), wherein an equal amount mixture of D-VMA and L-VMA (a racemic mixture of VMA) is used as the standard substance for preparing a calibration curve, a labeled anti-D-VMA antibody is used as the labeled anti-VMA antibody, and the concentration of D-VMA contained in the specimen is calculated by using the results obtained in the step (D):
(A) the step of conducting a competitive reaction of VMA in the specimen and VMA in solid phase against the labeled anti-VMA antibody by the use of the solid-phase VMA obtained by combining VMA with a solid phase and the labeled anti-VMA antibody;
(B) the step of separating the liquid phase from the solid phase and washing the solid phase, if necessary;
(C) the step of determining the quantity of labeling of the labeled anti-VMA antibody combined with the solid-phase VMA or the quantity of labeling of other substances; and
(D) the step of obtaining a curve (a calibration curve) or an equation exhibiting the relationship between the quantity of labeling and the VMA concentration by practicing the same procedure as in the above steps (A) to (C) except for using VMA of a known concentration (a standard substance) in place of the specimen, calculating the concentration of VMA corresponding to the quantity of labeling determined in the above step (C) by using the calibration curve or equation thus obtained, and defining the concentration thus calculated as the concentration of VMA contained in the spec
REFERENCES:
Yoshioka et al., Chem. Abst. vol. 107 (1987) p. 147,514g.
Kuroda Manami
Sugi Masahito
Rosen Sam
Yamasa Shoyu Kabushiki Kaisha
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