CN104730070A - Agent and method for jointly detecting sialic acid and hydroxyproline - Google Patents
Agent and method for jointly detecting sialic acid and hydroxyproline Download PDFInfo
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- CN104730070A CN104730070A CN201510120981.8A CN201510120981A CN104730070A CN 104730070 A CN104730070 A CN 104730070A CN 201510120981 A CN201510120981 A CN 201510120981A CN 104730070 A CN104730070 A CN 104730070A
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- hydroxyproline
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Abstract
The invention discloses an agent for jointly detecting sialic acid and hydroxyproline. The agent is prepared from a color developing agent and reference product liquid, wherein the color developing agent is prepared from the following components by concentration: 0.35mol/L of diethyl phthalate, 0.46mol/L of ethyl acetate, 0.07mol/L of 2, 4-dinitrophenylhydrazine, 0.006mol/L of hydrogen peroxide, 0.62mol/L of hydrochloric acid, 0.2% of benzoic acid, and water; the reference product liquid is prepared from sialic acid, L-hydroxyproline, methyl parahydroxybenzoats and water. According to the agent and the method, various tumor markers (sialic acid and hydroxyproline) can be reacted and subjected to color developing by one-time reaction; due to the color developing superposition, the detection sensitivity can be improved, and the requirement on joint detection of the tumor markers is met; in addition, the operation is simple and convenient; expensive instruments are saved.
Description
Technical field
The present invention relates to reagent and the detection method thereof of a kind of joint-detection sialic acid and hydroxyproline, belong to technical field of medical examination.
Background technology
Sialic acid, also known as N-acetyl-neuraminate, is a kind of natural neuraminic acid derivatives, and cyclic amino acids in its precursor structure is made up of nine carbon atoms is the general name of a compounds of group.Sialic acid is distributed widely in animal body, sialic acid in human serum, comprises free sialic acid and bound sialic acid, and wherein the former content is lower, is sialic main existence form in serum with the bound sialic acid of lipoprotein and Glycoprotein binding.Hydroxyproline is a kind of imino acid, is the product after proline hydroxylation, usually on the 4th with hydroxyl, but sometimes also on the 3rd.Owing to there being two asymmetric carbon atoms, so there are 4 kinds of steric isomers.Naturally occurring at occurring in nature is L-hydroxyproline, there is not the acid of D-hydroxyl dried meat.L-hydroxyproline is amino acid specific to collagen, and other protein content is very micro-, the free hydroxyproline discharged when the hydroxyproline in human serum is mainly collagen degradation.
During eubolism, in human serum, sialic acid, hydroxyproline content are stablized.When histocyte cancerates, cell surface glycoprotein all occurs obviously to change on 26S Proteasome Structure and Function, and being combined in the sialic acid on glycoprotein and coming off into blood with the glycoprotein of unconventionality expression, is one of reason that in blood, sialic acid content raises.And Serum oxyproline rising is because in tumorigenesis process, tumour cell infiltrates, transfer, destroys collagen tissue, elastin laminin bone tissue even, causes hydroxyproline to discharge caused by increase.Current multinomial research finds, in Serum of Cancer Patients, sialic acid, hydroxyproline content increase, therefore in clinical examination often using sialic acid, hydroxyproline as Testing index, in the middle of the diagnosis of malignant tumour.
Current sialic detection method mainly contains enzyme process, chemical colorimetry and high performance liquid chromatography.But enzymatic assays susceptibility is lower, and chemical colorimetry poor specificity, though efficient liquid-phase chromatography method has highly sensitive, that specificity is good advantage, but expensive equipment and be not suitable for clinical batches and detect.The detection method of hydroxyproline mainly contains chemical colorimetry, high performance liquid chromatography, automatic amino acid analyzer method.Toluene-sodium-sulfonchloramide colourimetry is the traditional assay method of hydroxyproline, but the method poor specificity, sensitivity are low, have applied few at present.High performance liquid chromatography, automatic amino acid analyzer method detect hydroxyproline and then there is expensive equipment with bar, detect the problems such as inconvenient, limit clinical batches application.
As can be seen here, although current sialic acid, hydroxyproline detection method are separately more, but all there is various problem, cannot routine be applied to clinical, lack the method for the combined detection especially, thus a kind of method of quick, accurate, with low cost, convenient applicable sialic acid, hydroxyproline joint-detection is sought, significant for susceptibility, the specificity improving diagnosing tumor.
Summary of the invention
For above-mentioned prior art, problem to be solved by this invention is to provide the reagent of a kind of quick, accurate, with low cost, convenient applicable joint-detection sialic acid and hydroxyproline, and detection method.
The present invention is achieved by the following technical solutions:
A reagent for joint-detection sialic acid and hydroxyproline, is made up of developer and reference product liquid, wherein, developer is by diethyl phthalate, ethyl acetate, 2,4-dinitrophenylhydrazine, hydrogen peroxide, hydrochloric acid, benzoic acid and water composition, wherein, the concentration of each component is as follows:
Reference product liquid is made up of sialic acid, hydroxyproline, methyl p-hydroxybenzoate and water, and the concentration of each component is as follows:
Sialic acid 0.3g/L
Hydroxyproline 0.25g/L
Methyl p-hydroxybenzoate 0.2% (mass percent).
The compound method of described developer and reference product liquid is conventional method, and after namely component described in developer and reference product adds distilled water respectively, mixing stirs evenly separately.
Apply joint-detection sialic acid of the present invention and hydroxyproline reagent measure sialic acid, hydroxyproline method as follows:
(1) in every secondary response, reference QC all makees three parallel pipes, measures pipe sample and does a pipe.
(2) by table 1 procedure operation:
Table 1
Abundant mixing, puts the middle accurate response of boiling water bath (can keep constantly boiling) 15 minutes, takes out rapidly and be placed in room temperature water (< 25 DEG C) cooling 5 minutes, cessation reaction.
(3) pipe is measured with 3000 revs/min, centrifugal 10 minutes.
(4) get supernatant colorimetric, wavelength 450nm, optical path 0.5cm, with distilled water zeroing, read each pipe absorbance.
The reagent of joint-detection sialic acid of the present invention and hydroxyproline measures sialic acid, hydroxyproline content calculates as follows:
Result calculates:
Detected value is greater than 95U/ml for positive.
In the reagent of joint-detection sialic acid of the present invention and hydroxyproline, benzoic acid, methyl p-hydroxybenzoate play antisepsis, make reagent can long storage periods, wherein, benzoic acid in acid condition, all has inhibiting effect to mould, yeast and bacterium, antibacterial optimum pH is 2.5 ~ 4.0, due to containing hydrochloric acid in developer of the present invention, in acid, therefore select benzoic acid as antiseptic.P-hydroxybenzoate has phenolic hydroxyl structure, and antibacterium performance is strong, and its mechanism of action is: the cell membrane of destroy microorganisms, makes intracellular protein denaturation, and can suppress the respiratory enzyme system of microbial cell and the activity of electron transmission enzyme system.The bacteriostatic activity of methyl p-hydroxybenzoate mainly molecular state works, and its intramolecular hydroxyl esterification, no longer ionize, so it all has good fungistatic effect in the scope of pH3 ~ 8.The security of methyl p-hydroxybenzoate is also very high.Reference liquid of the present invention is faintly acid, therefore selects methyl p-hydroxybenzoate as antiseptic.
The present invention adopts specific chromogenic assay technology, in same detection system, Diagnostic Value of Several Serum Tumor Markers (sialic acid, hydroxyproline) reaction solution is made by primary first-order equation, due to colour developing superposition, thus improve the sensitivity of detection, comply with the trend of tumor markers joint-detection, and easy and simple to handle, need not expensive instrument, easy to utilize.
Embodiment
Below in conjunction with embodiment, the present invention is further detailed.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Embodiment 1 prepares the reagent of joint-detection sialic acid and hydroxyproline
Be made up of developer and reference product liquid, wherein, developer is made up of diethyl phthalate, ethyl acetate, DNPH, hydrogen peroxide, hydrochloric acid, benzoic acid and water, and in developer, the concentration of each component is:
Reference product liquid is made up of sialic acid, hydroxyproline, methyl p-hydroxybenzoate and water, and the concentration of each component is as follows:
Sialic acid 0.3g/L
Hydroxyproline 0.25g/L
Methyl p-hydroxybenzoate 0.2% (mass percent).
The compound method of developer and reference product liquid is conventional method, and after namely component described in developer and reference product adds distilled water respectively, mixing stirs evenly separately.
The method of application mentioned reagent mensuration sialic acid, hydroxyproline is as follows:
(1) in every secondary response, reference QC all makees three parallel pipes, measures pipe sample and does a pipe.
(2) by table 1 procedure operation; Abundant mixing, puts the middle accurate response of boiling water bath (can keep constantly boiling) 15 minutes, takes out rapidly and be placed in room temperature water (< 25 DEG C) cooling 5 minutes, cessation reaction.
(3) pipe is measured with 3000 revs/min, centrifugal 10 minutes.
(4) get supernatant colorimetric, wavelength 450nm, optical path 0.5cm, with distilled water zeroing, read each pipe absorbance.
Mensuration sialic acid, hydroxyproline content calculate as follows:
Result calculates:
Example 1
Reagent and other amino acid of joint-detection sialic acid prepared by embodiment 1 and hydroxyproline all do not react, in table 2.
Table 2 amino acid and reagent reacting result of the present invention
Note: 1. " 0 " number represents without colour response, i.e. water white transparency; 2. counted word is absorbance.
The stability experiment of example 2 reagent
Detect reagent loading reagent bottle (nitrite ion, reference liquid separate packing) prepared by embodiment 1, sealed type storage, (storage requirement: temperature 25 ± 2 DEG C after storage 0 month, March, June, Dec, 18 months, 24 months, relative humidity 30 ± 10%), detect the concentration of each effective constituent, and impurity (insolubles) content (mass percent) in reagent, detect its performance for stability.Result: store after 24 months, keep stable, the concentration of each effective constituent has no significant change, and insolubles has no obvious increase, in table 3.
Table 3
Claims (2)
1. the reagent of a joint-detection sialic acid and hydroxyproline, it is characterized in that: be made up of developer and reference product liquid, wherein, developer is by diethyl phthalate, ethyl acetate, 2,4-dinitrophenylhydrazine, hydrogen peroxide, hydrochloric acid, benzoic acid and water composition, the concentration of each component is as follows:
Reference product liquid is made up of sialic acid, hydroxyproline, methyl p-hydroxybenzoate and water, and the concentration of each component is as follows:
Sialic acid 0.3g/L
Hydroxyproline 0.25g/L
Methyl p-hydroxybenzoate 0.2% (mass percent).
2. utilize the reagent of the joint-detection sialic acid described in claim 1 and hydroxyproline to detect the method for sialic acid, hydroxyproline, it is characterized in that: sample thief, add developer, abundant mixing, put the middle accurate response of boiling water bath (constantly boiling can be kept) 15 minutes, rapid taking-up is placed in room temperature water (< 25 DEG C) cooling 5 minutes, cessation reaction; Centrifugal 10 minutes with 3000 revs/min, get supernatant, measure the absorbance at wavelength 450nm, optical path 0.5cm place, return to zero with distilled water; Meanwhile, carry out same operation to reference product liquid, reference product liquid theoretical value is 100U/ml; According to following formulae discovery detected value:
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226062A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | A kind of method that the content of hydroxyproline in fish-skin is measured using spectrophotometry |
Citations (4)
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| CN101296692A (en) * | 2005-07-22 | 2008-10-29 | 药物研发中心 | Rh-epo nasal formulations with low sialic acid concentration for the treatment of diseases of the central nervous system |
| CN101871893A (en) * | 2009-04-21 | 2010-10-27 | 陈延霞 | Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder |
| CN103040688A (en) * | 2013-01-10 | 2013-04-17 | 林建广 | Hand-mouth baby wet tissue and preparation method thereof |
| CN103512880A (en) * | 2013-09-18 | 2014-01-15 | 山东大学齐鲁医院 | Joint detection kit for sialic acid and hydroxyproline |
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2015
- 2015-03-19 CN CN201510120981.8A patent/CN104730070A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101296692A (en) * | 2005-07-22 | 2008-10-29 | 药物研发中心 | Rh-epo nasal formulations with low sialic acid concentration for the treatment of diseases of the central nervous system |
| CN101871893A (en) * | 2009-04-21 | 2010-10-27 | 陈延霞 | Colorimetric method for determining free L-hydroxyproline (L-HYP) in milk powder |
| CN103040688A (en) * | 2013-01-10 | 2013-04-17 | 林建广 | Hand-mouth baby wet tissue and preparation method thereof |
| CN103512880A (en) * | 2013-09-18 | 2014-01-15 | 山东大学齐鲁医院 | Joint detection kit for sialic acid and hydroxyproline |
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| 师海波等: "《最新临床药物手册》", 31 January 2013, 军事医学科学出版社 * |
| 王高松: "《皮肤科应用药物学》", 28 February 1983, 第二军医大学第一附属医院 * |
| 罗明生等: "《中国药用辅料》", 30 April 2006, 化学工业出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108226062A (en) * | 2016-12-15 | 2018-06-29 | 江苏维赛科技生物发展有限公司 | A kind of method that the content of hydroxyproline in fish-skin is measured using spectrophotometry |
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