CN104865210A - Single liquid reagent for AFU (alpha-fucosidase) determination - Google Patents
Single liquid reagent for AFU (alpha-fucosidase) determination Download PDFInfo
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- CN104865210A CN104865210A CN201510267236.6A CN201510267236A CN104865210A CN 104865210 A CN104865210 A CN 104865210A CN 201510267236 A CN201510267236 A CN 201510267236A CN 104865210 A CN104865210 A CN 104865210A
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Abstract
The invention discloses a single liquid reagent for AFU (alpha-fucosidase) determination. The reagent comprises components with the concentration as follows: a buffer solution with the concentration being 50-200 mmol/L and pH being 4.5-5.5, an anti-interference agent with the concentration being 20-80 g/l and 2-chloro-4-nitrobenzene-alpha-L-fucoside with the concentration being 0.1-1 g/L. By means of the single liquid reagent, the space of a reagent disc of a fully automatic biochemical analyzer can be saved effectively, the utilization rate of the fully automatic biochemical analyzer is increased, and clinical popularization and application of the reagent are facilitated. Further, a substrate can be stabilized effectively by the special buffer solution; a heparin interference effect can be removed from a sample instantly with addition of the anti-interference agent, a heparin degradation or shielding function is realized, so that the single liquid reagent has good anti-heparin-interference performance, and the cost of the reagent is lower due to the low market price of the added anti-interference agent.
Description
Technical field
The present invention relates to technical field of medical examination, be specifically related to a kind of Alpha-Fucosidase and measure liquid single agents.
Background technology
Alpha-Fucosidase (α-fucosidase, AFU) is a kind of lysosomal acid hydrolytic enzyme, is distributed widely in various tissues, cell and the body fluid in human body, especially higher with liver, Shen Deng tissue activity.In recent years, along with the further investigation changed the blood AFU level of AFU detection method and other tumours or non-neoplastic disease, AFU vitality test has become a basic diagnosis index, and combine with other lab index, become the important indicator of the medicals diagnosis on disease such as liver cancer, cancer of the stomach, cancer of pancreas, colon cancer, carcinoma of mouth, leukaemia, ovarian neoplasm.
At present, the method measuring AFU vigor mainly contains fluorescence method, terminal colorimetric analysis, rate method.Fluorescence method be utilize fucoside compound to discharge after AFU hydrolysis can the 4-methyl umbellate form ketone of emitting fluorescence, by checking that fluorescence intensity carrys out enzyme analysis vigor, this method highly sensitive, can serum be used for, be also applicable to biopsy, cell, sample that cerebrospinal fluid equivalent is little, but this method be high to instrument requirements, gel filtration is not only needed to remove interfering material, and need fluorospectrophotometer, thus can not realize automated analysis, be difficult to carry out penetration and promotion; Terminal colorimetric analysis take PNP-AFU as substrate, the look group that substrate produces after being decomposed by enzyme is in the basic conditions in yellow, spectrophotometric method is utilized to carry out quantitative test, this law is easy, equipment requirement is not high, but the anti-interference of zymolyte itself is poor, serum icterus and the factor such as haemolysis, piarhemia can not be solved on the impact of measurement result, and complex operation, the required reaction time is long, and AFU need carry out enzymatic reaction under sour environment, and look group needs colour generation in the basic conditions, so be also not suitable for Aulomatizeted Detect, promote difficulty large.Rate method is hydrolyzed containing the specific substrates of colour generation group with AFU catalysis under suitable reaction condition, absorbance can be made under certain wavelength to increase, the change of continuous monitoring absorbance, the activity of AFU in sample can be calculated, this method is simple to operate, minute is short, sensitivity and anti-interference are all better, and be applicable to various half/automatical analysis instrument, because rate method has the advantages that to be convenient to clinical application, in recent years, rate method AFU vitality test is progressively widely used in clinical medicine.
In routine check work, due to liver cancer, liver cirrhosis patient separation of serum needs the time long, in order to energy Result fast, shorten the sample turnaround time and prevent instrument sample pin to be blocked, many hospitals use heparin lithium or liquaemin anticoagulant tube blood sample collection to measure, because heparin is that negative ion sticks polysaccharide, with a large amount of negative charge, in highly acid, easily and rate method detect AFU agents useful for same and react and form the gruff absorbance that makes and raise, thus affect accuracy in detection, therefore, AFU need take into full account the problem of heparin interference in sample when measuring reagent exploitation, simultaneously, if single agents can be prepared into, automatic clinical chemistry analyzer reagent disc space effectively can be saved, improve the utilization factor of automatic clinical chemistry analyzer, more be conducive to this reagent applying clinically.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind ofly has good antiheparin jamming performance, good stability, and lower-cost AFU measures liquid single agents.
In order to solve the problem, the technical solution used in the present invention is: a kind of AFU measures liquid single agents, and each component in reagent and component concentration ranges are:
Damping fluid (pH 4.5 ~ 5.5) 50-200mmol/L,
Anti-interference agent 20-80g/L,
2-chloro-4-nitrobenzene-alpha-L-fucosidase (CNP-Afu) 0.1-1g/L.
The present invention's anti-interference agent described above is the one in soluble calcium salt or magnesium salts, more preferably lime chloride or magnesium chloride or calcium sulphate or magnesium sulfate.
Wherein said damping fluid can be phosphate buffer, glycine-NaOH buffer, citric acid-trisodium citrate damping fluid, borate buffer, imidazole buffer, Acetic acid-sodium acetate damping fluid, the one in MES damping fluid.
The compound method of reagent of the present invention is conventional method, and after namely component formula consumption described in reagent adds distilled water, mixing stirs evenly separately.
The test condition of the AFU in kit measurement sample of the present invention is as follows: temperature: 30-37 DEG C; Cuvette optical path is 1.0cm.Detect predominant wavelength 405nm.
The method applying AFU in AFU of the present invention mensuration kit measurement sample is as follows: sample (calibration tube makees sample with calibration object) reagent adding R hatches 2min for 30-37 DEG C after mixing, and continuous detecting 2min absorbance changes, and calculates Δ A/min.Wherein sample consumption 25 μ l, reagent dosage 225 μ l.
In kit measurement sample of the present invention, AFU content calculates as follows:
Outstanding substantive distinguishing features of the present invention and marked improvement are mainly manifested in:
Reagent of the present invention is liquid single agents, effectively can save automatic clinical chemistry analyzer reagent disc space, improves the utilization factor of automatic clinical chemistry analyzer; For biochemical instruments reagent bit comparison anxiety user, single reagent of the present invention is a kind of ideal selection, is more conducive to this reagent applying clinically.And the specific damping fluid of the present invention (damping fluid kind, concentration and pH) effectively can realize stablizing substrate; In addition, heparin disturbing effect in sample can be removed by anti-interference agent of the present invention add instantaneously, realize the effect of degraded or shielding heparin, thus make liquid single agents of the present invention have good antiheparin jamming performance simultaneously, and because of the anti-interference agent market price that adds inexpensively, make the cost of reagent of the present invention comparatively cheap.
Reagent of the present invention can use Heparin plasma, and can meet the anti-interference requirement of the normal concentration that this area specifies.
Embodiment
To further illustrate the present invention by following non-limiting example below, as well known to those skilled in the art, without departing from the spirit of the invention, can make many amendments to the present invention, such amendment also falls into scope of the present invention.
The collocation method of following reagent if no special instructions, is conventional method, and the experiment material used if no special instructions, all can easily obtain from commercial company.
Embodiment 1:
Acetic acid-sodium acetate damping fluid (pH 4.5) 50mmol/L,
Lime chloride 20g/L,
The chloro-4-nitrobenzene-alpha-L-fucosidase 0.3g/L of 2-.
The compound method of reagent is conventional method, and after namely component described in reagent adds distilled water, mixing stirs evenly separately.
Embodiment 2:
Citric acid-trisodium citrate damping fluid (pH 5.5) 100mmol/L
Magnesium chloride 50g/L
The chloro-4-nitrobenzene-alpha-L-fucosidase 0.1g/L of 2-
The compound method of reagent is conventional method, and after namely component described in reagent adds distilled water, mixing stirs evenly separately.
The test condition of the AFU in kit measurement sample of the present invention is as follows: temperature: 30-37 DEG C; Cuvette optical path is 1.0cm.Detect predominant wavelength 405nm.
The method applying AFU in AFU of the present invention mensuration kit measurement sample is as follows: sample (calibration tube makees sample with calibration object) reagent adding R hatches 2min for 30-37 DEG C after mixing, and continuous detecting 2min absorbance changes, and calculates Δ A/min.Wherein sample consumption 25 μ l, reagent dosage 225 μ l.
In kit measurement sample of the present invention, AFU content calculates as follows:
Be described below in conjunction with the performance of form to the embodiment of the present invention 1 gained reagent.
1, reagent stability experiment:
As shown in table 1:
The experimental result of 1 year preserved by the reagent of table 1 embodiment of the present invention 1 at 4-8 DEG C
| Month | Low value sample (56.3U/L) | High level sample (72.9U/L) |
| 1 | 56.1 | 73.6 |
| 3 | 53.7 | 72.2 |
| 6 | 54.6 | 69.6 |
| 9 | 53.9 | 70.5 |
| 12 | 52.8 | 68.1 |
2, the interference--free experiments of kit:
(1) interference of cholerythrin, haemoglobin, ascorbic acid
Respectively according to the testing sample of ordinary skill in the art preparation containing cholerythrin, haemoglobin, ascorbic acid variable concentrations, measure the kit of embodiment 1 to the antijamming capability (each sample three parts, gets its mean value) of various chaff interference.Testing result (mean value) is as shown in table 2:
Table 2
(2) interference of heparin
Respectively according to the heparin testing sample of ordinary skill in the art preparation containing variable concentrations, measure the kit of embodiment 1 to the antijamming capability (each sample three parts, gets its mean value) of heparin.Testing result (mean value) is as shown in table 3:
Table 3:
From above-mentioned testing result, reagent of the present invention has good antiheparin jamming performance, and reagent has good stability, and kit 2-8 DEG C of preservation at least stablizes 12 months, can meet clinical examination requirement completely.
Claims (3)
1. Alpha-Fucosidase measures a liquid single agents, it is characterized in that: each component in this reagent and concentration of component are:
Damping fluid (pH 4.5 ~ 5.5) 50-200mmol/L,
Anti-interference agent 20-80g/L,
The chloro-4-nitrobenzene-alpha-L-fucosidase 0.1-1g/L of 2-.
Wherein, described anti-interference agent is the one in soluble calcium salt or magnesium salts.
2. Alpha-Fucosidase according to claim 1 measures liquid single agents, and it is characterized in that: described calcium salt is lime chloride or calcium sulphate, described magnesium salts is magnesium chloride or magnesium sulfate.
3. Alpha-Fucosidase according to claim 1 measures liquid single agents, it is characterized in that: described damping fluid is phosphate buffer, glycine-NaOH buffer, citric acid-trisodium citrate damping fluid, borate buffer, imidazole buffer, Acetic acid-sodium acetate damping fluid, the one in MES damping fluid.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510267236.6A CN104865210A (en) | 2015-05-22 | 2015-05-22 | Single liquid reagent for AFU (alpha-fucosidase) determination |
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| CN201510267236.6A CN104865210A (en) | 2015-05-22 | 2015-05-22 | Single liquid reagent for AFU (alpha-fucosidase) determination |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112986164A (en) * | 2021-05-18 | 2021-06-18 | 上海执诚生物科技有限公司 | Anti-heparin stable alpha-L-fucosidase detection kit and application thereof |
| CN114924088A (en) * | 2022-05-09 | 2022-08-19 | 安徽伊普诺康生物技术股份有限公司 | Strong anti-interference and stable AFU detection kit and preparation method thereof |
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| CN102864206A (en) * | 2012-09-11 | 2013-01-09 | 宁波美康生物科技股份有限公司 | Anti-heparin interference leucine aminopeptidase measuring reagent |
| CN102967571A (en) * | 2012-12-24 | 2013-03-13 | 北京利德曼生化股份有限公司 | Liquid single reagent for detecting alpha-L-fucosidase and preparation method thereof |
| CN103380377A (en) * | 2011-02-25 | 2013-10-30 | 三菱化学美迪恩斯株式会社 | Method for measuring myocardial troponin |
| CN104483487A (en) * | 2014-12-22 | 2015-04-01 | 宁波美康生物科技股份有限公司 | Kit for detecting 1, 5-anhydro-D-ghlcitol in human blood |
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2015
- 2015-05-22 CN CN201510267236.6A patent/CN104865210A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2002508839A (en) * | 1997-05-29 | 2002-03-19 | メディカル アナリシス システムズ，インコーポレイテッド | Covalently linked troponin complex |
| CN103380377A (en) * | 2011-02-25 | 2013-10-30 | 三菱化学美迪恩斯株式会社 | Method for measuring myocardial troponin |
| CN102864206A (en) * | 2012-09-11 | 2013-01-09 | 宁波美康生物科技股份有限公司 | Anti-heparin interference leucine aminopeptidase measuring reagent |
| CN102967571A (en) * | 2012-12-24 | 2013-03-13 | 北京利德曼生化股份有限公司 | Liquid single reagent for detecting alpha-L-fucosidase and preparation method thereof |
| CN104483487A (en) * | 2014-12-22 | 2015-04-01 | 宁波美康生物科技股份有限公司 | Kit for detecting 1, 5-anhydro-D-ghlcitol in human blood |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112986164A (en) * | 2021-05-18 | 2021-06-18 | 上海执诚生物科技有限公司 | Anti-heparin stable alpha-L-fucosidase detection kit and application thereof |
| CN112986164B (en) * | 2021-05-18 | 2021-08-24 | 上海执诚生物科技有限公司 | Anti-heparin stable alpha-L-fucosidase detection kit and application thereof |
| CN114924088A (en) * | 2022-05-09 | 2022-08-19 | 安徽伊普诺康生物技术股份有限公司 | Strong anti-interference and stable AFU detection kit and preparation method thereof |
| CN114924088B (en) * | 2022-05-09 | 2023-09-26 | 安徽伊普诺康生物技术股份有限公司 | Strong anti-interference and stable AFU detection kit and preparation method thereof |
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Address after: 315104 Ningbo, Yinzhou, Central District, No. Qiming South Road, No. 299 Applicant after: Meikang biological Polytron Technologies Inc Address before: 315104 Ningbo, Yinzhou, Central District, No. Qiming South Road, No. 299 Applicant before: Ningbo Meikang Biotechnology Co., Ltd. |
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Application publication date: 20150826 |