CN105203472B - A kind of stable free fatty acid determination reagent kit - Google Patents
A kind of stable free fatty acid determination reagent kit Download PDFInfo
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- CN105203472B CN105203472B CN201510378206.2A CN201510378206A CN105203472B CN 105203472 B CN105203472 B CN 105203472B CN 201510378206 A CN201510378206 A CN 201510378206A CN 105203472 B CN105203472 B CN 105203472B
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- 238000000034 method Methods 0.000 claims abstract description 33
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- -1 2- ethoxy Chemical group 0.000 claims description 23
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- KDTSHFARGAKYJN-UHFFFAOYSA-N dephosphocoenzyme A Natural products OC1C(O)C(COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCS)OC1N1C2=NC=NC(N)=C2N=C1 KDTSHFARGAKYJN-UHFFFAOYSA-N 0.000 claims description 18
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Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The present invention relates to a kind of kit of stable enzyme process free fatty acid, which is made of the 2 two kinds of separate liquid reagents of reagent 1 and reagent placed respectively, with good stability and anti-interference.Enzymatic assays free fatty acid, method are easy, easily operated.Novel chromogen has been selected in reagent of the present invention, joined the anti-interferences agent such as ascorbic acid oxidase, potassium ferrocyanide simultaneously in formula, reagent is made to have the characteristics that high sensitivity, strong interference immunity；Liquid stabilising technology is used simultaneously; it joined a series of special stabilizers, chelating agent and enzymatic protective reagent, make reagent that there is excellent stability, can be widely applied to most half/automatic clinical chemistry analyzers; body fat metabolic condition and blood lipid level etc. are monitored, clinical expansion is convenient for.
Description
Technical field
The invention belongs to technical field of medical examination, and in particular to a kind of stable free fatty acid determination reagent kit.
Background technique
Free fatty acid, i.e. non-esterified fatty acid (non-estesterfied fatty acid, NEFA), are in human body
The donor of the intermediate product of fat metabolism and the endocellular signal molecules such as the substrate of Cell membrane lipids structure and prostaglandin.
The energy needed for muscle activity --- when glycogen exhausts, adipose tissue can decompose neutral fat as free fatty acid to serve as energy
Source uses.Although free fatty acid only accounts for the seldom a part of body fat, the significant portion of energy requirement is met, therefore
But the important indicator of the metabolism of monitor's body fat, glycometabolism.Free fatty acid can not only reflect body fat metabolic condition and
Blood lipid level, evaluation blood glucose and auxiliary diagnosis diabetes, moreover it is possible to reflect various other pathology, physiological conditions, such as pancreas islet in human body
Plain resistance, obesity, malignant disease, Metabolic syndrome are sought peace cardiovascular disease etc..
High free fatty acid concentration is more common in obesity, diabetes, myocardial infarction, severe hepatopathy, thyroid gland function in serum
It can hyperfunction, acromegalia and starvation etc.；Free fatty acid content is low see hypothyroidism, pituitary insufficiency,
Addison's disease etc. can also cause the reduction of free fatty acid physiological after the meal.
It is increasingly multiple with modern diseases such as diabetes, hypertension, hyperlipidemias, the abnormal metabolism of carbohydrate and lipid is supervised
Control also becomes the material particular of clinically diagnosis and treatment and the detection state of an illness.The method of free fatty acid is more in measurement serum at present, such as
Enzyme process, titration, colorimetric method, atom spectrophotometry, high pressure liquid chromatography, gas chromatography etc..Since conventional method is grasped
It is poor to make cumbersome and precision, and enzyme process detection serum free fatty acid kit has specificity height, precision good, easy to operate
The advantages that, therefore in the automatic quick detection of clinical high-volume sample, enzyme process is applied the most extensive.
The key reaction principle of commercially available enzymatic assays free fatty acid kit is all based on greatly Trinder reaction pattern, tool
Precursor reactant process is as follows:
That is free fatty acid is in the presence of ATP, coacetylase, through fatty acyl-CoA synthetase (Acyl CoA
Synthetase, ACS) effect, generate acyl coenzyme A；Acyl coenzyme A is by acyl coenzyme A oxidizing ferment (Acyl CoA
Oxidase, ACOD) oxidation, generate trans--enoyl CoA (2,3-trans-Enoyl-CoA) and hydrogen peroxide；And generate
Hydrogen peroxide is in the presence of peroxidase (peroxidase, POD), with Trinder chromogen and 4-AA (4-
Aminoantipyrine, 4-APP) generate red quinone imines object (purple dye), the trip in the production quantity and sample of the substance
It is proportional from content of fatty acid, the concentration of free fatty acid in sample can be obtained by measuring its absorbance.
The kit of presently commercially available enzymatic assays free fatty acid is primarily present following problems:
1. enzymatic assays free fatty acid kit is initially from external import, import reagent is with good stability and excellent
Elegant analysis performance, but it is expensive, and testing cost is higher, is not suitable for large-scale application.Now, the biochemical inspection of free fatty acid
Survey is in the ascendant in the domestic market, and only a smaller number of producer can be realized the self-produced of free fatty acid kit, but its
Used chromogen reactive material activity is low, and color stability is poor, anti-interference is weak, so as to cause entire stabilization of kit
Difference, testing result inaccuracy, sensitivity are low.
2. reacting since the peroxidase of catalysis Trinder reaction is poor to Substratspezifitaet vulnerable to endogenous object
The interference of matter, such as ascorbic acid, bilirubin, haemolysis.The reducing substances such as ascorbic acid can also be with H2O2Compete peroxidase
(POD) or with chromogenic substance H is competed2O2, to make the H generated in oxidation process2O2It is consumed, the red quinone imines of generation
Compound is reduced, and causes measurement result inaccurate.
3. presently commercially available enzyme process free fatty acid kit selects single stabilizer, due to free fatty acid kit
Comparison of ingredients complexity enzyme containing there are many, stabilization of kit is directly related to enzymatic activity, and the activity of enzyme is vulnerable to various factors
Interference, such as pH, temperature, ultraviolet light, heavy metallic salt, inhibitor, activator etc., lead to kit deficient in stability, save
Time is short, influences clinical use.Single stabilizer has not been able to satisfy the requirement to stabilization of kit, therefore, group more and more
Conjunction, which selects two or more to prepare free fatty acid determination reagent kit simple, low in cost, more to enzyme class, to be had well surely
The stabilizer for determining effect, is very important.
Summary of the invention
The present invention is to overcome above-mentioned prior art defect, provides a kind of enzyme process free-fat acidity test examination of high stability
Agent box can eliminate the interference of many kinds of substance such as ascorbic acid in human serum, bilirubin, high sensitivity, at low cost, can not only
The demand for adapting to large-scale crowd generaI investigation is also able to satisfy the requirement that routine clinical is quick, largely checks, is suitble to industrialization, is convenient for
Clinical expansion.
In order to solve the above-mentioned technical problem, the technical solution used in the present invention are as follows: one kind is based on Trinder reaction principle
Enzymatic assays serum in free fatty acid kit, composition include 2 two kinds of independent liquid reagents of reagent 1 and reagent,
Middle reagent 1, each component and concentration range in reagent 2 are as follows:
Reagent 1:
Reagent 2:
The volumetric ratio of the reagent 1 and reagent 2 is 4:1.
Since enzyme process reaction needs the buffer environment of suitable pH range and suitable ionic strength, ionic strength is excessively high, electricity
Solution matter interferases and Binding Capacity, enzymatic activity will be gradually reduced, but ionic strength it is too low may also can not kinase activity, because
This, is typically chosen the ionic strength relatively with the body fluid of physiological environment.Preferably, reagent buffer choosing of the present invention
From the unrestraints effect such as reacting enzymology, GOOD ' the S buffer of biochemical reaction process is not interfered, such as 2- morpholino second
Sulfonic acid (MES), 3- morpholino propane sulfonic acid (MOPS), N- [three (methylol) methyl] -2- NSC 209983 (TES), 3- [4- (2-
One of methylol) -1- piperazine] propane sulfonic acid ((H) EPPS), preferably MOPS buffer, pH preferred scope are 6.0~8.5.Root
According to the requirement of enzymic catalytic reaction optimum condition, also need to be added a certain amount of ion-activated dose in enzymatic determination system, activator
It can be the activated centre of enzyme, the activity of other mechanism activation enzymes can also be passed through.Preferably, institute in kit of the present invention
Ion-activated dose is stated as MgCl2·6H2O, while certain density KCL is added and adjusts ionic strength.
Preservative of the present invention is imidazolidinyl urea, and more commercially available common proclin series preservative not only has good
Good antisepsis, and without reproducibility, to enzymatic activity unrestraint, do not influence to react.
Stabilizer of the present invention is simultaneously not specific to some, but has played stabilization jointly by two or more reagent
The effect of agent is a key point of the invention to keeping enzymatic activity, the steady in a long-term of maintenance reagent to play a significant role.
The stabilizer is selected from one of trehalose, sucrose, beta-cyclodextrin, mannitol or a variety of, and can not only play stably reagent
Effect, can be also used for maintain reagent in a variety of enzymes activity.It is more preferable to improve in order to reduce the cross influence between each reagent
The stability of reagent, the stabilizer add albumin simultaneously, especially select the nothing using the method manufacture on genetic engineering
Fatty syntonin, through special degreasing purification process, content of fatty acid is lower than 0.002% (w/w), comes compared with commercial product selection
From ox, horse, sheep, people albumin, eliminate its influence of the contained high concentration fatty acid to testing result, reduce detection background
Interference.
Enzymatic protective reagent is that acyl coenzyme A aoxidizes enzyme stabilizers in reagent 2 of the present invention, for maintaining the rouge in reagent 2
The high activity of acyl coenzyme A oxidizing ferment, helps to improve the stability of reagent.
The selection of chromogen substance of the present invention is particularly significant to the stability of reagent, sensitivity and accuracy, for this
Another key factor of invention.The present invention is by being comprehensively compared common chromogen both at home and abroad, a kind of final choice new color of amine
Former bis- propanesulfonate aniline (TODP) of 3- methyl-N, N- the advantage is that chromogen as enzymatic assays free fatty acid color developing agent
Synthesis be easy, performance stablize, molecular conjugation architectural characteristic sensitivity is greatly improved (reaction sensitivity can reach
0.01mmol/L), sample usage amount can be accordingly reduced, the amount of chaff interferent is finally effectively reduced；It is the sensitivity of the chromogen, steady
It is qualitative better than the color developing agents such as TOPS, TOOS, ADPS, rapidly with the colour developing of 4-AAP oxidative condensation, in colour-stable, can be effectively improved pair
The influence of enzyme stability in reaction system；The amine chromogen element maximum absorption band that its chromogenic reaction generates, can in 540nm or more
Effectively avoid jaundice, haemolysis interference.
What each group subassembly of the present invention was played goes interference effect, is that enzyme process Accurate Determining serum N EFA of the present invention is dense
Spend another key factor.
Surfactant of the present invention is selected from Brij35, Brij98, Tween20.Surfactant is the promotion of reaction
Agent can remove the influence of other lipid impurities, improve the sensitivity of detection.It is described in order to improve the jamproof ability of reagent
It joined potassium ferrocyanide and ascorbic acid oxidase in reagent 1, the combination of potassium ferrocyanide and surfactant can effectively disappear
Influence except bilirubin high in sample to measured value, ascorbic acid oxidase can effectively eliminate in sample high ascorbic acid to measurement sample
This interference, to ensure that the accuracy of clinical detection result.
Reagent 1 of the present invention selects EGTA chelating agent (bis- (the 2- amino-ethyl ether) tetraacethyls of ethylene glycol), can effectively prevent
Only remove Mg2+Outer other bivalent metal ions interference first step reaction.The reagent 2 has especially selected amino carboxy chelating agent DTPA chela
Mixture (diethylene triamine pentacetic acid (DTPA)), more common disodium EDTA (EDTA), bicine N- (DHG) etc.
Chelating agent performance is more excellent, can generate water soluble chelate compound with calcium, magnesium, iron, lead, copper plasma rapidly, effectively complexing subsequent reactions
In excessive magnesium ion and inhibit the active metal ion of acyl-CoA oxidase, improve the accuracy of measurement result；Simultaneously also
Can prevent reaction in various metal cations to H2O2Decomposition, as H2O2Stabilizer, with apparent synergy and surely
It is set for using.
4-AA (4-AAP) of the present invention is selected from high-purity product, acts on not anti-in first time enzyme
Middle performance is answered, but remains to participate in color reaction in the enzyme reaction of Catalyzed Synthesis By Peroxidase.
It is the auxiliary of acyl coenzyme A oxidizing ferment that Flavin Adenin Dinucleotide Sodium salt (FAD) is added in reagent 2 of the present invention
Enzyme plays the role of hydrogen carrier in the reaction, helps to improve the flexibility of reaction, keeps oxidation reaction more complete, ensure that anti-
Answer efficient, stable progress.
Fatty acyl-CoA synthetase and acyl coenzyme A oxidizing ferment of the present invention is selected to be mentioned by genetic engineering recombinant product
The high-purity product taken；The peroxidase (POD) is selected from the high stability product extracted by horseradish.
Heretofore described percent concentration is quality concentration of volume percent, i.e., as do not illustrated
The grams of contained solute in 100ml solution.
Term " high-purity " used herein refers to that the purity of product is greater than 95%.
Compared with prior art, the present invention has following remarkable advantage and beneficial outcomes:
Kit of the present invention and import reagent box correlation are good, testing result have high consistency (correlation coefficient r >=
0.990), linear fit related coefficient is better than import reagent, and detection data is accurate and reliable, can substitute import reagent box in clinic
It uses, significantly reduces testing cost.
Kit of the present invention selects liquid double reagent detection method, and each reactive material of reasonable distribution is in entire reaction system
Concentration avoids the appearance of spontaneous reaction, and the space structure for remaining resident in one of the various enzymes is complete, while being added to enzyme protection
Agent ensure that the long-term high activity of enzyme, effectively raise reagent performance.
Kit of the present invention selects novel chromogen substance, and performance is more excellent, is in colour-stable, while being added to anti-interference agent, compared with
Conventional free fatty acid determination reagent kit strong anti-interference performance, can effectively avoid haemolysis in sample, bad hematic acid, bilirubin and Huang
Interference of the subcutaneous ulcer to testing result, improves the sensitivity for analysis of kit.
Kit of the present invention selects compound stabilizer, effectively extends the holding time of kit, ensure that in reagent
The performance of a variety of reaction enzymes is stablized, and each reaction enzymes will not generate variation because environment temperature etc. changes in storage life, improves
The stability of kit.Kit of the present invention is stored under the conditions of 2-8 DEG C of temperature, is at least stablized 20 months；After reagent
2-8 DEG C can stablize preservation at least one moon and accuracy without significant change, and commercial reagent box validity period is generally 12 months, opens
It can only generally be saved 10-15 days after bottle, be long placed in rear accuracy and be substantially reduced.
The present invention is used for clinical detection serum free fatty acid content, simple and quick, of less demanding to medical facilities,
It is applied widely, it can be not only widely used in large and medium-sized hospital, but also may be directly applied to various full-automatic, semiautomatic biochemistries
Analyzer, it is only necessary to which a few minutes can complete to detect.
Detailed description of the invention
It is attached it is shown in FIG. 1 be that enzyme process free fatty acid determination reagent kit described according to embodiments of the present invention 1 and import try
Agent box detects the correlation comparison chart of 40 clinical samples.
It is attached it is shown in Fig. 2 be that enzyme process free fatty acid determination reagent kit described according to embodiments of the present invention 1 and import try
Agent box line comparison diagram.
It is attached it is shown in Fig. 3 be that enzyme process free fatty acid determination reagent kit described according to embodiments of the present invention 2 and import try
Agent box detects the correlation comparison chart of 40 clinical samples.
It is attached it is shown in Fig. 4 be that enzyme process free fatty acid determination reagent kit described according to embodiments of the present invention 2 and import try
Agent box line comparison diagram.
It is attached it is shown in fig. 5 be that enzyme process free fatty acid determination reagent kit described according to embodiments of the present invention 3 and import try
Agent box detects the correlation comparison chart of 40 clinical samples.
It is attached it is shown in fig. 6 be that enzyme process free fatty acid determination reagent kit described according to embodiments of the present invention 3 and import try
Agent box line comparison diagram.
Specific embodiment
The present invention is further described by the following embodiment, and still, the application is not limited to these embodiments, these
Embodiment can not be construed to the limitation to the application.
The preparation method of following reagents is conventional method unless otherwise instructed, and used test material is as without especially
Illustrate, can be obtained from commercial company.
Embodiment 1
One, a kind of stable enzyme process free fatty acid determination reagent kit, the kit according to following compositions and ratio by matching
The liquid double reagent of system forms, in which:
Reagent 1:
Reagent 2:
The volumetric ratio of the reagent 1 and reagent 2 is 4:1.
Two, operating method
The free fatty acid determination reagent kit of the present embodiment description, using automatic biochemistry analyzer, by taking Hitachi 7080 as an example,
Concrete operations are as follows:
The kit test method of the present invention of table 1
Test condition: 37 DEG C, cuvette optical path is 1.0cm, detects dominant wavelength 546nm, commplementary wave length 660nm.By following public affairs
Formula calculates free fatty acid content.
Three, the correlation of kit of the present invention and import enzyme process kit
Using kit described in the present embodiment and import reagent box (desai), while to the rouge that dissociates in 40 human serum samples
The testing result of fat acid carries out correlation analysis.Import reagent box is operated according to its specification, measurement result such as the following table 2 institute
Show.Import reagent box term of reference is 0.10-0.60mmol/L in following table, and the term of reference of kit of the present invention is 0.13-
0.77mmol/L。
The kit of the present invention of table 2 is compared with import reagent box is to the measurement result of free fatty acid
Coefficient R indicates the degree of correlation of two groups of numerical value, and R shows that the correlation of two groups of data is better closer to 1.Usually
Think that correlation of the R greater than 0.990 when is good.
The calculation method of the related coefficient is as follows:
Wherein, X is the NEFA content concn value that kit of the present invention measures,For the average of the above-mentioned X value measured；Y
For the NEFA content concn value for using import reagent box to measure,For the average of the above-mentioned Y value measured.
It can be seen from upper table statistical result in this 40 Freshman serum samples, occur altogether 29 negative findings and
11 positive findings, kit of the present invention and import enzyme process kit are one-to-one, the two correlation in result judgement
Well (R=0.9990), clinical meaning is significant.
Above test is carried out using 7080 model automatic clinical chemistry analyzers of Hitachi, Ltd's manufacture, but of the invention
The application of kit is not limited to above-mentioned instrument, applies also for other full-automatic or semi-automatic biochemical analyzers, and this field skill
Art personnel can according to circumstances make the appropriate adjustments location parameter.
Four, linear assessment
A certain high level sample is taken, respectively with kit of the present invention and 5 times times of high level sample of import reagent box (desai) measurement
Than dilution after it is linear, the linear of each kit is assessed from actual sample with this, the results are shown in Table 3.
The kit of the present invention of table 3 and import reagent box Linear Comparison
| Extension rate | Kit of the present invention | Import reagent box |
| 0 | 0.000 | 0.000 |
| 0.0313 | 0.061 | 0.064 |
| 0.0625 | 0.186 | 0.143 |
| 0.125 | 0.362 | 0.354 |
| 0.25 | 0.741 | 0.652 |
| 0.5 | 1.582 | 1.443 |
| 1 | 3.211 | 2.881 |
The result shows that kit of the present invention is linearly good, fitting correlation coefficient is better than import reagent box.
Five, stability study
Using kit described in the present embodiment, high and low two horizontal free-fat acid samples are measured, Mei Geshui
Flat sample replication 10 times, and the reagent measurement result placed 20 months with 2-8 DEG C respectively compares, and verifies measurement result
Accuracy calculate the average and standard deviation of each horizontal sample measurement result by regular statistics requirement, then by public affairs
Formula:
The coefficient of variation (CV)=standard deviation/average value × 100%；
Wherein, X is the free fatty acid content concentration value measured,For the average of the X value measured, n is replication
Number；
Coefficient of variation CV is calculated, is as a result listed in the table below in 4；
By formula
Wherein T is initial measured value mean value,20 months measured value mean values are placed for 2-8 DEG C.
The 2-8 DEG C of relative deviation (B%) for placing 20 months measured values and initial measured value mean value is calculated, table 4 is as a result listed in
In.
2-8 DEG C of the kit of the present invention of table 4 places 20 months testing results for a long time
Kit described in the present embodiment is placed 20 months at 2-8 DEG C as can be seen from Table 4, and measurement result repeatability CV is small
In 10%, and and the deviation of initial measured value be respectively less than 10%, meet " external diagnosis reagent General Requirement ", show examination of the present invention
Agent box repeatability is good, has preferable accuracy and good stability.
Six, the influence of enzymatic activity
Protective effect of the 0.5% acyl coenzyme A oxidation enzyme stabilizers to acyl coenzyme A oxidizing ferment is added.
Influence of 5 enzymatic protective reagent of table to enzymatic activity in reagent
| Time | Not enzyme protective agent remaining enzyme activity | 0.5% acyl coenzyme A oxidation enzyme stabilizers remaining enzyme activity is added |
| 0 month | 100% | 100% |
| 1 month | 96% | 98% |
| 3 months | 84% | 94% |
| 6 months | 73% | 88% |
| 12 months | 42% | 78% |
| 18 months | 26% | 66% |
| 20 months | 17% | 62% |
Kit of the present invention has very strong protective effect to the enzyme in reagent, over time, makees to the protection of enzyme
With that can become apparent from, compared with the reagent of not enzyme stabilizer, remaining enzyme activity rate increases the reagent of enzyme stabilizer after 20 months
45%.
Embodiment 2
One, a kind of stable enzyme process free fatty acid determination reagent kit, the kit according to following compositions and ratio by matching
The liquid double reagent of system forms, in which:
Reagent 1:
Reagent 2:
The volumetric ratio of the reagent 1 and reagent 2 is 4:1.
Two, the correlation of kit of the present invention and import reagent box
Using kit described in the present embodiment, by 1 the method for embodiment, and meanwhile it is right with commercially available import reagent box (desai)
Free fatty acid content in 40 human serum samples is measured, and import reagent box is operated according to its specification, measurement
As a result as shown in table 6 below.Import reagent box term of reference is 0.10-0.60mmol/L in following table, the reference of kit of the present invention
Range is 0.13-0.77mmol/L.
The kit of the present invention of table 6 is with import reagent box to the measurement result of NEFA
It can be seen from upper table statistical result in this 40 Freshman serum samples, occur altogether 30 negative findings and
10 positive findings, kit of the present invention and import enzyme process kit are one-to-one, the calculating present invention in result judgement
Correlation coefficient r=0.9989 of kit and import reagent box measurement result, the two correlation is good, and clinical meaning is significant.
Above test is carried out using 7080 model automatic clinical chemistry analyzers of Hitachi, Ltd's manufacture, but of the invention
The application of kit is not limited to above-mentioned instrument, applies also for other full-automatic or semi-automatic biochemical analyzers, and this field skill
Art personnel can according to circumstances make the appropriate adjustments location parameter.
Three, linear assessment
A certain high level sample is taken, respectively with kit of the present invention and 5 times times of high level sample of import reagent box (desai) measurement
Than dilution after it is linear, the linear of each kit is assessed from actual sample with this, the results are shown in Table 7.
The kit of the present invention of table 7 and import reagent box Linear Comparison
| Extension rate | Kit of the present invention | Import reagent box |
| 0 | 0.000 | 0.000 |
| 0.0313 | 0.131 | 0.143 |
| 0.0625 | 0.272 | 0.252 |
| 0.125 | 0.553 | 0.499 |
| 0.25 | 1.122 | 0.973 |
| 0.5 | 2.275 | 1.876 |
| 1 | 4.616 | 3.591 |
The result shows that kit of the present invention is linearly good, fitting correlation coefficient is better than import reagent box.
Four, stability study
Using kit described in the present embodiment, high and low two horizontal free fatty acid samples, each horizontal samples are measured
Replication 10 times, the reagent measurement result placed 20 months to 2-8 DEG C compares, and verifies the accuracy of measurement result, presses
Regular statistics requirement, calculates the average and standard deviation of each horizontal sample measurement result, then presses formula:
The coefficient of variation (CV)=standard deviation/average value × 100%
Wherein, X is the NEFA content concn value measured,For the average of the X value measured, n is replication number；
Coefficient of variation CV is calculated, is as a result listed in the table below in 8；
By formula
Wherein T is initial measured value mean value,20 months measured value mean values are placed for 2-8 DEG C.
The 2-8 DEG C of relative deviation (B%) for placing 20 months measured values and initial measured value mean value is calculated, table 8 is as a result listed in
In.
2-8 DEG C of the kit of the present invention of table 8 places 20 months testing results for a long time
Kit described in the present embodiment is placed 20 months at 2-8 DEG C as can be seen from Table 8, and measurement result repeatability CV is small
In 10%, and and the deviation of initial measured value be respectively less than 10%, meet " external diagnosis reagent General Requirement ", show examination of the present invention
Agent box repeatability is good, has preferable accuracy and good stability.
Six, the influence of enzymatic activity
Protective effect of the 2.0% acyl coenzyme A oxidation enzyme stabilizers to acyl coenzyme A oxidizing ferment is added.
Influence of 9 enzymatic protective reagent of table to enzymatic activity in reagent
| Time | Not enzyme protective agent remaining enzyme activity | 2.0% acyl coenzyme A oxidation enzyme stabilizers remaining enzyme activity is added |
| 0 month | 100% | 100% |
| 1 month | 96% | 99% |
| 3 months | 87% | 94% |
| 6 months | 70% | 85% |
| 12 months | 41% | 79% |
| 18 months | 24% | 67% |
| 20 months | 11% | 60% |
Kit of the present invention has very strong protective effect to the enzyme in reagent, over time, makees to the protection of enzyme
With that can become apparent from, compared with the reagent of not enzyme stabilizer, remaining enzyme activity rate increases the reagent of enzyme stabilizer after 20 months
49%.
Embodiment 3
One, a kind of stable enzyme process free fatty acid determination reagent kit, the kit according to following compositions and ratio by matching
The liquid double reagent of system forms, in which:
Reagent 1:
Reagent 2:
The volumetric ratio of the reagent 1 and reagent 2 is 4:1.
Two, the correlation of kit of the present invention and import reagent box
Using kit described in the present embodiment, by 1 the method for embodiment, while commercially available import reagent box (desai) is used
Free fatty acid content in 40 human serum samples is measured, import reagent box is operated according to its specification, is surveyed
It is as shown in the following table 10 to determine result.Import reagent box term of reference is 0.10-0.60mmol/L in following table, the ginseng of kit of the present invention
Examining range is 0.13-0.77mmol/L.
The kit of the present invention of table 10 is with import reagent box to the measurement result of NEFA
It can be seen from upper table statistical result in this 40 Freshman serum samples, occur altogether 28 negative findings and
12 positive findings, kit of the present invention and import enzyme process kit are correspondingly, by 1 institute of embodiment in result judgement
Method is stated, correlation coefficient r=0.9992 of kit and import reagent box measurement result of the present invention is calculated, the two correlation is good
Good, clinical meaning is significant.
Above test is carried out using 7080 model automatic clinical chemistry analyzers of Hitachi, Ltd's manufacture, but of the invention
The application of kit is not limited to above-mentioned instrument, applies also for other full-automatic or semi-automatic biochemical analyzers, and this field skill
Art personnel can according to circumstances make the appropriate adjustments location parameter.
Three, linear assessment
A certain high level sample is taken, measures 5 times of high level sample with kit of the present invention and import reagent box (desai) respectively
It is linear after doubling dilution, the linear of each kit is assessed from actual sample with this, the results are shown in Table 11.
The kit of the present invention of table 11 and import reagent box Linear Comparison
| Extension rate | Kit of the present invention | Import reagent box |
| 0 | 0 | 0 |
| 0.0313 | 0.091 | 0.082 |
| 0.0625 | 0.233 | 0.216 |
| 0.125 | 0.488 | 0.441 |
| 0.25 | 0.962 | 0.917 |
| 0.5 | 2.031 | 1.933 |
| 1 | 4.119 | 3.676 |
The result shows that kit of the present invention is linearly good, fitting correlation coefficient is better than import reagent box.
Four, stability study
Using kit described in the present embodiment, high and low two horizontal free-fat acid samples, each horizontal repetition are measured
Measurement 10 times, the reagent measurement result placed 20 months to 2-8 DEG C compare, and the accuracy of measurement result are verified, by regular
Statistics requirement, calculates the average and standard deviation of each horizontal samples measurement result, then presses formula:
The coefficient of variation (CV)=standard deviation/average value × 100%
Wherein, X is the NEFA content concn value measured,For the average of the X value measured, n is replication number；
Coefficient of variation CV is calculated, is as a result listed in the table below in 12；
By formula
Wherein T is initial measured value mean value,20 months measured value mean values are placed for 2-8 DEG C.
The 2-8 DEG C of relative deviation (B%) for placing 20 months measured values and initial measured value mean value is calculated, table 12 is as a result listed in
In.
Table 12 is 2-8 DEG C of kit of the present invention long-term to be placed 20 months
Kit described in the present embodiment is placed 20 months at 2-8 DEG C as can be seen from the above results, measurement result repeatability
CV is respectively less than 10%, and and the deviation of initial measured value be respectively less than 10%, meet " external diagnosis reagent General Requirement ", show this
Invention enzyme process free-fat acid detection kit accuracy with higher and good stability.
Five, the influence of enzymatic activity
Protective effect of the 1% acyl coenzyme A oxidation enzyme stabilizers to acyl coenzyme A oxidizing ferment is added.
Influence of 13 enzymatic protective reagent of table to enzymatic activity in reagent
| Time | Not enzyme protective agent remaining enzyme activity | 1% acyl coenzyme A oxidation enzyme stabilizers remaining enzyme activity is added |
| 0 month | 100% | 100% |
| 1 month | 94% | 98% |
| 3 months | 80% | 95% |
| 6 months | 71% | 89% |
| 12 months | 40% | 81% |
| 18 months | 25% | 72% |
| 20 months | 15% | 66% |
Kit of the present invention has very strong protective effect to the enzyme in reagent, over time, makees to the protection of enzyme
With that can become apparent from, compared with the reagent of not enzyme stabilizer, remaining enzyme activity rate increases the reagent of enzyme stabilizer after 20 months
51%.
Claims (4)
1. a kind of stable enzyme process free fatty acid determination reagent kit, it is characterised in that: including 2 two kinds of difference of reagent 1 and reagent
The liquid reagent of placement, each component and concentration range are as follows:
Reagent 1:
Reagent 2:
The volumetric ratio of the reagent 1 and reagent 2 is 4:1；
The buffer is selected from 2- morpholino ethanesulfonic acid (MES), 3- morpholino propane sulfonic acid (MOPS), N- [three (methylol) first
Base] -2-aminoethanesulfonic acid (TES), one of 3- [4- (2- ethoxy) -1- piperazine] propane sulfonic acid (HEPPS)；
MOPS buffer, pH range are 6.0~8.5；
Stabilizer combination one or more in trehalose, sucrose, beta-cyclodextrin, mannitol, while adding without rouge
Fat syntonin；
The preservative is imidazolidinyl urea；
The surfactant is selected from Brij35, Brij98, Tween20；
The enzymatic protective reagent is that acyl coenzyme A aoxidizes enzyme stabilizers；
The chromogen is bis- propanesulfonate aniline of 3- methyl-N, N-；
Percent concentration is quality concentration of volume percent as do not carried out specified otherwise, i.e., contained solute in 100ml solution
Grams.
2. kit according to claim 1, it is characterised in that: the albumin is to utilize legal system above genetic engineering
The no fatty acids albumin made, purity are greater than 98%, and content of fatty acid is lower than 0.002% weight percent.
3. kit according to claim 1 or 2, it is characterised in that: the fatty acyl-CoA synthetase and acyl coenzyme A
Oxidizing ferment is selected from high-purity product of the purity greater than 95% extracted by genetic engineering recombinant product.
4. kit according to claim 3, it is characterised in that: the peroxidase is the high stability that horseradish extracts
Product.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510378206.2A CN105203472B (en) | 2015-06-30 | 2015-06-30 | A kind of stable free fatty acid determination reagent kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510378206.2A CN105203472B (en) | 2015-06-30 | 2015-06-30 | A kind of stable free fatty acid determination reagent kit |
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| Publication Number | Publication Date |
|---|---|
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| CN105203472B true CN105203472B (en) | 2019-03-08 |
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| CN106191206B (en) * | 2016-07-07 | 2019-05-07 | 宏葵生物（中国）有限公司 | Free-fat acid detection kit and preparation method |
| CN106191213B (en) * | 2016-09-19 | 2019-10-11 | 中拓生物有限公司 | A kind of free fatty acid determination reagent kit and preparation method thereof |
| CN106442496A (en) * | 2016-09-20 | 2017-02-22 | 上海科华生物工程股份有限公司 | Free fatty acid detection kit |
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| CN113418877A (en) * | 2021-06-09 | 2021-09-21 | 东软威特曼生物科技(南京)有限公司 | Free fatty acid detection kit and method |
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| CN115932239B (en) * | 2022-12-22 | 2024-02-20 | 广州市进德生物科技有限公司 | Oxidized low-density lipoprotein protective agent, detection kit for determining oxidized low-density lipoprotein and application of detection kit |
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Denomination of invention: A Stable Free Fatty Acid Determination Kit Effective date of registration: 20231222 Granted publication date: 20190308 Pledgee: Zhongguancun Branch of Bank of Beijing Co.,Ltd. Pledgor: BEIJING WANTAI DRD CO.,LTD. Registration number: Y2023110000546 |
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