CN111982841B - Free fatty acid detection kit - Google Patents
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Abstract
The invention discloses a free fatty acid detection kit, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent contains buffer solution, fatty acyl coenzyme A synthetase, coenzyme A, ATP, 4-aminoantipyrine, an enzyme protective agent, a surfactant and a preservative, the R2 reagent contains buffer solution, fatty acyl coenzyme A oxidase, peroxidase, chromogen substrate, an enzyme protective agent, a surfactant and a preservative, the chromogen substrate in the R2 reagent is a mixture of DAOS and TOOS, and the concentration ratio of DAOS to TOOS is 1. The invention optimizes the chromogen substrate in the free fatty acid detection kit, widens the linear range of the kit, and solves the problem that ascorbic acid and bilirubin in a serum sample interfere the detection of free fat, namely, the free fatty acid detection kit with wider linear range and stronger anti-interference capability is obtained; furthermore, the invention also preferably selects the type and proportion of the enzyme protective agent, thereby enhancing the stability of the kit.
Description
Technical Field
The invention belongs to the technical field of clinical chemical detection, and particularly relates to a free fatty acid detection kit.
Background
Free fatty acids, i.e., non-esterified fatty acids (NEFA), are intermediates of fat metabolism in humans, and are also donors of intracellular signaling molecules such as substrates of cell membrane lipid structures and prostaglandins. The NEFA in serum has extremely high metabolic activity, and is easily influenced by fat metabolism, carbohydrate metabolism and endocrine metabolism. NEFA has been proved to be closely related to the occurrence and development of cardiovascular and cerebrovascular diseases, respiratory diseases, digestive diseases, endocrine diseases, immune diseases and other system diseases, and energy metabolism such as traumatic stress.
The detection principle applied by the free fatty acid determination kit (enzyme method) is that the free fatty acid in a sample generates fatty acyl-CoA under the action of fatty acyl-CoA synthetase under the condition that coenzyme A and Adenosine Triphosphate (ATP) coexist, the generated fatty acyl-CoA simultaneously generates hydrogen peroxide under the oxidation action of fatty acyl-CoA oxidase, and a colored product is generated under the action of Peroxidase (POD) through a Trinder substrate. And (3) quantitatively detecting the content of the free fatty acid in the sample by comparison with the similarly treated calibration solution at a certain wavelength.
At present, most free fatty acid detection kits in the market have a narrow linear range, which can only achieve about 3.0mmol/L, for example, patent CN109521013A discloses a free fatty acid detection kit and a production process, and the highest detection range of the kit can only achieve 4.2mmol/L. And the sample exceeding the upper limit of the linear range needs to be diluted and retested, and the retested result is multiplied by the dilution multiple to obtain the sample result. If the upper linear range limit of the kit is higher, less sample needs to be diluted for review, thereby reducing hospital testing costs and manpower requirements. In addition, after the off-fatty acid determination kit in the market is transported or stored for a long time, the reagent is easy to precipitate, so that the stability of the reagent is influenced; it is also susceptible to ascorbic acid and bilirubin.
Disclosure of Invention
The invention aims to provide a free fatty acid detection kit, which is wider in linear range, higher in stability and stronger in anti-interference capability by replacing the traditional chromogen substrate with a mixture of DAOS and TOOS, adjusting the concentration ratio of the two chromogen substrates and adding a special enzyme protective agent.
In order to realize the purpose, the invention adopts the technical scheme that:
a free fatty acid detection kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent contains buffer solution, fatty acyl coenzyme A synthetase, coenzyme A, ATP, 4-aminoantipyrine, an enzyme protective agent, a surfactant and a preservative, and the R2 reagent contains buffer solution, fatty acyl coenzyme A oxidase, peroxidase, chromogen substrate, enzyme protective agent, surfactant and preservative, and is characterized in that the chromogen substrate in the R2 reagent is a mixture of DAOS and TOOS, and the concentration ratio of DAOS to TOOS is 1.
The invention creatively selects N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline sodium salt (DAOS) with higher sensitivity and N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt (TOOS) with stronger anti-jamming capability from the chromogen substrate, and adjusts the proportion of the two substances, so that the linear range of the free fatty acid detection kit is wider, and simultaneously, the problem of interference of ascorbic acid and bilirubin in serum on the detection of the free fatty acid is also avoided.
Preferably, the concentration ratio of DAOS and toss is 1.
Preferably, the enzyme protective agent in the R1 reagent and the R2 reagent is EDTA-2K (ethylene diamine tetraacetic acid dipotassium salt), FAD (flavin adenine dinucleotide), mgSO 4 、CaCl 2 、CoCl 2 A mixture of any two of. The enzyme protective agent is added into the R1 and R2 reagents, so that enzyme substances such as fatty acyl-CoA synthetase, fatty acyl-CoA oxidase and the like in the reagents can be protected, the influence of temperature change and long-term storage on the enzyme activity is avoided, and the stability of the reagents is enhanced.
Preferably, the enzyme protecting agent is EDTA-2K and CoCl 2 1 in a concentration ratio of 1. The stability of the reagent is further enhanced by the preference for the type of enzyme protecting agent and its ratio.
Preferably, the buffer solution in the R1 reagent and the R2 reagent is any one of PBS buffer solution, phosphate buffer solution, GOOD's buffer solution, MES buffer solution, and HEPES buffer solution.
Preferably, the surfactant is one or more of Tween20, EMULGEN A-60, EMULGEN A-90, EMULGEN B-66, EMULGEN709, polyoxyethylene lauryl ether (Brij-35), triton X-100, GENAPOLX-080 and dodecyl-tetradecyl dimethyl betaine.
Preferably, the preservative is one or more of sodium azide, potassium sorbate, sodium benzoate, proclin series.
Preferably, the R1 reagent comprises:
the R2 reagent comprises:
preferably, the R1 reagent comprises:
the R2 reagent comprises:
compared with the prior art, the invention has the beneficial effects that: the free fatty acid detection kit has the advantages that the chromogen substrate in the free fatty acid detection kit is optimized, so that the linear range of the free fatty acid detection kit is widened, the problem that ascorbic acid and bilirubin in a serum sample interfere the detection result of the free fatty acid is solved, and the free fatty acid detection kit with a wider linear range and stronger anti-interference capability is obtained; furthermore, the invention also preferably selects the type and proportion of the enzyme protective agent, thereby enhancing the stability of the free fatty acid detection kit.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
EXAMPLE 1 optimization of chromogen substrate
The free fatty acid detection kit provided by the embodiment comprises an R1 reagent and an R2 reagent, wherein the R1 reagent (with pH of 7.2) comprises the following components in parts by weight:
the R2 reagent (with the pH value of 7.2) comprises the following components in percentage by weight:
| PBS buffer | 100mmol/L |
| acyl-CoA oxidase | 50KU/L |
| Peroxidase enzymes | 25KU/L |
| Chromogen substrates | 0.5g/L |
| Enzyme protective agent | 0.5g/L |
| Brij-35 | 10mL/L |
| Sodium azide | 5mL/L。 |
Wherein, the enzyme protective agent in the R1 reagent and the R2 reagent is EDTA-2K and CoCl 2 1, the selection of chromogen substrate is arranged according to the following grouping, wherein TOPS is N-ethyl-N- (3-sulfopropyl) -3-toluidine sodium salt, DHBS is 3, 5-dichloro-2-hydroxybenzene sodium sulfonate, MAOS is N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethylaniline sodium salt, ADPS is N-ethyl-N- (3-sulfopropyl) -3-methoxyaniline sodium salt.
| Group of | Chromogen substrate and concentration ratio thereof |
| 1 | DAOS:TOOS＝1:1 |
| 2 | DAOS |
| 3 | TOOS |
| 4 | DAOS:topS＝1:1 |
| 5 | DHBS:TOOS＝1:1 |
| 6 | MAOS:ADPS＝1:1 |
| 7 | DAOS:TOOS＝1:0.5 |
| 8 | DAOS:TOOS＝1:2 |
| 9 | DAOS:TOOS＝1:0.3 |
| 10 | DAOS:TOOS＝1:3 |
The evaluation method of the kit comprises the following steps:
(1) An experimental instrument: HITACHI 7180
(2) Experimental parameters: an end point method; temperature: 37 ℃; dominant wavelength: 546nm; sample size: 5 mu L of the solution; r1 reagent: 240 mu L of the solution; r2 reagent: 60 mu L of the solution; the reaction direction is as follows: forward direction; reaction time: 10min; the calibration mode comprises the following steps: two-point scaling.
Calibration was performed using raw source free fatty acid calibrators and zeroed with blank tubes. Adding the R1 reagent into the sample, measuring the absorbance A1 at the wavelength of 546nm, then adding the R2 reagent, uniformly mixing, reacting at the constant temperature of 37 ℃ for 10 minutes, and measuring the absorbance A2 at the wavelength of 546 nm. Calculating delta A = A2-A1 to obtain the content of free fatty acid in the sample.
(3) Performance evaluation indexes are as follows: the kits obtained from the above groups were tested as follows:
(1) linear range: measuring a free fatty acid sample with the concentration of 0-30mmol/L, and calculating a correlation coefficient r and the deviation of the measured mean value and the theoretical concentration, wherein r is less than or equal to 0.99, and the deviation is within +/-10 percent, so that the requirement is met;
(2) interference evaluation: diluting the interference substances by using a purified water gradient, adding the diluted interference substances into blank control serum according to the proportion of 1. And if the interference degree is less than 10%, judging to be anti-interference, otherwise, judging to be non-anti-interference. Among the substances in serum that interfere with the determination of free fatty acids are: 0.03-0.5g/L ascorbic acid and 0.03-0.4g/L bilirubin.
(4) Results and analysis: among them, the evaluation data of the linear range is shown in table 1, the evaluation data of ascorbic acid interference is shown in table 2, and the evaluation data of bilirubin interference is shown in table 3.
TABLE 1 Linear Range evaluation data
TABLE 2 ascorbic acid interference evaluation data
TABLE 3 evaluation data of bilirubin interference
From the results shown in table 1, it can be seen that when the concentration ranges of DAOS and toss are higher or lower than 1 (groups 9 and 10) and DAOS or toss alone (groups 2 to 3), or DAOS and toss are replaced by other commonly used chromogen substrates (groups 4 to 6), the highest detection value of the free fatty acid detection reagent cannot reach 32mmol/L, the correlation coefficient is less than 0.99, and the deviation value is greater than ± 10%, i.e., the linear range is narrow and the linearity is poor, and the clinical use requirements cannot be met. Only when the chromogen substrate is DAOS and TOOS with the concentration ratio of 1.5-2 (groups 1, 7 and 8), the linear range value of the free fatty acid detection kit is wide, the highest detection value can reach 32mmol/L, the correlation coefficient is greater than 0.99, and the deviation value is within +/-10%, namely the linear correlation is good, the measurement is accurate, and the kit meets the clinical use requirement.
From the results of tables 2 to 3, it was found that when the concentration range of DAOS and toss is higher or lower than 1 (groups 9 and 10) or either DAOS and toss is used alone (groups 2 to 3), or DAOS and toss are replaced with other commonly used chromogen substrates (groups 4 to 6), the free fatty acid detection reagent has no anti-interference ability with respect to ascorbic acid and bilirubin in the sample or its anti-interference ability gradually decreases as the concentration of the interfering substances ascorbic acid and bilirubin in the sample gradually increases. The detection reagent has strong anti-interference capability to 0.03-0.5g/L ascorbic acid and 0.03-0.4g/L bilirubin, and the interference degree is less than 5% only when the chromogen substrate is DAOS and TOOS (groups 1, 7 and 8) with the concentration ratio of 1.5-2.
EXAMPLE 2 preference of enzyme protecting Agents
The free fatty acid detection kit provided by the embodiment comprises an R1 reagent and an R2 reagent, wherein the components and the ratio of the R1 reagent and the R2 reagent are similar to those of the embodiment 1, and the difference is that: the chromogen substrates in the R2 reagent are DAOS and tos at a concentration ratio of 1. The selection of enzyme protective agents in the R1 reagent and the R2 reagent is set according to the grouping of the following table:
the method for evaluating the stability of the kit comprises the following steps:
a. transportation stability: oscillating the reagent on a 42-degree shaking table for 3 days, and then evaluating the property, blank absorbance and accuracy of the reagent;
b. long-term stability: placing the reagent at 2-8 ℃ for 12 months, and evaluating the property, blank absorbance and accuracy of the reagent every several months;
wherein, the evaluation of the reagent character: mainly judging whether the reagent is precipitated or not;
evaluation of blank absorbance: at the wavelength of 546nm and under the optical path of 10mm, measuring the absorbance of the R1 and R2 reagents, and taking the average value as blank absorbance, wherein the blank absorbance is less than or equal to 1.0000;
evaluation of accuracy: and measuring a third party quality control product as a target value, and calculating the deviation value of the measured average value and the target value of the kit disclosed by the invention as the accuracy, wherein the accuracy is less than or equal to 10% and meets the requirement.
The measurement results are shown in Table 4.
Table 4 stability data
According to the measurement results shown in Table 4, the reagents of groups 2, 3, and 8-10 all showed precipitation and significantly decreased accuracy after shaking at 42 ℃ for 3 days, and the reagents of groups 4-7 showed precipitation and significantly decreased accuracy when stored at 2-8 ℃ for 6 months, while the reagents of groups 4-7 showed precipitation and significantly decreased accuracy when stored at 2-8 ℃ for 12 monthsThe precipitation and the accuracy are remarkably reduced, only after the group 1 is stored at 2-8 ℃ for 12 months or is shaken at 42 ℃ for 3 days, the precipitation does not occur, and the accuracy is not obviously changed, and the results show that the EDTA-2K and the CoCl 2 When the free fatty acid detection reagent is mixed as an enzyme protective agent according to the concentration ratio of 1.
Example 3
The embodiment provides a free fatty acid detection kit, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent (with pH of 7.2) comprises the following components in parts by weight:
| phosphate buffer | 150mmol/L |
| Fatty acyl-CoA synthetase | 10KU/L |
| Coenzyme A | 8g/L |
| ATP | 10g/L |
| 4-aminoantipyrine | 5g/L |
| Enzyme protective agent | 1g/L |
| Tween 20 | 20mL/L |
| Proclin 300 | 10mL/L |
The R2 reagent (with the pH value of 7.2) comprises the following components in percentage by weight:
| phosphate buffer | 120mmol/L |
| acyl-CoA oxidase | 100KU/L |
| Peroxidase enzymes | 100KU/L |
| Chromogen substrates | 1g/L |
| Enzyme protective agent | 1g/L |
| Triton X-100 | 20mL/L |
| Proclin 300 | 10mL/L。 |
Wherein the enzyme protecting agent in the R1 and R2 reagents is EDTA-2K and CoCl 2 The concentration ratio of (1) to (1), and the chromogen substrate in the R2 reagent is a mixture of DAOS and tos at a concentration ratio of 1.
Example 4
The embodiment provides a free fatty acid detection kit, which comprises an R1 reagent and an R2 reagent, wherein the R1 reagent (with pH of 7.2) comprises the following components in parts by weight:
| MES buffer | 30mmol/L |
| acyl-CoA synthetase | 2KU/L |
| Coenzyme A | 1g/L |
| ATP | 1g/L |
| 4-aminoantipyrine | 0.5g/L |
| Enzyme protective agent | 0.1g/L |
| GENAPOLX-080 | 5mL/L |
| Sodium benzoate | 1mL/L |
The R2 reagent (with the pH value of 7.2) comprises the following components in percentage by weight:
| MES buffer | 30mmol/L |
| acyl-CoA oxidase | 20KU/L |
| Peroxidase enzymes | 20KU/L |
| Chromogen substrates | 0.3g/L |
| Enzyme protective agent | 0.1g/L |
| EMULGEN A-60 | 5mL/L |
| Potassium sorbate | 1mL/L。 |
Wherein the enzyme protecting agent in the R1 and R2 reagents is EDTA-2K and CoCl 2 The concentration ratio of (1) to (1), and the chromogen substrate in the R2 reagent is a mixture of DAOS and tos at a concentration ratio of 1.
The linear range evaluation data, ascorbic acid interference evaluation data, bilirubin interference evaluation data, and stability evaluation data of the free fatty acid detection reagents of examples 3-4 were measured as described in examples 1-2 and the results are shown in tables 5-8.
TABLE 5 Linear Range evaluation data for examples 3-4
TABLE 6 ascorbic acid interference evaluation data for examples 3-4
TABLE 7 evaluation data of bilirubin interference for examples 3-4
Table 8 stability data for examples 3-4
From the results in tables 5-8, it can be seen that the linear range, interference rejection and stability of the reagent are relatively little affected by changes in the components and amounts of the reagent other than the enzyme protectant chromogen substrate.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.
Claims (8)
1. A free fatty acid detection kit comprises an R1 reagent and an R2 reagent, wherein the R1 reagent contains buffer solution, fatty acyl-CoA synthetase, coenzyme A, ATP,The reagent R2 comprises a buffer solution, acyl coenzyme A oxidase, peroxidase, a chromogen substrate, an enzyme protective agent, a surfactant and a preservative, and is characterized in that the chromogen substrate in the reagent R2 is a mixture of DAOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline sodium salt) and TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt), the concentration ratio of DAOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline sodium salt) to TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt) is 1.5 to 2, and the enzyme protective agent is EDTA-2K and CoCl 2 1 in a concentration ratio of 1.
2. The free fatty acid detection kit according to claim 1, wherein the concentration ratio of DAOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3, 5-dimethoxyaniline sodium salt) and TOOS (N-ethyl-N- (2-hydroxy-3-sulfopropyl) -3-methylaniline sodium salt) is 1.
3. The free fatty acid detection kit according to claim 1, wherein the enzyme protecting agent in the R1 reagent and the R2 reagent is EDTA-2K (dipotassium ethylenediaminetetraacetate), FAD (flavin adenine dinucleotide), mgSO 4 、CaCl 2 、CoCl 2 A mixture of any two of.
4. The free fatty acid detection kit according to claim 1, wherein the buffer solution of the R1 reagent and the R2 reagent is any one of a PBS buffer solution, a phosphate buffer solution, a GOOD's buffer solution, a MES buffer solution, and a HEPES buffer solution.
5. The free fatty acid detection kit of claim 1, wherein the surfactant is one or more of Tween20, EMULGENA-60, EMULGENA-90, EMULGENB-66, EMULGEN709, brij-35, triton x-100, GENAPOLX-080, dodecyl-tetradecyl dimethyl betaine.
6. The free fatty acid detection kit according to claim 1, wherein the preservative is one or more of sodium azide, potassium sorbate, sodium benzoate, and Proclin 300.
7. The free fatty acid detection kit according to any one of claims 1 to 6, wherein the R1 reagent comprises:
buffer solution 30 to 150mmol/L
Fatty acyl-CoA synthetase 1-10KU/L
Coenzyme A0.5 to 8g/L
ATP 0.5~10g/L
0.2 to 5g/L of 4-aminoantipyrine
0.1-1g/L enzyme protective agent
5-20mL/L surfactant
Preservative 1-10mL/L
The R2 reagent comprises:
buffer solution 30 to 150mmol/L
Fatty acyl-CoA oxidase 20 to 100KU/L
Peroxidase 20 to 100KU/L
0.3 to 1g/L of chromogen substrate
0.1-1g/L enzyme protective agent
5-20mL/L surfactant
1-10 mL/L preservative.
8. The free fatty acid detection kit according to claim 7, wherein the R1 reagent comprises:
buffer 50mmol/L
acyl-CoA synthetase 5KU/L
Coenzyme A4 g/L
ATP 6g/L
4-aminoantipyrine 1g/L
Enzyme protective agent 0.5g/L
Surfactant 10mL/L
Preservative 5mL/L
The R2 reagent comprises:
buffer solution 100mmol/L
acyl-CoA oxidase 50KU/L
Peroxidase 25KU/L
0.5g/L of chromogen substrate
Enzyme protective agent 0.5g/L
Surfactant 10mL/L
Preservative 5mL/L.
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| CN112763731B (en) * | 2020-12-29 | 2023-02-28 | 广州市伊川生物科技有限公司 | Lipoprotein (a) determination kit and detection method thereof |
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| KR101943673B1 (en) * | 2011-07-29 | 2019-01-29 | 교와 메덱스 가부시키가이샤 | Sphingomyelin measurement method and measurement kit |
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