CN112710853B - Anti-interference and stable serum direct bilirubin (enzyme method) determination kit and preparation method and application thereof - Google Patents
Anti-interference and stable serum direct bilirubin (enzyme method) determination kit and preparation method and application thereof Download PDFInfo
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/728—Bilirubin; including biliverdin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/52—Use of compounds or compositions for colorimetric, spectrophotometric or fluorometric investigation, e.g. use of reagent paper and including single- and multilayer analytical elements
Abstract
The invention provides a Direct Bilirubin (DB) determination kit, which comprises a reagent R1 and a reagent R2; the reagent R1 contains the following components: phosphate buffer solution, NaCl, N-acetylcysteine, sodium fluoride, disodium ethylene diamine tetraacetate, ascorbic acid oxidase, surfactant and preservative. Reagent R2: trihydroxymethyl aminomethane-hydrochloric acid buffer solution, bilirubin oxidase, stabilizer and preservative. The invention also provides a preparation method and application of the kit, the kit adopts different types of stabilizers and surfactants, can effectively avoid the interference of blood fat, and is a liquid kit with strong anti-interference capability, high sensitivity, good stability and low cost.
Description
Technical Field
The invention relates to the technical field of biochemical reagent determination, in particular to a direct bilirubin determination kit, and also relates to a preparation method and application of the direct bilirubin determination kit.
Background
Bilirubin is one of the bile pigments and is the major pigment in human bile. Bilirubin is a major metabolite of the iron porphyrin compound in the body, is toxic and causes irreversible damage to the brain and nervous system, but also has antioxidant activity and inhibits the oxidation of linoleic acid and phospholipids. Bilirubin is an important basis for clinically judging jaundice and also an important index of liver function.
Direct bilirubin: indirect bilirubin is higher than direct bilirubin, which indicates hepatocellular jaundice, damage to liver cells, hypohepatia, incomplete conversion of indirect bilirubin into direct bilirubin by the liver, excretory disorder caused by the compression of intrahepatic bile ducts, incomplete excretion of direct bilirubin into biliary tracts, and possible acute icteric hepatitis, chronic active hepatitis, liver cirrhosis, liver cancer and other diseases.
Direct bilirubin: this is due to obstructive jaundice. Indirect bilirubin: it is probably caused by hemolytic jaundice, and the direct increase of bilirubin may also cause blood group incompatibility and anemia during blood transfusion.
The existing methods for detecting direct bilirubin mainly comprise a diazo method, a vanadate method and an oxidase method. The diazo method for detecting bilirubin is widely adopted in clinical laboratories, but the method is easily interfered by a plurality of factors, and a reagent cannot be stable for a long time, so that the method is influenced to a certain extent in disease diagnosis. The vanadic acid oxidation method is easy to pollute the instrument and has influence on other tests. The oxidase method has high specificity and high accuracy, but the existing kit has the defects of poor anti-interference capability, poor stability, easy influence by blood fat and the like.
Disclosure of Invention
In order to solve the problems, the invention provides a direct bilirubin determination kit, a preparation method and application thereof.
The invention is realized by the following technical scheme:
a direct bilirubin determination kit comprises a reagent R1 and a reagent R2;
the reagent R1 contains the following components:
buffer solution is 20-50 mmol/L;
NaCl 1-9g/L；
1g/L of N-acetylcysteine;
sodium fluoride0.1g /L；
0.1 g/L of disodium ethylene diamine tetraacetate;
ascorbic acid oxidase 1-5 KU/L;
0.1 to 2 percent of surfactant;
0.5-1g/L of preservative;
the reagent R2 contains the following components:
buffer solution is 20-50 mmol/L;
10-20g/L of stabilizer;
2-6KU/L of bilirubin oxidase;
0.1 to 2 percent of surfactant;
0.5-1g/L of preservative;
wherein the percentages are by volume.
Preferably, the pH of the reagent R1 is 5.0-5.5.
Preferably, the pH of the reagent R2 is 8.5-9.0.
Preferably, the surfactant in the reagent R1 and R2 is selected from one or more of sorbitan laurate, triton x-100 and polyoxypropylene mannitol dioleate. More preferably, the surfactant in reagent R1 is sorbitan laurate and polyoxypropylene mannitol dioleate, and the surfactant in reagent R2 is triton x-100.
Preferably, the stabilizer in the reagent R2 is one or more of bovine serum albumin, polyvinylpyrrolidone k30 and mannitol. More preferably, the stabilizer consists of bovine serum albumin and mannitol.
Preferably, the preservative in the reagents R1 and R2 is one or more of sodium azide, proclin300, MIT, and sodium benzoate, more preferably, the preservative is proclin 300.
Preferably, the volume ratio of the reagent R1 to the reagent R2 is 1-5: 1. More preferably, the volume ratio of reagent R1 to reagent R2 is 3: 1.
The preparation method of the direct bilirubin determination kit comprises the following steps: adding trihydroxymethyl aminomethane-hydrochloric acid buffer solution into the reagents R1 and R2, adjusting the pH value with hydrochloric acid or sodium hydroxide, adjusting the pH value of the reagent R1 to 5.0-5.5 and the pH value of the reagent R2 to 8.5-9.0, and adding other substances according to the proportion to dissolve the reagents to prepare the direct bilirubin determination kit.
The invention also discloses the application of the direct bilirubin determination kit, which is used for determining the concentration of the direct bilirubin in serum for the purposes of non-disease diagnosis and treatment.
The kit adopts a colorimetric method, and has the reaction principle that the direct bilirubin in a sample is oxidized into biliverdin in a buffer solution with the pH value of 7.2 under the action of Bilirubin Oxidase (BOD), and the content of the direct bilirubin can be obtained by measuring the absorbance change caused by the reaction at the wavelength of 450 nm. The reaction has specificity, the full-automatic biochemical analyzer is used for detection, the operation is simple, the automation degree is high, the human error can be reduced, and the method is suitable for most clinical laboratories.
Advantageous effects
1) The stable direct bilirubin determination kit is a liquid double reagent, is prepared without re-dissolving, and can be directly used after opening a bottle.
2) The composite stabilizer is formed by adding the bovine serum albumin and the mannitol into the reagent R2, so that the stability of the enzyme in the reagent is effectively improved, the stability of the reagent is excellent, and the reagent is further promoted in the market.
3) The sorbitan laurate and the polyoxypropylene mannitol dioleate are added to act together, so that the interference of blood fat can be effectively removed, the anti-interference capability of the reagent is greatly enhanced, and the stability of the reagent is improved to a certain extent.
4) The reagent has excellent performance indexes such as accuracy, repeatability, analysis sensitivity, linear range, stability and the like, is low in price and convenient to use, and is favorable for further popularization of the reagent in the market.
Drawings
FIG. 1 is a correlation curve for the reagents of example 1 and comparative example 1;
FIG. 2 is a linear curve of example 1;
FIG. 3 is a graph showing the change in concentration of the direct bilirubin assay reagent of example 1 and comparative examples 1, 2 and 3 in the stability test.
Detailed Description
The invention is further illustrated by the following specific examples:
in the use of the kit of this embodiment, the determination method is performed by using a michael 800 full-automatic biochemical analyzer with double reagent functions and using a rate method, and the following operations are performed to detect the main wavelength of 450nm and the sub-wavelength of 600 nm:
adding 8 muL of physiological saline, a sample or a calibrator, adding 225 muL of R1 reagent, pre-incubating for 5min, adding 75 muL of R2 reagent, mixing uniformly, delaying for 1min, reading absorbance A1, reading absorbance A2 after 5min,
calculating Δ A = (A2-A1).
Direct bilirubin content (μmol/L) = (Δ a sample ÷ Δ a calibrator) × calibrator concentration.
Sample requirements:
1. blood serum is not dissolved and is stored in dark.
2. Sample stability: the specimen can be stored stably for 3 days at the temperature of 2-8 ℃ and for 2 weeks at the temperature of-20 ℃.
Example 1
A conventional direct bilirubin assay kit comprises reagent R1 and reagent R2.
The reagent R1 contains the following components:
25mmol/L of phosphate buffer;
NaCl 1g/L；
1g/L of N-acetylcysteine;
sodium fluoride0.1g /L；
0.1 g/L of disodium ethylene diamine tetraacetate;
1% of sorbitan laurate;
1% of polyoxypropylene mannitol dioleate;
proclin300 1 ml/L。
the pH of reagent R1 was 5.2.
The reagent R2 contains the following components:
piperazine-1, 4-diethylsulfonic acid (PIPES) buffer 50 mmol/L;
bovine Serum Albumin (BSA) 5 g/L;
10g/L of mannitol;
triton 1001%;
1KU/L bilirubin oxidase;
proclin300 1ml/L。
the pH of the reagent R2 was 9.0.
Wherein the percentages are by volume.
Comparative example 1
Commercial imported Sigma direct bilirubin assay kit.
Comparative example 2
The reagent kit is different from the direct bilirubin determination reagent kit in the example 1 only in that all stabilizers in the reagent R2 are bovine serum albumin, and the bovine serum albumin is 10 g/L; the rest is the same as in example 1.
Comparative example 3
The reagent kit is different from the direct bilirubin assay kit in example 1 only in that the stabilizers in the reagent R2 are all mannitol, and 20g/L mannitol is the same as that in example 1.
Comparative example 4
The reagent kit is different from the direct bilirubin assay kit in example 1 only in that the reagent R1 does not contain sorbitan laurate or polyoxypropylene mannitol dioleate, and the other steps are the same as those in example 1.
Comparative example 5
The reagent kit was different from the direct bilirubin assay kit of example 1 only in that the reagent R1 contained no sorbitan laurate and that the amount of polyoxypropylene mannitol dioleate was 2%, and the other points were the same as in example 1.
Comparative example 6
The reagent kit was the same as in example 1 except that the reagent R1 contained no polyoxypropylene mannitol dioleate and that sorbitan laurate contained 2%.
Comparative example 7
The reagent kit for total bilirubin determination in example 1 is different from the reagent kit for total bilirubin determination in example 1 only in that the reagent R1 does not contain sorbitan laurate or polyoxypropylene mannitol dioleate, and that the reagent kit is 2% SDS, and the reagent kit is otherwise the same as in example 1.
Comparative example 8
The reagent kit for total bilirubin determination in example 1 was different from the reagent kit for total bilirubin determination in example 1 only in that the reagent R1 contained no sorbitan laurate and no polyoxypropylene mannitol dioleate, and that the reagent kit contained 2% of trialkylammonium chloride, and the other components were the same as those in example 1.
Comparative example 9
The difference from the total bilirubin determination kit in example 1 is that the reagent R1 does not contain sorbitan laurate or polyoxypropylene mannitol dioleate, and Tween 80 is 2%, and the other points are the same as those in example 1.
Performance verification
Test No.)
Correlation experiments: example 1 and comparative example 1, 40 clinical serum samples were tested simultaneously, correlation analysis was performed on the two sets of test results, and a correlation coefficient r was calculated; the relative deviation (r) of 40 pairs of data was calculated using the test results of comparative example 1 as control values, respectively. It is required that r is not less than 0.990 and the relative deviation is not more than. + -. 10%.
The results are shown in table 1, and correlation curves (shown in fig. 1) were obtained for the reagents of example 1 and comparative example 1.
TABLE 1 correlation comparative experiment results
| Sample number | Example 1 (mu mol/L) | Comparative example 1 (mu mol/L) | Example 1 relative deviation from comparative example 1 |
| 1 | 2.44 | 2.49 | -2.01% |
| 2 | 0.28 | 0.27 | 3.70% |
| 3 | 5.05 | 4.78 | 5.65% |
| 4 | 6.54 | 6.20 | 5.48% |
| 5 | 3.20 | 3.09 | 3.56% |
| 6 | 1.77 | 1.79 | -1.12% |
| 7 | 8.73 | 8.51 | 2.57% |
| 8 | 6.22 | 6.33 | -1.74% |
| 9 | 2.17 | 2.27 | -4.41% |
| 10 | 6.67 | 6.60 | 1.06% |
| 11 | 2.63 | 2.52 | 4.37% |
| 12 | 2.97 | 3.12 | -4.81% |
| 13 | 3.19 | 3.01 | 5.98% |
| 14 | 2.89 | 2.82 | 2.48% |
| 15 | 1.75 | 1.80 | -2.78% |
| 16 | 6.83 | 7.23 | -5.53% |
| 17 | 1.54 | 1.50 | 2.67% |
| 18 | 6.07 | 6.08 | -0.16% |
| 19 | 3.11 | 3.21 | -3.12% |
| 20 | 5.99 | 6.18 | -3.07% |
| 21 | 7.69 | 7.56 | 1.72% |
| 22 | 5.62 | 5.69 | -1.23% |
| 23 | 5.47 | 5.45 | 0.37% |
| 24 | 8.98 | 8.90 | 0.90% |
| 25 | 1.26 | 1.24 | 1.61% |
| 26 | 0.82 | 0.85 | -3.53% |
| 27 | 2.07 | 1.95 | 6.15% |
| 28 | 5.10 | 4.99 | 2.20% |
| 29 | 8.96 | 9.11 | -1.65% |
| 30 | 3.61 | 3.42 | 5.56% |
| 31 | 4.60 | 4.63 | -0.65% |
| 32 | 3.26 | 3.24 | 0.62% |
| 33 | 2.14 | 2.17 | -1.38% |
| 34 | 1.32 | 1.40 | -5.71% |
| 35 | 0.55 | 0.54 | 1.85% |
| 36 | 3.78 | 3.57 | 5.88% |
| 37 | 4.71 | 4.67 | 0.86% |
| 38 | 4.24 | 4.26 | -0.47% |
| 39 | 8.21 | 8.63 | -4.87% |
| 40 | 8.52 | 8.33 | 2.28% |
As can be seen from Table 1 and FIG. 1, the maximum value of the deviation of the low values of the serum test of the kits of example 1 and comparative example 1 is 6.15%, the correlation coefficient of the two reagents is 0.998, and the detection results of example 1 and comparative example 1 are very close, so that the detection reagent of example 1 provided by the invention has good correlation with the imported detection reagent, and can completely replace the imported reagent for clinical examination.
Test No. two
And (3) precision test: in example 1 and comparative example 1, 20 tests were performed on clinical samples, and the mean, standard deviation, and coefficient of variation of the 20 tests were calculated. The results are shown in Table 2.
TABLE 2 precision testing data table
| Number of tests | Example 1 (mu mol/L) | Comparative example 1 (mu mol/L) |
| 1 | 13.83 | 13.83 |
| 2 | 13.90 | 14.06 |
| 3 | 14.30 | 14.53 |
| 4 | 13.99 | 13.84 |
| 5 | 14.07 | 14.02 |
| 6 | 14.13 | 13.72 |
| 7 | 14.03 | 14.55 |
| 8 | 14.03 | 14.06 |
| 9 | 14.05 | 14.55 |
| 10 | 13.87 | 13.90 |
| 11 | 13.82 | 14.12 |
| 12 | 13.90 | 14.07 |
| 13 | 13.76 | 13.71 |
| 14 | 13.99 | 13.37 |
| 15 | 14.00 | 14.19 |
| 16 | 14.09 | 13.71 |
| 17 | 14.12 | 13.47 |
| 18 | 13.99 | 13.63 |
| 19 | 14.03 | 13.58 |
| 20 | 13.92 | 13.95 |
| Mean value (X) | 13.99 | 13.94 |
| Standard deviation (S) | 0.13 | 0.34 |
| Coefficient of Variation (CV) | 0.90% | 2.43% |
As can be seen from Table 2, the standard deviation of the detection values of the example 1 and the comparative example 1 is small, the coefficient of variation is small, the repeatability is good, the coefficient of variation of 0.90% of the example 1 is smaller than the coefficient of variation of 2.43% of the comparative example 1, the precision of the example 1 is high and is better than that of an imported reagent, and the reagent of the example 1 can completely replace the imported reagent.
Experiment three
Linear experiments: the direct bilirubin high value sample is 428 mu mol/L and diluted, 6 samples with different concentrations are prepared, the samples with the concentrations of 428 mu mol/L, 214 mu mol/L, 107 mu mol/L, 53.5 mu mol/L, 26.75 mu mol/L and 0 mu mol/L are sequentially detected by the reagent in the embodiment 1, each sample of each concentration level is respectively measured for three times, and the average value of each sample is respectively taken. The results are shown in Table 3.
TABLE 3 table of testing data of linear correlation verification experiment
| Theoretical concentration (mu mol/L) | Example 1 (mu mol/L) |
| 0.00 | 0.00 |
| 26.75 | 26.30 |
| 53.50 | 53.07 |
| 107.00 | 105.87 |
| 214.00 | 208.53 |
| 428.00 | 426.60 |
| Coefficient of correlation r | 0.999 |
As can be seen from Table 3 and FIG. 2, the linear variation of example 1 of the present invention with diluted concentration is 0.999, which is greater than 0.990, and meets the standard requirement, and the value is greater than that of other control groups. The linear range is better in example 1. The better linear variation of the reagent of example 1 is more consistent with the requirements of clinical case samples.
Experiment four
Stability test: the stability test was performed on the direct bilirubin assay reagents provided in example 1 and comparative examples 1, 2 and 3 according to the following protocol: the reagents provided in example 1 and comparative examples 1, 2 and 3, each of which comprises 10 groups in parallel, are stored in a 37 ℃ incubator together, an accelerated test is performed, a quality control product with a target value of 9.80 +/-0.5 mu mol/L is detected every day, each group is tested for 5 times, the average value is calculated, the change of the measured value of the quality control product is monitored, and the detection result is shown in Table 4.
Table 4 reagent thermal stability verification data
| Time (sky) | Example 1 reagent μmol/L | Comparative example 1 reagent mu mol/L | Comparative example 2 reagent mu mol/L | Comparative example 3 reagent μmol/ |
| 1 | 9.80 | 9.75 | 9.59 | 9.58 |
| 2 | 9.81 | 9.55 | 9.26 | 9.00 |
| 3 | 9.75 | 9.53 | 9.17 | 8.84 |
| 4 | 9.49 | 9.22 | 9.01 | 8.71 |
| 5 | 9.54 | 9.16 | 9.04 | 8.63 |
| 6 | 9.82 | 8.84 | 8.84 | 8.43 |
| 7 | 9.48 | 8.73 | 8.57 | 8.36 |
| 8 | 9.84 | 8.46 | 8.25 | 8.24 |
| 9 | 9.57 | 8.32 | 8.14 | 8.02 |
| 10 | 9.49 | 8.23 | 8.03 | 7.86 |
As can be seen from Table 4 and FIG. 3, the reagent of example 1 provided by the present invention has substantially no change within 10 days at 37 ℃, and has good stability; whereas the comparative examples 2, 3 show significant change in the agents over 10 days, example 1 with two stabilizers is superior to comparative examples 2, 3 with a single stabilizer. The stability of the kit of example 1 is superior to that of the kits of comparative examples 1, 2 and 3, which shows that the stability of the direct bilirubin determination kit is significantly improved by the combined action of the buffer solution and the two stabilizers.
Experiment five
Interference experiments: and (4) taking the traceability quality control substances, and adding the median quality control substances (the target value is 9.80 +/-0.5 mu mol/L) into the contents of the fat emulsion in the table respectively. Then, the content of direct bilirubin in the sample was measured using the reagent of example 1 simultaneously with the reagents of comparative example 1, comparative example 4, comparative example 5 and comparative example 6, and the measurement results of each group are shown in Table 5.
TABLE 5 anti-interference verification results of reagents
As can be seen from Table 5, when the fat milk is 1mL/L, the reagent of example 1 is not disturbed significantly, the reagents of comparative example 1, comparative example 4, comparative example 5 and comparative example 6 are disturbed to a certain extent, and the disturbance of comparative example 4 is more obvious. When the fat milk was 4mL/L, the example 1 reagent was not significantly disturbed, the comparative examples 1,4, 5, 6 reagents were significantly disturbed. Example 1 has a stronger interference resistance than comparative examples 4, 5 and 6, and example 1 having two components has a stronger interference resistance. The reagents of comparative examples 7 and 8 show that the ionic surfactant has a remarkable effect on the anti-hyperlipidemia effect of the reagents, but has a remarkable interference on the accuracy of the reagents. The interference rejection of the nonionic surfactant Tween 80 is obviously inferior to that of the surfactant in example 1. This shows that after the sorbitan laurate and the polyoxypropylene mannitol dioleate are added, the anti-interference performance of the reagent in the example 1 is obviously improved due to the synergistic effect of the sorbitan laurate and the polyoxypropylene mannitol dioleate, which is superior to that of the reagent in the comparative example 1 and meets the clinical requirements.
In conclusion, the kit adopts sorbitan laurate, polyoxypropylene mannitol dioleate, different stabilizers and a good buffer solution system, and is a liquid kit with high interference resistance, high stability, high sensitivity, good repeatability and low cost. Provides good development space for the kit and simultaneously enhances the market competitiveness of the kit.
Claims (3)
1. A direct bilirubin assay kit, comprising a reagent R1 and a reagent R2;
the reagent R1 contains the following components:
25mmol/L of phosphate buffer;
NaCl 1g/L；
1g/L of N-acetylcysteine;
0.1 g/L of sodium fluoride;
0.1 g/L of disodium ethylene diamine tetraacetate;
ascorbic acid oxidase 1 KU/L;
1% of sorbitan laurate;
1% of polyoxypropylene mannitol dioleate;
proclin300 1ml/L；
the reagent R2 contains the following components:
piperazine-1, 4-diethylsulfoacid buffer 50 mmol/L;
5g/L of bovine serum albumin;
10g/L of mannitol;
triton 1001%;
1KU/L bilirubin oxidase;
proclin300 1ml/L；
wherein the percentages are by volume;
the pH of the reagent R1 is 5.2, and the pH of the reagent R2 is 9.0.
2. A method for preparing a direct bilirubin assay kit according to claim 1 which comprises the steps of: adding phosphate buffer solution into a reagent R1, adding piperazine-1, 4-diethylsulfonic acid buffer solution into a reagent R2, adding other substances according to a proportion for dissolving, adjusting the pH value by using hydrochloric acid or sodium hydroxide, adjusting the pH value of the reagent R1 to be 5.2, adjusting the pH value of the reagent R2 to be 9.0, and finally adding bilirubin oxidase to prepare the direct bilirubin determination kit.
3. Use of the direct bilirubin assay kit of claim 1 in the determination of serum direct bilirubin levels for non-disease diagnostic and therapeutic purposes.
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