Background technology
Total bilirubin is combination or the bilirubinic general name of non-binding type that is produced by the protoheme metabolism in the human body, the unusual higher disease in the liver and gallbladder that is common in of total bilirubin, and it is normal relevant with coronary heart disease to studies show that more in recent years blood mesobilirubin is lower than normal value.Therefore, to the mensuration of total bilirubin in serum, blood plasma or the urine of human body, be one of the most conventional project of medical test.
The kit of measuring total bilirubin mainly contains following three kinds at present: the diazonium method is measured kit, the kit of enzymatic assays total bilirubin and the kit of mensurating total bilirubin through chemistry oxidation process of total bilirubin.
The diazonium method is measured the kit of total bilirubin: history under strict control operation environment temperature, time, step, adopt this kit higher to the higher sample recall rate of total bilirubin level, and reagent is cheap at most.So still widespread use at present.But adopt this kit is puzzlement medical worker's a difficult problem to problems such as the detecting operation complexity of total bilirubin, reagent instability, reaction are not single-minded always, though people did many times improvement over more than 100 year, does not still have the result who is satisfied with so far.
The kit of enzymatic assays total bilirubin: adopt this kit measurement total bilirubin to have simple, special characteristics.But because enzymatic cholerythrin reaction velocity is slow, especially be difficult to the reaction of catalysis δ cholerythrin, therefore actually be measured as glucuronic acid combined with bilirubin and the bilirubinic value of non-binding type, and can not truly reflect total bilirubin.And the photobilirubin that produces after some especial patient sample such as the light therapy (not belonging to cholerythrin) also can participate in this reaction, causes measured value inaccurate, and these shortcomings have limited its application greatly.The kit enzyme heat stability of enzymatic assays total bilirubin is poor in addition, and the holding time is short, and selling at exorbitant prices is unfavorable for applying.So, though can enter clinical practice at present, be far from perfect, also need continue research.
The kit of mensurating total bilirubin through chemistry oxidation process:
From the nineties in last century, the kit that vanadic acid oxidizing process in the chemical oxidization method is measured total bilirubin begins practical application abroad, and the kit of measuring total bilirubin since vanadic acid oxidizing process in 1998 enters China.Measure the kit of total bilirubin compares with the kit of enzymatic assays total bilirubin with the diazonium method, the kit that the vanadic acid oxidizing process is measured total bilirubin demonstrates progressively when measuring total bilirubin that it is simple to operation, stable reagent, holding time long (at room temperature can stablize 1 year as the double reagent formulation, place the reagent storehouse of instrument to stablize more than one month) and advantage such as cheap.But when this kit was surveyed the low value sample, subsidiary reaction obviously influenced the result, and certain toxicity is arranged, and especially discarded object may be reduced into hypertoxic metavanadate and contaminated environment.
The current research result shows both at home and abroad, the reagent (box) of the potassium persulfate oxidizing process mensuration total bilirubin in the chemical oxidization method had both kept the original advantage of kit of vanadic acid oxidizing process mensuration total bilirubin, had the advantages such as kit that the vanadic acid oxidizing process is measured the environmentally safe that kit did not have of total bilirubin, total bilirubin is measured in subsidiary reaction when surveying the low value sample less than the vanadic acid oxidizing process again.But still there is following shortcoming in it: 1, the linear upper limit is little, can not cover whole pathological changes fully; 2, anti-interference is poor; 3, the influence of subsidiary reaction is still more obvious, and response curve is lack of standardization, and does not arrive terminal point; 4, the accuracy of Ce Dinging, precision and sensitivity are still low, and especially obvious when surveying low value.Because the existence of above shortcoming is not so the kit that the potassium persulfate oxidizing process is measured total bilirubin still obtains practical application up to now.
Summary of the invention
The technical problem to be solved in the present invention is, provide a kind of potassium persulfate oxidizing process that had both had prior art to measure the advantage of total bilirubin kit, the kit that the linear again upper limit is big, strong interference immunity, response curve standard, the accuracy that can reach home rapidly and measure, precision and highly sensitive and chemical oxidization method that can practical application (potassium persulfate oxidizing process, down with) are measured total bilirubin.
Technical scheme of the present invention is, a kind of kit of mensurating total bilirubin through chemistry oxidation process is provided, and this kit is made up of reagent 1 and reagent 2, and wherein the each component of reagent 1 and concentration range thereof (concentration is g/L or milliliter/liter be that ml/L represents with grams per liter) are as follows:
Potassium dihydrogen phosphate 0.5~3g/L
Sodium hydrogen phosphate 16~82g/L
Sodium chloride 2.5~12.5g/L
Polyvinylpyrrolidone 1~30g/L
Disodium ethylene diamine tetraacetate 0.2~1g/L
Triton x-100 0.5~3ml/L
Thimerosal 0.05~0.25g/L
It is 7.6~8.4 that above sodium hydrogen phosphate mixes back accent pH value with the potassium dihydrogen phosphate dissolving;
Wherein the each component of reagent 2 and concentration range thereof (concentration is g/L with grams per liter) are as follows:
Sodium hydrogen phosphate 1~5.5g/L
Potassium dihydrogen phosphate 0.25~1.5g/L
Potassium persulfate 12.5~65g/L
Sodium chloride 4.5~22.g/L
Thimerosal 0.05~0.25g/L
It is 6.8~7.6 that above sodium hydrogen phosphate mixes back accent pH value with the potassium dihydrogen phosphate dissolving.
Wherein the preferable range of the each component of reagent 1 and concentration thereof (concentration is g/L or milliliter/liter be that ml/L represents with grams per liter) is as follows:
Potassium dihydrogen phosphate 1~2g/L
Sodium hydrogen phosphate 25~50g/L
Sodium chloride 4~7.5g/L
Polyvinylpyrrolidone 5~20g/L
Disodium ethylene diamine tetraacetate 0.3~0.7g/L
Triton x-100 0.75~1.75ml/L
Thimerosal 0.08~0.15g/L
It is 7.8~8.2 that above sodium hydrogen phosphate mixes back accent pH value with the potassium dihydrogen phosphate dissolving;
Wherein the each component of reagent 2 and concentration range thereof (concentration is g/L with grams per liter) are as follows:
Sodium hydrogen phosphate 1.6~3.3g/L
Potassium dihydrogen phosphate 0.4~0.8g/L
Potassium persulfate 20~40g/L
Sodium chloride 7~15g/L
Thimerosal 0.08~0.15g/L
It is 7.0~7.4 that above sodium hydrogen phosphate mixes back accent pH value with the potassium dihydrogen phosphate dissolving.
Wherein the optimum value of the each component of reagent 1 and concentration thereof (concentration is g/L or milliliter/liter be ml/L with grams per liter) is as follows:
Potassium dihydrogen phosphate 1.5g/L
Sodium hydrogen phosphate 35g/L
Sodium chloride 5g/L
Polyvinylpyrrolidone 15g/L
Disodium ethylene diamine tetraacetate 0.5g/L
Triton x-100 1.2ml/L
Thimerosal 0.1g/L
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 8.0;
Wherein the optimum value of the each component of reagent 2 and concentration thereof (concentration is g/L with grams per liter) is as follows:
Sodium hydrogen phosphate 2.5g/L
Potassium dihydrogen phosphate 0.6g/L
Potassium persulfate 30g/L
Sodium chloride 10g/L
Thimerosal 0.1g/L
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 7.2.
In above component, potassium dihydrogen phosphate and sodium hydrogen phosphate form stable damping fluid, are beneficial to the dispersion stabilization of various compositions in the reagent.Disodium ethylene diamine tetraacetate, but metallic ions such as complexing trace element reduce bilirubinic autoxidation.Sodium chloride, real biochemical environment in the simulation human body keeps the former structure picture that has living space for sample mesobilirubin composition, and the ionic strength that strengthens solution is arranged, and produces primary salt effect, the effect of accelerated reaction speed.Polyvinylpyrrolidone is a kind of non-ionic water-soluble macromolecular compound, can improve sensitivity.The triton x-100 surfactant is the solubilizer of water-insoluble courage, is promoter, the lubricant of reaction, and it improves the sensitivity that detects, and promotes various substance dissolves in the sample, reduces the influence of the turbid grade of sample fat to measuring.Potassium persulfate mainly plays oxidisability.Thimerosal mainly plays antiseptic.
The collocation method of reagent 1 of the present invention and reagent 2 is a conventional method, and promptly reagent 1 and reagent 2 add respectively to mix to stir evenly separately behind entry such as the distilled water (amount of distilled water adds according to actual needs) and gets final product.
Kit with mensurating total bilirubin through chemistry oxidation process of the present invention is measured total bilirubin based on following principle: total bilirubin is in the presence of surfactant, by the chemical oxidizing agent oxidation, generate dehydrobilirubin, the minimizing that can be measured to 350-550nm place absorbance is directly proportional with the concentration of total bilirubin.
30~45 ℃ of temperature; Cuvette optical path 1.0cm.
(commplementary wave length can be eliminated the interference of other matrix in the sample to wavelength 350-550nm (master)/450-650nm (pair), improves detection sensitivity, and promptly the absorbance A that reads of instrument is A
Predominant wavelength-A
Commplementary wave length)。
Sample (standard) 8 μ l;
Reagent 1 (R1) 180~320 μ l; Reagent 2 (R2) 40~120 μ l
Sample (calibration tube is made sample with calibration object) adds the R1 mixing, and 30-40 ℃ is read absorbance A after hatching 3~5min
0Add the R2 mixing, 30-45 ℃ hatch 5min after, read absorbance A
1, Δ A=A
1-A
0
Calculate
Embodiment
Below in conjunction with the drawings and specific embodiments the present invention is described in further detail, but the present invention far is not limited only to following examples.
Embodiment 1
Reagent 1 (R
1)
Potassium dihydrogen phosphate (KH
2PO
4) 3g
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 82g
Sodium chloride (NaCl) 2.5g
Polyvinylpyrrolidone [(C
6H
9NO) n] 30g
Disodium ethylene diamine tetraacetate (EDTA-Na
2) 0.2g
Triton x-100 (TritonX-100) 3ml
Thimerosal (C
9H
9O
2HgNaS) 0.05g
H
2O (distilled water, down together) adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 8.4.
Reagent 2 (R
2)
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 5.5g
Potassium dihydrogen phosphate (KH
2PO
4) 1.5g
Potassium persulfate (K
2S
2O
8) 12.5g
Sodium chloride (NaCl) 22.5g
Thimerosal (C
9H
9O
2HgNaS) 0.05g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 7.6.
Reagent 1 mixed to stir evenly with distilled water according to a conventional method get final product.Reagent 2 mixed to stir evenly with distilled water equally according to a conventional method get final product.
Kit with the mensurating total bilirubin through chemistry oxidation process of present embodiment is measured total bilirubin based on following principle: total bilirubin is in the presence of surfactant, by the chemical oxidizing agent oxidation, generate dehydrobilirubin, the minimizing that can be measured to 350-550nm place absorbance is directly proportional with the concentration of total bilirubin.
37 ℃ of temperature; Cuvette optical path 1.0cm.
(commplementary wave length can be eliminated the interference of other matrix in the sample to wavelength 450nm (master)/546nm (pair), improves detection sensitivity, and promptly the absorbance A that reads of instrument is A
Predominant wavelength-A
Commplementary wave length)。
Sample (standard) 8 μ l;
Reagent 1 (R1) 240 μ l; Reagent 2 (R2) 60 μ l
Sample (calibration tube is made sample with calibration object) adds the R1 mixing, and 37 ℃ are read absorbance A after hatching 3~5min
0Add the R2 mixing, 37 ℃ hatch 5min after, read absorbance A
1, Δ A=A
1-A
0
Calculate
Embodiment 2
Reagent 1 (R
1)
Potassium dihydrogen phosphate (KH
2PO
4) 0.5g
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 16g
Sodium chloride (NaCl) 12.5g
Polyvinylpyrrolidone [(C
6H
9NO) n] 1g
Disodium ethylene diamine tetraacetate (EDTA-Na
2) 1g
Triton x-100 (TritonX-100) 0.5ml
Thimerosal (C
9H
9O
2HgNaS) 0.25g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 7.6.
Reagent 2 (R
2)
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 1g
Potassium dihydrogen phosphate (KH
2PO
4) 0.25g
Potassium persulfate (K
2S
2O
8) 65g
Sodium chloride (NaCl) 4.5g
Thimerosal (C
9H
9O
2HgNaS) 0.25g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 6.8.
The preparation method of present embodiment 2 and with the method for the kit measurement total bilirubin of the mensurating total bilirubin through chemistry oxidation process of present embodiment with embodiment 1.
Embodiment 3
Reagent 1 (R
1)
Potassium dihydrogen phosphate (KH
2PO
4) 2g
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 50g
Sodium chloride (NaCl) 4g
Polyvinylpyrrolidone [(C
6H
9NO) n] 20g
Disodium ethylene diamine tetraacetate (EDTA-Na
2) 0.3g
Triton x-100 (TritonX-100) 1.75ml
Thimerosal (C
9H
9O
2HgNaS) 0.15g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 7.8.
Reagent 2 (R
2)
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 1.6g
Potassium dihydrogen phosphate (KH
2PO
4) 0.4g
Potassium persulfate (K
2S
2O
8) 40g
Sodium chloride (NaCl) 7g
Thimerosal (C
9H
9O
2HgNaS) 0.15g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 7.0.
The preparation method of present embodiment 3 and with the method for the kit measurement total bilirubin of the mensurating total bilirubin through chemistry oxidation process of present embodiment with embodiment 1.
Embodiment 4
Reagent 1 (R
1)
Potassium dihydrogen phosphate (KH
2PO
4) 1g
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 25g
Sodium chloride (NaCl) 7.5g
Polyvinylpyrrolidone [(C
6H
9NO) n] 5g
Disodium ethylene diamine tetraacetate (EDTA-Na
2) 0.7g
Triton x-100 (TritonX-100) 0.75ml
Thimerosal (C
9H
9O
2HgNaS) 0.15g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 8.2.
Reagent 2 (R
2)
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 3.3g
Potassium dihydrogen phosphate (KH
2PO
4) 0.8g
Potassium persulfate (K
2S
2O
8) 20g
Sodium chloride (NaCl) 15g
Thimerosal (C
9H
9O
2HgNaS) 0.08g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 7.4.
The preparation method of present embodiment 4 and with the method for the kit measurement total bilirubin of the mensurating total bilirubin through chemistry oxidation process of present embodiment with embodiment 1.
Embodiment 5
Reagent 1 (R
1)
Potassium dihydrogen phosphate (KH
2PO
4) 1.5g
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 35g
Sodium chloride (NaCl) 5g
Polyvinylpyrrolidone [(C
6H
9NO) n] 15g
Disodium ethylene diamine tetraacetate (EDTA-Na
2) 0.5g
Triton x-100 (TritonX-100) 1.2ml
Thimerosal (C
9H
9O
2HgNaS) 0.1g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 8.0.
Reagent 2 (R
2)
Sodium hydrogen phosphate (contains 12H
2O) (Na
2HPO
4.12H
2O) 2.5g
Potassium dihydrogen phosphate (KH
2PO
4) 0.6g
Potassium persulfate (K
2S
2O
8) 30g
Sodium chloride (NaCl) 10g
Thimerosal (C
9H
9O
2HgNaS) 0.1g
H
2O adds to 1000ml
Sodium hydrogen phosphate mixes back accent pH value with potassium dihydrogen phosphate be 7.2.
The preparation method of present embodiment 5 and with the method for the kit measurement total bilirubin of the mensurating total bilirubin through chemistry oxidation process of present embodiment with embodiment 1.
The performance of the potassium persulfate oxidizing process of prior art being surveyed bilirubinic kit (the existing kit of following abbreviation) and the kit (following abbreviation kit of the present invention) of mensurating total bilirubin through chemistry oxidation process of the present invention below in conjunction with accompanying drawing and form compares.The relative merits that compare these two kinds of kits by the influence of measuring precision, accuracy and various chaff interferences etc.
Principle:
Existing kit: potassium persulfate as preferential under the reaction promoter and total bilirubin reaction, causes the decline of 450nm wavelength place absorbance at polyoxyethylene nonylphenol ether, and its drop-out value is directly proportional with total bilirubin concentration.
Kit of the present invention: in phosphate buffer, under the condition of triton x-100 as sensitizer and reaction promoter, improvement chemical oxidizing agent and total bilirubin reaction cause the decline of 450nm wavelength place absorbance, and its drop-out value is directly proportional with total bilirubin concentration.
Reagent:
Existing kit is pressed following formulated:
Reagent 1: the physiological saline that contains 0.05% polyoxyethylene nonylphenol ether;
Reagent 2: potassium persulfate 73.98mmol/L aqueous solution.
Kit of the present invention: reagent 1 and reagent 2.
Instrument:
Hitachi's 7020 automatic clinical chemistry analyzers; Tianjin, island 2450 ultraviolet spectrophotometers.
Experiment parameter:
Hitachi's 7020 experiment parameters: 2 end-point methods, reacted 10 minutes, wavelength: predominant wavelength 450nm, commplementary wave length 546nm, sample size 8u L mixes with R1 240uL in advance hatches 5 minutes, surveys A1, and the back adds R2,60uL hybrid reaction 5 minutes (35 point) is surveyed A2, and the correcting fluid absorbance comparison under A2-A1 and the same terms just can be measured sample value.37 ℃ of the temperature of reaction, cuvette optical path l 0cm, the above test parameters of two kinds of reagent is identical.
Tianjin, island 2450 ultraviolet spectrophotometer experiment parameters: wavelength 450nm, 37 ℃ of the temperature of reaction, cuvette optical path l 0cm.
The comparison of the range of linearity of two kinds of kits:
Preparation 0,50,100,200,300,400,500,600,700 total bilirubin standard solution (the μ mol/L of unit), measure on biochemical instruments with two kinds of kits:
Theoretical value (μ mol/L) |
0 |
50 |
100 |
200 |
300 |
400 |
500 |
600 |
700 |
Kit of the present invention |
0.1 |
50.4 |
101.2 |
202.3 |
303.5 |
403.6 |
506.1 |
607.6 |
697.6 |
Existing kit |
0.1 |
49.2 |
98.6 |
192.5 |
289.4 |
384.7 |
476.6 |
567.4 |
620.6 |
In conjunction with Fig. 1 (existing kit), Fig. 2 (kit of the present invention) as can be seen, the range of linearity of kit of the present invention is bigger than the range of linearity of existing kit.
The comparison of two kinds of kit real time reaction curves:
During the curve of existing kit wave phenomenon is arranged, and do not arrive terminal point (referring to Fig. 3).
The real time reaction curve of kit of the present invention no matter be high and low value sample, is its maximum characteristics in range of linearity internal reaction curve standard (referring to Fig. 4), and good response curve is to guarantee result basis accurately.
The comparison of the interference free performance of two kinds of kits:
Add four kinds of chaff interferences and form pooled serum in Luo Shi quality-control product (68.4 μ mol/L), measurement result is as follows:
Chaff interference and concentration thereof |
Heparin (1250U/L) |
Fat milk (6.5mmol/L) |
Hb (7.4g/L) |
VitC (1.0g/L) |
Kit measured value of the present invention |
68.8 |
68.1 |
69.1 |
68.2 |
Kit deviation of the present invention |
0.58% |
0.44% |
1.02% |
0.29% |
Existing kit measured value |
67.2 |
67.3 |
69.6 |
67.9 |
Existing kit deviation |
1.75% |
1.61% |
1.75% |
0.73% |
Kit of the present invention as can be seen from the above table is than the good in anti-interference performance of existing kit.
The comparison of the sensitivity of two kinds of kits:
Sensitivity is low during existing kit measurement, what monitored is bilirubinic absorbance, and various compositions are quite complicated in the blood, many materials all have absorption at the 450nm place, although the commplementary wave length that has adopted 546nm is to remove the interference of haemoglobin, but being descended by the absorbance that produces after the potassium persulfate oxidation is still sensitively inadequately, is subject to disturb.When low value (about 10 μ mol/L) are fairly obvious for this phenomenon, and high value sample exists equally, just because the mensuration total absorbance is bigger, this phenomenon flooded.This phenomenon has also influenced the accuracy and the precision performance of potassium persulfate oxidizing process.
Contain triton x-100 and polyvinylpyrrolidone in the kit of the present invention, can strengthen absorbance with the cholerythrin complexing, the influence of the absorbance of various materials can be ignored comparatively speaking.
The precision of two kinds of kits and accuracy are compared:
Proofread and correct the product calibration with Luo Shi, measure the high value of Luo Shi (68.4 μ mol/L) and low value (23.4 μ mol/L) quality-control product.Carry out accuracy relatively with two kinds of kits continuously, the result is as follows:
TB1L average (n=20) |
Kit of the present invention |
Existing kit |
The high value actual measurement of Luo Shi average |
68.5 μ mol/L (deviation 0.15%) |
66.8 μ mol/L (deviation 2.34%) |
Precision of measurement |
1.5% |
2.6% |
Luo Shi low value actual measurement average |
23.6 μ mol/L (deviation 0.85%) |
20.5 μ mol/L (deviation 12.39%) |
Precision of measurement |
2.2% |
6.8% |
For measuring the low value quality-control product, the precision of existing kit and accuracy are all relatively poor, and the measured value of kit of the present invention is accurate.
In sum, the kit of the mensuration total bilirubin of (in the chemical oxidization method) potassium persulfate oxidizing process of the kit of the mensuration total bilirubin of chemical oxidization method of the present invention and prior art is compared, kit of the present invention has kept the advantage of kit of mensuration total bilirubin of the potassium persulfate oxidizing process of prior art: it is simple to operation when measuring total bilirubin, stable reagent, holding time is long, cheap (above also is the advantage of vanadic acid oxidizing process kit), and environmentally safe, have good Environmental Safety performance, the kit shortcoming of vanadic acid oxidizing process kit (more than overcome) of total bilirubin is measured in subsidiary reaction when surveying the low value sample less than the vanadic acid oxidizing process.