CN102944683A - Double reagent method for detecting indirect bilirubin kit and preparation method - Google Patents

Double reagent method for detecting indirect bilirubin kit and preparation method Download PDF

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CN102944683A
CN102944683A CN2012104600369A CN201210460036A CN102944683A CN 102944683 A CN102944683 A CN 102944683A CN 2012104600369 A CN2012104600369 A CN 2012104600369A CN 201210460036 A CN201210460036 A CN 201210460036A CN 102944683 A CN102944683 A CN 102944683A
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reagent
bilirubin
indirect
serum
azobilirubin
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李立和
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Abstract

The invention discloses a double reagent method for detecting indirect bilirubin in serum, which belongs to a material detecting method based on color change in the result of a test reaction by means of visible light. The technical scheme is that a diazo reagent is added in a reagent I, and the reagent I only contains active ingredients of an accelerator. The detecting method comprises the following steps: before use, a reagent A is mixed with a reagent B in the volume ratio of 20:1 to obtain the reagent I, warm bath is performed firstly on serum and the reagent I at 37 degrees C for 3 to 5 minutes, and direct bilirubin in the serum reacts with diazo acid chloride in the reagent I to obtain azobilirubin; then, a reagent II is added, warm bath is performed at 37 degrees C for 4 to 7 minutes; under the action of the accelerator, hydrogen bonds of the indirect bilirubin are broken, and then, the indirect bilirubin reacts with the diazo acid chloride to obtain the azobilirubin. The instrument is detected at the wavelength of 540 to 550 nm, and the azobilirubin obtained by the reaction of the reagent I is blank. Therefore, the content of the indirect bilirubin is calculated based on the azobilirubin obtained by the reaction of the reagent II.

Description

The double reagent method detects indirect bilirubin kit and preparation method
Technical field
The present invention relates to a kind of kit of measuring serum, blood plasma or urine mesobilirubin; Or utilizing visible light, the result by test reaction produces the method that change color is come test material, particularly relates to a kind ofly detecting the bilirubinic double reagent assay method of serum indirect with Biochemical Analyzer.
Background technology
Indirect bilirubin (english abbreviation IBIL) mainly is by hematoclasis, does not pass through the indirect bilirubin that is called of glucuronidation in liver.Indirect bilirubin can be changed into again bilirubin direct through liver metabolism, enters biliary tract with bile, discharges finally by stool.
Clinical meaning: indirect bilirubin reference value: 1.0～20.0 μ mol/L.The reason that indirect bilirubin is higher: it is higher that 1, some malignant diseases of liver disease also can cause the indirect bilirubin in the blood, such as acute icteric hepatitis, acute hepatic necrosis, chronic active hepatitis, cirrhosis etc.2, red blood cell considerable damage in the hemolytic anemia human body; discharge indirect bilirubin; when blood indirect hyperbilirubinemia; the conversion capability that has surpassed liver; indirect bilirubin is detained in blood; thereby cause that blood indirect cholerythrin is higher, this situation also is referred to as hemolytic jaundice, and the patient has yellowing of the skin, sclera jaundice, urine look jaundice symptom usually.3, incompatible blood transfusion can cause haemolysis when the blood of input blood group incompatibility, makes red blood cell considerable damage in the body, thereby causes the indirect bilirubin in the blood higher.4, icterus neonatorum is after neonate's birth, jaundice appearred in 48～72 hours, off color, and do not disappear in 2 weeks, this is mainly because mother and sons' blood group or Neonatal Congenital anomalies of biliary system etc. cause that this situation also can cause the indirect bilirubin in the blood higher.
At present the determination of bilirubin method has improvement J-G method, Bilirubin Oxidase method, high performance liquid chromatography, measures the assay methods such as cholerythrin method, vanadate oxidizing process through skin, wherein, improvement J-G method is the laboratory determination method that recommend at the World Health Organization (WHO) and China Ministry of Public Health clinical examination center, and these methods are used for measuring total bilirubin and bilirubin direct.
Take dimethyl sulfoxide method as example total bilirubin (TBIL) assaying reaction formula as:
Sulfanilic acid+HCl+NaNO 2→ diazonium chloride benzene sulfonic acid+NaCl
Bilirubin direct+diazonium chloride benzene sulfonic acid → azobilirubin
Figure BSA00000805828100011
What direct bilirubin detecting adopted is not add dimethyl sulfoxide in total bilirubin, that measures is bilirubin direct, experimental determination indirect bilirubin method is and measures first total bilirubin at present, then measuring bilirubin direct, both results subtract each other the value that is indirect bilirubin, but this method workload and reagent cost double, and are subjected to simultaneously the impact of two experimental errors, therefore, the invention use the method that the double reagent method once records indirect bilirubin.In same reaction system, after bilirubin direct is finished with the reaction of diazonium chloride benzene sulfonic acid generation azobilirubin first, add dimethyl sulfoxide, indirect bilirubin generates azobilirubin with the diazonium chloride benzene sulfonic acid under the dimethyl sulfoxide effect, instrument reacts the azobilirubin that generates and is blank take the first step, calculate the value of indirect bilirubin as assaying reaction take the azobilirubin of second step reaction generation, be not subjected to the interference of bilirubin direct, therefore, measurement result is the value of indirect bilirubin.
Summary of the invention:
Can't directly measure serum indirect cholerythrin in order to solve prior art, it is convenient and easy to the invention provides a kind of economy, the double reagent assay method that accuracy is higher, can directly measure the serum indirect bilirubin.
The technical scheme that solves this technical problem employing is: sulfanilic acid, hydrochloric acid and sodium nitrite coexist among the reagent I, after bilirubin direct is finished with the reaction of diazonium chloride benzene sulfonic acid generation azobilirubin first, add reagent II, indirect bilirubin is under the dimethyl sulfoxide effect, generate azobilirubin with the diazonium chloride benzene sulfonic acid, instrument detects at 540nm wavelength place, reacting the azobilirubin that generates take the first step is blank, is calculated the content of indirect bilirubin by the azobilirubin of reagent II reaction generation.
Reaction equation:
First step reaction
Sulfanilic acid+HCl+NaNO 2→ diazonium chloride benzene sulfonic acid+NaCl
Bilirubin direct+diazonium chloride benzene sulfonic acid → azobilirubin
The second step reaction
Among the mentioned reagent I: sulfanilic acid 60mmol/L, hydrochloric acid 280mmol/L, sodium nitrite 60mmol/L, 0.2%Proclin 300; Among the reagent II: dimethyl sulfoxide (DMSO) 24mmol/L, 0.2%Proclin 300.
The volume ratio of reactant is in the said determination: S: R I: R II=5: 150: 50, wherein reagent A and reagent B formed reagent I by 20: 1, though the reagent of this method is identical with original method reagent constituent, just dimethyl sulfoxide were placed among the reagent II, and it is different that it measures successful.
Dimethyl sulfoxide also can select glucuronyl transferase or caffeine and Sodium Benzoate to replace among the present invention.
The present invention compared with prior art has the following advantages: the bilirubinic double reagent method of serum indirect of the present invention is not subjected to the impact of bilirubin direct when detecting, measure the range of linearity and can reach 220.0 μ mol/L.Its using method is identical with original total bilirubin and bilirubin direct, can not increase experimenter's burden, does not increase reagent cost, and is economical convenient and easy, is the higher indirect bilirubin detection method of a kind of accuracy.
Description of drawings
Accompanying drawing 1 is the synoptic diagram directly perceived that the present invention measures the indirect bilirubin kit.
Accompanying drawing 2 is real time reaction curve maps that the present invention measures indirect bilirubin.
Embodiment:
Below by embodiment and accompanying drawing the present invention is described in further details.
Embodiment 1
The preparation of reagent:
A. reagent A: take by weighing 10.96 gram sulfanilic acid and be dissolved in 800 ml distilled waters, add 18.83 milliliter of 36% concentrated hydrochloric acid, add 200 μ L Proclin-300, supply 1 liter with distilled water and be sulfanilic acid 63.0mmol/L, hydrochloric acid 294.0mmol/L, 0.2%Proclin-300.
B. reagent B: take by weighing 86.94 gram sodium nitrites and be dissolved in 1 liter of distilled water, add 200 μ LProclin-300, be sodium nitrite 1260.0mmol/L, 0.2%Proclin-300.
C. reagent II: take by weighing dimethyl sulfoxide 1.87 grams and be dissolved in 1 liter of distilled water, add 200 μ LProclin-300, be dimethyl sulfoxide (DMSO) 24.0mmol/L.
Using method: at first reagent A and reagent B mixed by 20: 1 and form reagent I, temperature was bathed 3 minutes after reagent I and the sample mix, then add reagent II after 5.1 minutes, the mensuration wavelength is 540nm, end-point method carries out colorimetric, react the azobilirubin that generates take the first step and be blank, calculated the content of indirect bilirubin by the azobilirubin that adds reagent II reaction generation.The assaying reaction volume ratio is: S: R I: R II=5: 150: 50.Each component final concentration is in the reactant liquor: sulfanilic acid 45mmol/L, hydrochloric acid 210mmol/L, dimethyl sulfoxide (DMSO) 6mmol/L, sodium nitrite 45mmol/L.
Total bilirubin concentration=Δ A Sample/ Δ A Calibration* calibrate concentration
Embodiment 2.
Among the reagent II dimethyl sulfoxide changed into caffeine and Sodium Benzoate or glucuronyl transferase.Content is constant, and other compositions are all constant.
Embodiment 3
The mensuration program
Double reagent method: on Japanese OLYMPUS AU 2700 full-automatic Biochemical Analyzers, instrument joins 5 μ l samples mixing among the 150 μ l reagent I automatically, hatched 3 minutes for 37 ℃, add 50 μ l reagent II mixings, hatched 5.1 minutes for 37 ℃, fully-automatic analyzer detects at 540nm wavelength place, and instrument calculates the indirect bilirubin result automatically.Specifically see Table 1
Table 1. robotization Biochemical Analyzer of the present invention test condition
Figure BSA00000805828100031
OD IBIL＝OD 27-OD 10×[(SV+R IV I)/(SV+R IV I+R IIV II)]
Indirect bilirubin concentration=F * OD IBIL
OD wherein IBILThe absorbance that indirect bilirubin produces, OD 10That sample joins the absorbance that records after the working fluid reaction, OD 27Be that sample adds the absorbance that records after the reagent II reaction, SV is the volume of blood serum sample, R IV IThe volume of reagent I, R IIV IIBe the volume of reagent II, F is correction factor.
OD behind the adding reagent II 10Extension rate be [(SV+R IV I)/(SV+R IV I+ R IIV II)], the absorbance behind the adding reagent II is OD 10* [(SV+R IV I)/(SV+R IV I+ R IIV II)].So the absorbance of the azobilirubin that is produced by indirect bilirubin is: OD IBIL=OD 27-OD 10* [(SV+R IV I)/(SV+R IV I+ R IIV II)], i.e. OD IBIL=OD 27-(5+150)/(5+150+50) * OD 10As long as measure OD 10And OD 27Can calculate the concentration of indirect bilirubin.
1. detected object: outpatient service and inpatient 82 people, the male sex 42 people wherein, 42.5 years old mean age; Women 40 people, 39.5 years old mean age, on an empty stomach blood sampling adopts respectively total bilirubin and bilirubin direct subtractive method and the inventive method to measure indirect bilirubin.
2. the present laboratory of classic method method commonly used, adopt the value of Beijing Leaderman Biochemistry Co., Ltd's total bilirubin (TBIL) and bilirubin direct (DBIL) kit measurement patients serum's total bilirubin and bilirubin direct, and by formula IBIL=TBIL-DBIL calculates the value of indirect bilirubin.
3. the inventive method adopts the double reagent method, at first the reaction of the diazonium chloride benzene sulfonic acid among the bilirubin direct in the serum and the reagent I generates azobilirubin, after reaction reaches balance, add reagent II, under the dimethyl sulfoxide effect, diazonium chloride benzene sulfonic acid reaction in indirect bilirubin and the working fluid generates azobilirubin, the azobilirubin that bilirubin direct and indirect bilirubin generate is colour generation successively, instrument reacts the azobilirubin of (bilirubin direct) generation as blank take the first step, the azobilirubin that generates with second step reaction (indirect bilirubin) calculates indirect bilirubin.
Two kinds of experimental technique indirect bilirubin measurement results relatively see Table 1:
Two kinds of methods of table 1. are measured the patient and are organized serum bilirubin result relatively (μ mol/L).
Figure BSA00000805828100041
As can be seen from Table 1, (t=1.24, P＞0.05 n=82), can be used for clinical labororatory's indirect bilirubin assay method for the present invention and traditional comparison there was no significant difference.
Change dimethyl sulfoxide into caffeine and Sodium Benzoate or glucuronyl transferase among the reagent II, it is measured effect and uses dimethyl sulfoxide method identical.
Can find out through above comparison, though the present invention forms basic identical with original formulation, but because dimethyl sulfoxide and sulfanilic acid and sodium nitrite successively add in reaction, the azobilirubin priority colour generation that bilirubin direct and indirect bilirubin are generated, and the azobilirubin that makes instrument react (bilirubin direct) generation take the first step is blank, and the azobilirubin that generates with second step reaction (indirect bilirubin) calculates indirect bilirubin.Actual effect is also fully different.Therefore, the present invention measures the value of indirect bilirubin by the addition sequence that changes reagent.

Claims (6)

1. bilirubinic double reagent assay method of serum indirect, it is characterized in that diazo reagent is in reagent I, reagent II only contains accelerator effective constituent, its assay method is: serum was bathed 3～5 minutes in 37 ℃ of temperature with reagent I first, the reaction of bilirubin direct and reagent I generates azobilirubin in the serum, bathed 4～7 minutes in 37 ℃ of temperature after adding again reagent II, indirect bilirubin is under the effect of accelerator, the indirect bilirubin hydrogen bond is destroyed, generate azobilirubin with the diazo reagent reaction, instrument detects at 540nm wavelength place, react the azobilirubin that produces take reagent I and be blank, react the content that the azobilirubin that produces calculates indirect bilirubin by reagent II, computing formula is:
OD IBiL＝OD 27-OD 10×[(SV+R IV I)/(SV+R IV I+R IIV II)]
Indirect bilirubin concentration=F * OD IBiL
OD wherein IBiLThe absorbance that indirect bilirubin produces, OD 10That sample adds the absorbance that records after the reagent I reaction, OD 27Be that sample adds the absorbance that records after the reagent II reaction, SV is the volume of blood serum sample, R IV IThe volume of reagent I, R IIV IIBe the volume of reagent II, F is correction factor.
2. the bilirubinic double reagent assay method of serum indirect according to claim 1 is characterized in that containing among the reagent I hydrochloric acid, sulfanilic acid, sodium nitrite and antiseptic.
3. the bilirubinic double reagent assay method of serum indirect according to claim 1 is characterized in that only containing among the reagent II dimethyl sulfoxide or glucuronyl transferase or caffeine and Sodium Benzoate.
4. the bilirubinic double reagent assay method of serum indirect according to claim 1 is characterized in that each constituent is among the reagent I: sulfanilic acid 40～80mmol/L, hydrochloric acid 240～320mmol/L, sodium nitrite 40～80mmol/L, Proclin 300 antiseptics 100 μ l～300 μ l.
5. the bilirubinic double reagent assay method of serum indirect according to claim 1 is characterized in that only having among the reagent II dimethyl sulfoxide (DMSO) 20～28mmol/L, Proclin 300 antiseptics 100 μ l～300 μ l.
6. the bilirubinic double reagent assay method of serum indirect according to claim 1 is characterized in that the volume ratio of measuring is: sample: reagent I: reagent II=3～7: 120～180: 30～70.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103278652A (en) * 2013-05-24 2013-09-04 宁波美康生物科技股份有限公司 Total bilirubin detection reagent
CN104406971A (en) * 2014-11-28 2015-03-11 山东博科生物产业有限公司 Direct bilirubin detection reagent
CN106841078A (en) * 2017-03-31 2017-06-13 福建师范大学 A kind of method for quantitatively determining of aqueous solution mesobilirubin concentration
CN107884397A (en) * 2017-10-24 2018-04-06 黄世业 Neonatal bilirubin quickly determines pipe
CN110261625A (en) * 2019-07-15 2019-09-20 三诺生物传感股份有限公司 A kind of bilirubin multiplexed detection reagents box and its application method

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US5872009A (en) * 1994-12-02 1999-02-16 Nitto Boseki Co., Ltd. Method for measuring bilirubin
CN101055271A (en) * 2006-04-12 2007-10-17 上海复星医药（集团）股份有限公司 Enzyme method reagent kit for detecting DBil
CN102023158A (en) * 2010-10-19 2011-04-20 李立和 Method for enzymatically determining creatinine in serum by two steps

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US5872009A (en) * 1994-12-02 1999-02-16 Nitto Boseki Co., Ltd. Method for measuring bilirubin
US5804405A (en) * 1996-11-27 1998-09-08 Research Corporation Technologies, Inc. Bilirubin detection
US5935805A (en) * 1996-11-27 1999-08-10 Research Corporation Technologies, Inc. Measurement of bilirubin albumin binding
CN101055271A (en) * 2006-04-12 2007-10-17 上海复星医药（集团）股份有限公司 Enzyme method reagent kit for detecting DBil
CN102023158A (en) * 2010-10-19 2011-04-20 李立和 Method for enzymatically determining creatinine in serum by two steps

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103278652A (en) * 2013-05-24 2013-09-04 宁波美康生物科技股份有限公司 Total bilirubin detection reagent
CN103278652B (en) * 2013-05-24 2015-04-15 宁波美康生物科技股份有限公司 Total bilirubin detection reagent
CN104406971A (en) * 2014-11-28 2015-03-11 山东博科生物产业有限公司 Direct bilirubin detection reagent
CN104406971B (en) * 2014-11-28 2017-03-08 山东博科生物产业有限公司 A kind of bilirubin direct detectable
CN106841078A (en) * 2017-03-31 2017-06-13 福建师范大学 A kind of method for quantitatively determining of aqueous solution mesobilirubin concentration
CN107884397A (en) * 2017-10-24 2018-04-06 黄世业 Neonatal bilirubin quickly determines pipe
CN110261625A (en) * 2019-07-15 2019-09-20 三诺生物传感股份有限公司 A kind of bilirubin multiplexed detection reagents box and its application method

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Application publication date: 20130227