CN103048282A - Detection method of bilirubin and detection kit - Google Patents
Detection method of bilirubin and detection kit Download PDFInfo
- Publication number
- CN103048282A CN103048282A CN2012104144690A CN201210414469A CN103048282A CN 103048282 A CN103048282 A CN 103048282A CN 2012104144690 A CN2012104144690 A CN 2012104144690A CN 201210414469 A CN201210414469 A CN 201210414469A CN 103048282 A CN103048282 A CN 103048282A
- Authority
- CN
- China
- Prior art keywords
- detection method
- bilirubin
- damping fluid
- sample
- concentration
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a detection method of bilirubin. A velocity measurement method is adopted; the reducing velocity of the absorbancy of a reaction system is directly proportional to the concentration of the bilirubin in a sample at the position with 440-465nm of wavelength; and the reducing velocity of the absorbancy within 30-180s is measured after delaying for 30-60s, so as to obtain the content of the bilirubin. The reaction velocity is measured by an enzyme velocity measurement method; reading is carried out during a linear phase in a reaction process; and interference of a background of a serum sample can be completely removed; the accuracy of a result is improved; the detection method is short in delay time, and short in reading duration; the entire reaction time is greatly shortened; the efficiency is improved; removal of the background of the serum sample does not need to be considered; the detection method is simple and convenient to operate, parameters of an instrument are simple to set, and a complicated computational formula is not needed; and either a dual-reagent model or a single-reagent mode can be adopted for measurement, and the detection method is suitable for various automatic and self-automatic biochemical analyzers and spectrophotometers, thus being wide in application range.
Description
Technical field
The present invention relates to a kind of bilirubinic detection method, specifically a kind of bilirubinic detection method of enzyme rate method human serum and detection kit, be used for the bilirubinic concentration of external quantitative measurement human serum, blood plasma, belong to medical and clinical biochemical diagnosis reagent field.
Background technology
Cholerythrin is the complicated linear organic compound that is linked to each other and formed by the 2-methine bridge of four pyrrole rings by molecule central authorities, is the metabolic product of protoheme.In blood, bilirubinic combined with bilirubin and two kinds of forms of unconjugated bilirubin of having, the absorption peak of combined with bilirubin is 422nm, the absorption peak of unconjugated bilirubin is 459nm.Combined with bilirubin is for to form non-covalent Reversible binding with albumin under the normal physiological conditions, but forms absorption peak as the intermediate material of 433nm at part hepatopathy human blood mesobilirubin and albumin take covalent bonds.
Total bilirubin is that being determined at clinically of serum total bilirubin has great significance, and mainly at liver metabolism, is an important conventional liver functional testing index in conjunction with (directly) cholerythrin and non-binding (indirectly) bilirubinic summation.Total bilirubin increases, normally because biliary obstruction, hepatitis, cirrhosis, hepatic failure, hemolytic syndrome and some heredity azymia.The indirect bilirubin increase be common in before the liver or liver in reason, such as the hemolytic syndrome, cause cholerythrin picked-up, transportation or in conjunction with damaged liver diseases.Bilirubinic physiological reduction sees the pregnant woman.The reduction of serum total bilirubin is relevant with the dangerous increase of coronary heart disease.Bilirubin monitoring is for the neonate, and especially the premature is very important.The interior bilirubin metabolism of liver cell this moment is usually immature, and the jaundice that causes owing to the unconjugated bilirubin rising is very common.Unconjugated bilirubin is combined with albumin as failing, and then very easily by blood-brain barrier, increases the danger of brain damage.
The bilirubinic method of clinical mensuration commonly used has diazo reagent method, chemical oxidization method, bilirubin oxidase end-point method etc. at present.The diazo reagent method is used the earliest, but linearity is lower, poor quality's control, and result difference is large; The chemical oxidization method developed recently is rapider, but also easily is disturbed, and negative value often appears in the result; Enzymatic assays is state-of-the-art method, and is linear high, enjoys in recent years industry to praise highly, but the bilirubin oxidase end-point method computing formula of using at present is loaded down with trivial details, and the reaction time is long, also is subject to the serum background and disturbs, only in high-end market use is arranged, do not have spread to use.Above method is used all more extensive, is end-point method and measures, and has a common shortcoming to be subject to exactly the serum background and disturbs, and has greatly affected result's accuracy.
Summary of the invention
The technical problem to be solved in the present invention is for above deficiency, a kind of bilirubinic detection method of enzyme rate method human serum and detection kit are provided, the bilirubinic concentration that is used for external quantitative measurement human serum, blood plasma, overcome existing detection method and be subject to the defective that the serum background disturbs, detection method of the present invention is with the enzymatic oxidation method, use the rate determination method, got rid of the interference of serum background fully, shortened the reaction time, easy and simple to handle, the result is applicable to various biochemical analyzers accurately and reliably.
For overcoming the above problems, the technical solution used in the present invention is: a kind of bilirubinic detection method, it is characterized in that: described detection method is for adopting the rate determination method, according at wavelength 440～465nm place, the changing down of reaction system absorbance is directly proportional with the concentration of sample mesobilirubin, postpone to measure the descend rate of absorbency in 30～180 seconds after 30～60 seconds, obtain bilirubinic content;
A kind of prioritization scheme, described descend rate of absorbency determination step: at first get needed blank tube, standard pipe and sample tube according to mensuration, add respectively damping fluid; Then correspondence adds sample to be tested, distilled water and titer in sample tube, blank tube and standard pipe, and mixing is incubated 3～10 minutes under 37 ℃ of conditions, adds enzyme reagent again, and mixing obtains reactant liquor; Or first damping fluid and enzyme reagent are made into the reagent working fluid, stablized 3～10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively obtains reactant liquor again;
Reactant liquor reacts under 37 ℃ of conditions, postpone after 30～60 seconds, the absorbance that reads reactant liquor at wavelength 440～465nm place changes, reading interval time is 30～180 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and obtained per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid.
Another kind of prioritization scheme, calculate bilirubinic content according to following formula:
Normal concentration is the concentration value of titer.
Using method: according to the instrument requirement, use the single reagent pattern at semi-automatic biochemical analyzer; Use the single reagent pattern at spectrophotometer; On automatic clinical chemistry analyzer, can use the double reagent pattern, also can use the single reagent pattern, be preferably the single reagent pattern.
Another prioritization scheme, the volume ratio of described damping fluid and enzyme reagent is 20～3:1; The volume ratio of damping fluid, enzyme reagent sum and sample is 20:1～10:1.
Further prioritization scheme, described damping fluid are the Tris-HCl damping fluid of 0.1mol/L that contains the SDS of 4mmol/L sodium taurocholate and 15mmol/L; Or
Damping fluid is the EDTA-Na that contains 1.86g/L
2The phosphate buffer of 0.2mol/L.
Prioritization scheme further, described enzyme reagent is bilirubin oxidase solution, concentration is 1～20U/ml.
Prioritization scheme further, described titer is unconjugated bilirubin solution or taurine combined with bilirubin solution, concentration is 5～40 μ mol/L.
Based on above cholerythrin detection method, the invention provides a kind of detection kit that realizes the cholerythrin detection method, comprise packing box, damping fluid, enzyme reagent, titer;
Described damping fluid is the Tris-HCl damping fluid of 0.1mol/L that contains the SDS of 4mmol/L sodium taurocholate and 15mmol/L, perhaps for the EDTA-Na of 1.86g/L is arranged
2The phosphate buffer of 0.2mol/L;
Described enzyme reagent is bilirubin oxidase solution, and concentration is 1～20U/ml;
Described titer is unconjugated bilirubin solution or taurine combined with bilirubin solution, and concentration is 5～40 μ mol/L.
Condition of storage and the term of validity are: 2～8 ℃ of preservations of former installed reagents, the term of validity 12 months; After damping fluid, enzyme reagent were made into the reagent working fluid, room temperature can be stablized 8 hours, can stablize 7 days under 2～8 ℃ of refrigerations, lucifuge condition.
Applicable instrument is for being applicable to various semi-automatic and automatic clinical chemistry analyzer and spectrophotometers.
Sample requires: sample can be serum or blood plasma, and EDTA can be used as anti-coagulants; Sample is answered lucifuge; Preferably measure immediately after the sampling; Under the lucifuge condition, sample can be preserved 2 days in room temperature, can preserve 4～7 days at 2～8 ℃, can stablize 3 months for-20 ℃.
The present invention adopts above technical scheme, compares with the prior art scheme, has the following advantages:
The present invention uses enzyme rate determination method, mensuration be the reaction speed, the linear phase in course of reaction is carried out reading, is different from the end assay method of additive method fully, can get rid of the interference of blood serum sample background fully, the accuracy that has improved the result;
After the present invention adopted rate method, time delay was short, and the reading duration is also very short, and the whole reaction time is shortened greatly, shortened in 3 minutes by original 5～10 minutes, had improved efficient;
The present invention is because need not to consider the removal of blood serum sample background, so easy and simple to handle, the instrument parameter setting is also very simple, need not complicated computing formula;
The present invention can use the double reagent pattern to measure, and also can use the single reagent pattern to measure, therefore go for various automatically, semi-automatic biochemical analyzer device and spectrophotometer, applied widely.
The invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Accompanying drawing 2 is that embodiment of the invention mesobilirubin concentration is certain, the asynchronous reaction absorbance of bilirubin oxidase concentration curve.
Embodiment
Below the preferred embodiments of the present invention are described, should be appreciated that preferred embodiment described herein only is used for description and interpretation the present invention, is not intended to limit the present invention.
Damping fluid is the Tris-HCl damping fluid of 0.1mol/L that contains the SDS of 4mmol/L sodium taurocholate and 15mmol/L, perhaps for the EDTA-Na of 1.86g/L is arranged
2The phosphate buffer of 0.2mol/L.
Enzyme reagent is bilirubin oxidase solution, and concentration is 1～20U/ml, and concrete concentration guarantees that according to determining with the volume ratio of damping fluid the enzyme final concentration is 0.05～2U/ml in the reactant liquor.
The volume ratio of damping fluid and enzyme reagent is 20～3:1.
Titer is unconjugated bilirubin solution or taurine combined with bilirubin solution, and concentration is 5～40 μ mol/L, calibrates with the national standard assignment.
Condition of storage and the term of validity are: 2～8 ℃ of preservations of former installed reagents, the term of validity 12 months; After damping fluid, enzyme reagent were made into working fluid, room temperature can be stablized 8 hours, can stablize 7 days under 2～8 ℃ of refrigerations, lucifuge condition.
Applicable instrument is for being applicable to various semi-automatic and automatic clinical chemistry analyzer and spectrophotometers.
Based on above detection kit, bilirubinic detection method is carried out according to following steps, detection side's ratio juris is the zymetology rate method, according at wavelength 440～465nm place, the changing down of absorbance is directly proportional with the concentration of sample mesobilirubin, postpone after 30～60 seconds, measure the descend rate of absorbency in 30～180 second time period, calculate bilirubinic content according to formula.
Measuring principle is according to as follows:
Shown in Fig. 1, in the situation that enzyme concentration is certain, the bilirubinic response curve of variable concentrations was linear relation in 0～5 minute substantially, further, in 0～4 minute, the linearity of reaction is good, so determine that the linear phase is 0～4 minute, the reading duration should be in this scope.
Shown in Fig. 2, in the certain situation of cholerythrin, the response curve of variable concentrations enzyme has notable difference, by carrying out changing the response curve that enzyme concentration obtains in the variable concentrations cholerythrin situation, the result is presented in enzyme concentration 0.05U/ml～2U/ml scope, and the linearity in 0～4 minute is good, surpasses 2U/ml then without the linear phase, can't realize uniform speed response, so determine that the enzyme concentration scope is 0.05U/ml～2U/ml.
Concrete operation method is as follows:
(1) descend rate of absorbency detects
Double reagent pattern operation steps:
At first get needed blank tube, standard pipe and sample tube according to mensuration, add respectively damping fluid; Then in sample tube, the corresponding sample to be tested that adds in blank tube and the standard pipe, distilled water and titer, mixing, mixing, be incubated 3～10 minutes under 37 ℃ of conditions, be preferably 5 minutes, and then adding enzyme reagent, mixing, postpone to obtain reactant liquor after 30～60 seconds, enzyme concentration is 0.05～2U/ml in the reactant liquor, the absorbance that reads reactant liquor at wavelength 440～465nm place changes, reading interval time is 30～180 seconds, calculates average per minute absorbance rate of change Δ A/ minute, obtains blank tube, the per minute absorbance rate of change Δ of standard pipe and sample tube internal reaction liquid A/ minute;
Or,
Single reagent pattern operation steps: first damping fluid and enzyme reagent are made into the reagent working fluid, stablized 10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively again, 37 ℃ of reactions, postpone after 30～60 seconds, obtain reactant liquor, the absorbance that reads reactant liquor at wavelength 440～465nm place changes, reading interval time is 30～180 seconds, calculates average per minute absorbance rate of change Δ A/ minute, obtains per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid;
The volume ratio of damping fluid, enzyme reagent sum and sample is 20:1～10:1.
(2) result of calculation:
Calculate bilirubinic content according to following formula:
Normal concentration is the concentration value of titer.
The preparation method of sample is: sample adopts serum or blood plasma, and EDTA is as anti-coagulants; According to hospital's common accepted standard method blood sample collection, then separation of serum or blood plasma; Measure immediately after the sampling; Sample is gathering, is transporting and preserving link and should select lucifuge; Under the lucifuge condition, sample can be preserved 2 days in room temperature, can preserve 4～7 days at 2～8 ℃, can stablize 2～3 months for-20 ℃.
Sample requires: sample can be serum or blood plasma, and EDTA can be used as anti-coagulants; Sample is answered lucifuge; Preferably measure immediately after the sampling; Under the lucifuge condition, sample can be preserved 2 days in room temperature, can preserve 4～7 days at 2～8 ℃, can stablize 3 months for-20 ℃.
Adopt above detection kit that total bilirubin is detected, detection method is carried out according to following steps: the descend rate of absorbency determination step carries out according to the operation of single reagent pattern, at first damping fluid and enzyme reagent is made into the reagent working fluid, stablizes 10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively again, 37 ℃ of reactions postpone to obtain reactant liquor after 30 seconds, and enzyme concentration is 0.05U/ml in the reactant liquor; The absorbance that reads reactant liquor at wavelength 450nm place changes, reading interval time is 30 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and measured 10 times, obtain per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid; The concrete amount of each composition sees the following form, and all the other steps are identical with the detection method of embodiment 1.
Analytical approach: rate method; The Direction of Reaction: negative reaction.
Calculate the content of total bilirubin according to following formula:
Δ A is the absorbance rate of change;
Normal concentration is the concentration value of titer.
The total bilirubin term of reference is:
Adult: 2～20 μ mol/L or 0.1～1.2 mg/dL, 37 ℃;
Neonate: 20～200 μ mol/L or 1.2～12 mg/dL, 37 ℃.
The explanation of assay: total bilirubin is the summation of combined with bilirubin and unconjugated bilirubin.Total bilirubin increases, normally because biliary obstruction, hepatitis, cirrhosis, hemolytic syndrome and some heredity azymia.The indirect bilirubin increase be common in before the liver or liver in reason, such as the hemolytic syndrome, cause cholerythrin picked-up, transportation or in conjunction with damaged liver diseases.
Bilirubin monitoring is for the neonate, and especially the premature is very important.The interior bilirubin metabolism of liver cell this moment is usually immature, and the jaundice that causes owing to the unconjugated bilirubin rising is very common.Unconjugated bilirubin is combined with albumin as failing, and then very easily by blood-brain barrier, increases the danger of brain damage.
The range of linearity: greater than 427 μ mol/L(25 mg/dL) (37 ℃) are up to 35mg/dL.
Points for attention
1, use the single reagent mode of operation at automatic clinical chemistry analyzer, the result can be more accurately and reliably;
2, according to the requirement of different instruments, reagent and sample consumption can change in proportion;
3, sample and standard must keep in Dark Place;
4, measuring wavelength can select between 440～465nm;
5, international unit converts: μ mol/L * 0.0585=mg/dL.
Embodiment 3, a kind of detection kit, and except following and embodiment 1 were different, all the other were identical with detection kit among the embodiment 1; Damping fluid is the Tris-HCl damping fluid of 0.1mol/L, contains the SDS of 4mmol/L sodium taurocholate and 15mmol/L, and the pH value is 8.2; Enzyme reagent is bilirubin oxidase solution, and concentration is 1U/ml, and the volume ratio of damping fluid and enzyme reagent is 3:1; Titer is unconjugated bilirubin solution, and concentration is 20 μ mol/L, calibrates with the national standard assignment.
Adopt above detection kit that total bilirubin is detected, detection method is carried out according to following steps: the descend rate of absorbency determination step carries out according to the operation of single reagent pattern, at first damping fluid and enzyme reagent is made into the reagent working fluid, stablizes 10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively again, 37 ℃ of reactions postpone to obtain reactant liquor after 60 seconds, and enzyme concentration is 0.2U/ml in the reactant liquor; The absorbance that reads reactant liquor at wavelength 450nm place changes, reading interval time is 60 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and measured 10 times, obtain per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid; Each composition and all the other steps are identical with the detection method of embodiment 2.
Embodiment 4, a kind of detection kit, and except following and embodiment 1 were different, all the other were identical with detection kit among the embodiment 1; Damping fluid is the Tris-HCl damping fluid of 0.1mol/L, contains the SDS of 4mmol/L sodium taurocholate and 15mmol/L, and the pH value is 8.2; Enzyme reagent is bilirubin oxidase solution, and concentration is 2U/ml, and the volume ratio of damping fluid and enzyme reagent is 4:1; Titer is unconjugated bilirubin solution, and concentration is 40 μ mol/L, calibrates with the national standard assignment.
Adopt above detection kit that total bilirubin is detected, detection method is carried out according to following steps: the descend rate of absorbency determination step carries out according to the operation of single reagent pattern, at first damping fluid and enzyme reagent is made into the reagent working fluid, stablizes 10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively again, 37 ℃ of reactions postpone to obtain reactant liquor after 30 seconds, and enzyme concentration is 0.4U/ml in the reactant liquor; The absorbance that reads reactant liquor at wavelength 450nm place changes, reading interval time is 180 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and measured 10 times, obtain per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid; Each composition and all the other steps are identical with the detection method of embodiment 2.
Adopt above detection kit that bilirubin direct is detected, detection method is carried out according to following steps: the descend rate of absorbency determination step carries out according to the operation of single reagent pattern, at first damping fluid and enzyme reagent is made into the reagent working fluid, stablizes 10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively again, 37 ℃ of reactions postpone to obtain reactant liquor after 30 seconds, and enzyme concentration is 0.5U/ml in the reactant liquor; The absorbance that reads reactant liquor at wavelength 450nm place changes, reading interval time is 60 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and measured 10 times, obtain per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid; Specifically seeing the following form of each composition, all the other steps are identical with the detection method of embodiment 1.
Analytical approach: rate method; The Direction of Reaction: negative reaction.
Calculate the content of bilirubin direct according to following formula:
Δ A is the absorbance rate of change;
Normal concentration is the concentration value of titer.
Normal person's bilirubin direct term of reference is: 0～6 μ mol/L or 0～0.3 mg/dL, 37 ℃.
The reference range of quoting is only for reference, advises this term of reference of each laboratory proofing or sets up the reference range of oneself.
Performance index of the present invention are: the range of linearity is greater than 171 μ mol/L(10 mg/dL) (37 ℃).
Points for attention are:
1, use the single reagent mode of operation at automatic clinical chemistry analyzer, the result can be more accurately and reliably;
2, according to the requirement of different instruments, reagent and sample consumption can change in proportion;
3, sample and standard must keep in Dark Place;
4, measuring wavelength selects between 440～465nm;
5, international unit converts: μ mol/L * 0.0585=mg/dL.
The explanation of assay: total bilirubin is in conjunction with (directly) cholerythrin and non-binding (indirectly) bilirubinic summation.The indirect bilirubin increase be common in before the liver or liver in reason, such as the hemolytic syndrome, cause cholerythrin picked-up, transportation or in conjunction with damaged liver diseases.
Bilirubin monitoring is for the neonate, and especially the premature is very important.The interior bilirubin metabolism of liver cell this moment is usually immature, and the jaundice that causes owing to the unconjugated bilirubin rising is very common.Unconjugated bilirubin is combined with albumin as failing, and then very easily by blood-brain barrier, increases the danger of brain damage.
Embodiment 6, a kind of detection kit, and except following and embodiment 1 were different, all the other were identical with detection kit among the embodiment 1; Damping fluid is the phosphate buffer of 0.2mol/L, contains 1.86g/L EDTA-Na2, and PH is 6.0; Enzyme reagent is bilirubin oxidase solution, and concentration is 5U/ml, and the volume ratio of damping fluid and enzyme reagent is 4:1; Titer is unconjugated bilirubin solution, and concentration is 8 μ mol/L, calibrates with the national standard assignment.
Adopt above detection kit that bilirubin direct is detected, detection method is carried out according to following steps: the descend rate of absorbency determination step carries out according to the operation of single reagent pattern, at first damping fluid and enzyme reagent is made into the reagent working fluid, stablizes 10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively again, 37 ℃ of reactions postpone to obtain reactant liquor after 30 seconds, and enzyme concentration is 1U/ml in the reactant liquor; The absorbance that reads reactant liquor at wavelength 450nm place changes, reading interval time is 60 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and measured 10 times, obtain per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid; Each composition and all the other steps are identical with the detection method of embodiment 5.
Embodiment 7, a kind of detection kit, and except following and embodiment 1 were different, all the other were identical with detection kit among the embodiment 1; Damping fluid is the phosphate buffer of 0.2mol/L, contains 1.86g/L EDTA-Na2, and PH is 7.0; Enzyme reagent is bilirubin oxidase solution, and concentration is 8U/ml, and the volume ratio of damping fluid and enzyme reagent is 3:1; Titer is unconjugated bilirubin solution, and concentration is 10 μ mol/L, calibrates with the national standard assignment.
Adopt above detection kit that bilirubin direct is detected, detection method is carried out according to following steps: the descend rate of absorbency determination step carries out according to the operation of single reagent pattern, at first damping fluid and enzyme reagent is made into the reagent working fluid, stablizes 10 minutes; Then get needed blank tube, standard pipe and sample tube according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively again, 37 ℃ of reactions postpone to obtain reactant liquor after 30 seconds, and enzyme concentration is 2U/ml in the reactant liquor; The absorbance that reads reactant liquor at wavelength 450nm place changes, reading interval time is 180 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and measured 10 times, obtain per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid; Each composition and all the other steps are identical with the detection method of embodiment 5.
The descend rate of absorbency determination step also can carry out according to the operation of double reagent pattern among the above embodiment, at first gets needed blank tube, standard pipe and sample tube according to mensuration, adds respectively damping fluid; Then correspondence adds sample to be tested, distilled water and titer in sample tube, blank tube and standard pipe, mixing, and mixing, insulation is 5 minutes under 37 ℃ of conditions, and then adds enzyme reagent, mixing, all the other steps are identical with each embodiment; Specifically seeing the following form of each composition, acquired results is consistent with the result of each embodiment.
Use the single reagent pattern at semi-automatic biochemical analyzer; On automatic clinical chemistry analyzer, can use the double reagent pattern, also can use the single reagent pattern, recommendation single reagent pattern.
The above embodiment of the present invention 2 to embodiment 4 is carried out the detection of total bilirubin, result such as following table with traditional enzyme end-point method and vanadate oxidizing process:
The above embodiment of the present invention 5 to embodiment 7 is carried out the detection of bilirubin direct, result such as following table with traditional enzyme end-point method and vanadate oxidizing process:
The result shows: carry out bilirubinic detection by embodiments of the invention, and compare with the testing result of enzyme end-point method and vanadate oxidizing process, without significant difference, but find out from data, method of the present invention removed the impact of increasing of background on measured value, so measured value is slightly on the low side because got rid of the interference of serum factor of background fully, near actual value, the result more more; Can find out also that from the result relative deviation is little, show that precision is high; The present invention has also shortened the reaction time, and makes operation easier, is applicable to various biochemical analyzers.
It should be noted that at last: the above only is the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment the present invention is had been described in detail, for a person skilled in the art, it still can be made amendment to the technical scheme that aforementioned each embodiment puts down in writing, and perhaps part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (8)
1. bilirubinic detection method, it is characterized in that: described detection method is for adopting enzyme rate determination method, according at wavelength 440～465nm place, the changing down of reaction system absorbance is directly proportional with the concentration of sample mesobilirubin, postpone after 30～60 seconds, measure the descend rate of absorbency in 30～180 seconds, obtain bilirubinic content.
2. a kind of bilirubinic detection method as claimed in claim 1 is characterized in that: described descend rate of absorbency determination step: at first get needed sample tube, blank tube and standard pipe according to mensuration, add respectively damping fluid; Then correspondence adds sample to be tested, distilled water and titer in sample tube, blank tube and standard pipe, and mixing is incubated 3～10 minutes under 37 ℃ of conditions, adds enzyme reagent again, and mixing obtains reactant liquor;
Or first damping fluid and enzyme reagent are made into the reagent working fluid, stablized 3～10 minutes; Then get needed sample tube, blank tube and standard pipe according to mensuration, add respectively the reagent working fluid; Corresponding sample to be tested, distilled water and the titer of adding in sample tube, blank tube and standard pipe respectively obtains reactant liquor again;
Reactant liquor reacts under 37 ℃ of conditions, postpone after 30～60 seconds, the absorbance that reads reactant liquor at wavelength 440～465nm place changes, reading interval time is 30～180 seconds, calculate average per minute absorbance rate of change Δ A/ minute, and obtained per minute absorbance rate of change Δ A/ minute of blank tube, standard pipe and sample tube internal reaction liquid.
3. a kind of bilirubinic detection method as claimed in claim 2 is characterized in that: described
Normal concentration is the concentration value of titer.
4. a kind of bilirubinic detection method as claimed in claim 2, it is characterized in that: the volume ratio of described damping fluid and enzyme reagent is 20:1～3:1; The volume ratio of damping fluid, enzyme reagent sum and sample is 20:1～10:1.
5. a kind of bilirubinic detection method as claimed in claim 2 is characterized in that: described damping fluid is the Tris-HCl damping fluid of 0.1mol/L that contains the SDS of 4mmol/L sodium taurocholate and 15mmol/L; Or damping fluid is the EDTA-Na that contains 1.86g/L
2The phosphate buffer of 0.2mol/L.
6. a kind of bilirubinic detection method as claimed in claim 2, it is characterized in that: described enzyme reagent is bilirubin oxidase solution, concentration is 1～20U/ml.
7. a kind of bilirubinic detection method as claimed in claim 2, it is characterized in that: described titer is unconjugated bilirubin solution or taurine combined with bilirubin solution, and concentration is 5～40 μ mol/L.
8. a realization is such as the detection kit of claim 1-7 cholerythrin detection method as described in one of them, and it is characterized in that: described detection kit comprises packing box, damping fluid, enzyme reagent, titer;
Described damping fluid is the Tris-HCl damping fluid of 0.1mol/L that contains the SDS of 4mmol/L sodium taurocholate and 15mmol/L, perhaps for the EDTA-Na of 1.86g/L is arranged
2The phosphate buffer of 0.2mol/L;
Described enzyme reagent is bilirubin oxidase solution, and concentration is 1～20U/ml;
Described titer is unconjugated bilirubin solution or taurine combined with bilirubin solution, and concentration is 5～40 μ mol/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210414469.0A CN103048282B (en) | 2012-10-26 | 2012-10-26 | Detection method of bilirubin and detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210414469.0A CN103048282B (en) | 2012-10-26 | 2012-10-26 | Detection method of bilirubin and detection kit |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103048282A true CN103048282A (en) | 2013-04-17 |
| CN103048282B CN103048282B (en) | 2015-04-15 |
Family
ID=48061001
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210414469.0A Active CN103048282B (en) | 2012-10-26 | 2012-10-26 | Detection method of bilirubin and detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103048282B (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837487A (en) * | 2014-03-19 | 2014-06-04 | 潍坊鑫泽生物科技有限公司 | Uric acid detection method and detection kit |
| CN103837685A (en) * | 2014-03-19 | 2014-06-04 | 潍坊鑫泽生物科技有限公司 | Blood glucose detection method and detection kit |
| CN107828855A (en) * | 2017-10-31 | 2018-03-23 | 南京欣迪生物药业工程有限责任公司 | A kind of refining neutrality α glucosidases detection kit and its application |
| CN108918435A (en) * | 2018-04-17 | 2018-11-30 | 武汉景川诊断技术股份有限公司 | Determination of bilirubin method and kit |
| CN110088628A (en) * | 2016-12-14 | 2019-08-02 | 豪夫迈·罗氏有限公司 | Determination to chaff interferent in sample |
| CN114277088A (en) * | 2021-12-02 | 2022-04-05 | 深圳市锦瑞生物科技股份有限公司 | Total bilirubin determination reagent, preparation method of reagent ball and determination chip |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4571383A (en) * | 1982-12-29 | 1986-02-18 | Nippon Shoji Kaisha, Ltd. | Reagent for measuring direct bilirubin by enzymatic method and method for measurement thereof |
| CN1967252A (en) * | 2006-10-30 | 2007-05-23 | 宁波美康生物科技有限公司 | Direct bilirubin detecting kit |
-
2012
- 2012-10-26 CN CN201210414469.0A patent/CN103048282B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4571383A (en) * | 1982-12-29 | 1986-02-18 | Nippon Shoji Kaisha, Ltd. | Reagent for measuring direct bilirubin by enzymatic method and method for measurement thereof |
| CN1967252A (en) * | 2006-10-30 | 2007-05-23 | 宁波美康生物科技有限公司 | Direct bilirubin detecting kit |
Non-Patent Citations (4)
| Title |
|---|
| BASIL T. DOUMAS ET AL.: "Determination of the Sum of Bilirubin Sugar Conjugates in Plasma by Bilirubin Oxidase", 《CLINICAL CHEMISTRY》, vol. 45, no. 8, 31 December 1999 (1999-12-31), pages 1255 - 1260, XP008111644 * |
| 宋耀忠 等: "半自动生化分析仪测定血清肌酐探讨", 《中国卫生检验杂志》, vol. 18, no. 10, 31 October 2008 (2008-10-31), pages 2166 * |
| 王琰 等: "《生物化学和临床生物化学检验实验教程》", 30 September 2005, article "生物化学和临床生物化学检验实验教程", pages: 215-218 * |
| 郑铁生 等: "《临床生物化学检验》", 28 February 2010, article "临床生物化学检验", pages: 120-128 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103837487A (en) * | 2014-03-19 | 2014-06-04 | 潍坊鑫泽生物科技有限公司 | Uric acid detection method and detection kit |
| CN103837685A (en) * | 2014-03-19 | 2014-06-04 | 潍坊鑫泽生物科技有限公司 | Blood glucose detection method and detection kit |
| CN103837685B (en) * | 2014-03-19 | 2016-06-29 | 潍坊鑫泽生物科技有限公司 | The detection method of a kind of blood glucose and detection kit |
| CN110088628A (en) * | 2016-12-14 | 2019-08-02 | 豪夫迈·罗氏有限公司 | Determination to chaff interferent in sample |
| CN107828855A (en) * | 2017-10-31 | 2018-03-23 | 南京欣迪生物药业工程有限责任公司 | A kind of refining neutrality α glucosidases detection kit and its application |
| CN107828855B (en) * | 2017-10-31 | 2020-08-21 | 南京欣迪生物药业工程有限责任公司 | Seminal plasma neutral alpha-glucosidase detection kit and application thereof |
| CN108918435A (en) * | 2018-04-17 | 2018-11-30 | 武汉景川诊断技术股份有限公司 | Determination of bilirubin method and kit |
| CN114277088A (en) * | 2021-12-02 | 2022-04-05 | 深圳市锦瑞生物科技股份有限公司 | Total bilirubin determination reagent, preparation method of reagent ball and determination chip |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103048282B (en) | 2015-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103048282B (en) | Detection method of bilirubin and detection kit | |
| CN104198408A (en) | Detection kit for determining content of creatinine in serum by enzymic method | |
| CN202404019U (en) | Kit for detecting content of homocysteine | |
| WO2006109279A3 (en) | Analyte determination method and analyte meter | |
| CN104198473A (en) | Stable uric acid detection kit | |
| CN201689054U (en) | Kit for detecting glycated albumin content in blood | |
| CN102809651B (en) | Creatine phosphokinase isozyme chemiluminescence immune quantitative detection kit and preparation method thereof | |
| CN109856128A (en) | A kind of urine glucose detection test paper and preparation method thereof of ascorbic acid interference | |
| WO2022095576A1 (en) | NOVEL N-ACETYL-β-D GLUCOSAMINIDASE DETECTION AGENT | |
| CN102650591A (en) | Kit for determining glycated serum protein | |
| CN101226198A (en) | Enzymatical detection method of saccharify blood albumen as well as liquid stabilising agent | |
| CN104198407A (en) | Detection kit for detecting content of beta-hydroxybutyrate in serum by adopting stable enzymatic method | |
| CN104714040B (en) | Glucose in serum oxidase double reagent assay method | |
| CN102507482B (en) | Detection method and reagents for quantitatively detecting 6-methyl-2-thiopyridyl-N-acetyl-beta-D-glucosaminide (MPT-NAG) | |
| Cheng et al. | A smartphone-connected point-of-care photochemical biosensor for the determination of whole blood creatinine by differential optical signal readout | |
| CN103773833A (en) | Creatinine measurement reagent | |
| CN104048928A (en) | Detection kit for direct bilirubin | |
| CN103837685B (en) | The detection method of a kind of blood glucose and detection kit | |
| CN107153044A (en) | The homocysteine kit and its detection method of a kind of modified form | |
| CN105842437A (en) | Kit for detecting D-3-hydroxybutyric acid and preparation method of kit | |
| CN104673878A (en) | Kit for measuring concentration ratio of glycated albumin and albumin by virtue of single system | |
| CN115267229A (en) | Kit and preparation method thereof | |
| CN103837487A (en) | Uric acid detection method and detection kit | |
| CN110849870A (en) | Detection reagent for N-acetyl- β -D-glucosaminidase | |
| CN106338606A (en) | Stable phospholipid oxidase method detection reagent kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 261061, Shandong, Weifang province hi tech Zone, Health East Street, south of high-tech two Road East, bio pharmaceutical science and Technology Industrial Park academic exchange center room 519 Patentee after: Weifang Ze Cheng Biotechnology Co., Ltd. Address before: 261061, Shandong, Weifang province hi tech Zone, Health East Street, south of high-tech two Road East, bio pharmaceutical science and Technology Industrial Park academic exchange center room 519 Patentee before: Weifang Xinze Biotechnology Co., Ltd. |
|
| CP01 | Change in the name or title of a patent holder |