CN1858239A - Method for producing glucose-6-phosphate dehydrogenase detecting reagent - Google Patents
Method for producing glucose-6-phosphate dehydrogenase detecting reagent Download PDFInfo
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- CN1858239A CN1858239A CN 200610034659 CN200610034659A CN1858239A CN 1858239 A CN1858239 A CN 1858239A CN 200610034659 CN200610034659 CN 200610034659 CN 200610034659 A CN200610034659 A CN 200610034659A CN 1858239 A CN1858239 A CN 1858239A
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Abstract
The method of making glucose-6-phosphate dehydrogenase (G6PD) detecting reagent belongs to the field of biochemistry and detection reagent. The present invention detects enzyme activity directly with packed red blood cells without the influence of the patient's anemia degree and the interference of the patient's G6PD and has raised G6PD heterozygote detecting rate. The G6PD detecting reagent consists of reagent I and reagent II. The reagent I consists of magnesium chloride 0.2-2.0%, disodium EDTA 0.02-0.2%, oxide type coenzyme II 0.02-0.2%, trisodium hexaphosphate gluconate 0.01-0.1%, trihydrox7ymethyl aminomethane 1.5-2.0%, hydrochloric acid 0.8-2.0%, emulsifier 0.2-0.5%, and distilled water 97.25-93%. The reagent II contains disodium hexaphosphate gluconate, rather than trisodium hexaphosphate gluconate, 0.2-0.5%. The reagent I and the reagent II are colorless transparent liquid and are packed separately.
Description
[technical field]
The present invention relates to a kind of glucose six phosphate dehydrogenases (G6PD) and measure the manufacture method of reagent, belong to biological chemistry, zymetology and check diagnostic reagent field.
[background technology]
Glucose six phosphate dehydrogenases (G6PD) are the key enzymes of red corpuscle phosphopentose pathways metabolism, it is the unique channel that red corpuscle produces DPNH I (NADPH), reduced coenzyme (NADPH) is the coenzyme of glutathione reductase, and reduced glutathion (GSH) is the prerequisite that keeps the integrity of erythrocyte membrane.The shortage of glucose six phosphate dehydrogenases (G6PD) causes reduced coenzyme (NADPH) to generate minimizing; causing reduced glutathion (GSH) to generate indirectly reduces; make erythrocyte membrane lose the protection of sulfydryl and the impaired generation haemolysis of function, cause unconjugated bilirubin to raise indirectly.In the medical diagnosis process, glucose six phosphate dehydrogenases (G6PD) activity in the check patient red corpuscle, can judge the haemolysis that hemolytic anemia and men and women are pre-marital and women's prenatal care causes with the shortage of preventing heredity glucose six phosphate dehydrogenases (G6PD), this kind check also is the important medical science index of pedicterus classification.The reagent product operation of checking glucose six phosphate dehydrogenases (G6PD) in the market is not suitable for automatic clinical chemistry analyzer and uses than more complicated; Six PDGs (6PGD) detect glucose six phosphate dehydrogenases (G6PD) to rate method just to be had and disturbs, be a stubborn problem in the clinical chemistry technology, the diagnosis of lacking patient's heterozygote for clinical glucose six phosphate dehydrogenases (G6PD) brings very big difficulty always.Do not see that both at home and abroad having the author to report gets rid of this interferential method.The present invention is in order to fill a hole in the market, and exploitation adapts to the automatic clinical chemistry analyzer schedule of operation, utilizes the blank method of sample speed to get rid of the interference of 6PGD, develops the present invention.
[summary of the invention]
The purpose of this invention is to provide a kind of glucose six phosphate dehydrogenases (G6PD) and measure the manufacture method of reagent.The present invention need not measure hemoglobin concentration in checkout procedure; Without the normal saline solution Washed Red Blood Cells, directly measure the activity of enzyme with packed red cells, can not be subjected to the influence of patient's severity of anemia; In testing process, the 6PGD that can get rid of patient itself simultaneously disturbs, and improves the recall rate of heterozygote; Store down at 2～8 ℃ under the reagent liquid state, the steady quality time is long.
(1) inventive principle: technical solution scheme of the present invention be such according to principle: glucose six phosphoric acid (G6P) in glucose six phosphate dehydrogenases (G6PD) catalytic reagent in the test sample generate six phosphogluconates (6PG), simultaneously with the oxidized form of nicotinamide-adenine dinucleotide I (NADP in the reagent
+) be converted into DPNH I (NADPH).Because oxidized form of nicotinamide-adenine dinucleotide I (NADP
+) be reduced into deoxidizing type enzyme II (NADPH), thus the absorbancy at 340nm place in the instrument is risen.By the speed that monitoring 340nm place absorbancy rises, can calculate the activity of glucose six phosphate dehydrogenases in the sample.Owing to contain six PDGs (6PGD) in the sample, it can generate five ribulose monophosphates by catalysis six phosphogluconates (6PG), simultaneously also NADP
+Be reduced into NADPH, absorbancy is risen, make the false rising of detected result.The present invention adopts two kinds of reagent, and promptly Double bottom thing six phosphogluconates (6PG) that contain in reagent (1) and the reagent (2) and glucose six phosphoric acid (G6P) act on the rate variation that is produced behind the detected sample simultaneously and deduct independent use six phosphogluconates (6PG) rate variation that is produced and obtain the activity of real glucose six phosphate dehydrogenases.Its reaction process is in following:
First reaction is the first step, is second step together with second reaction, and Biochemical Analyzer can deduct the rate of change of the first step reaction automatically after finishing second reaction, read measured result.
(2) combination material and weight percentage
The present invention is made up of three kinds of reagent: reagent 1, reagent 2, reagent 3,
1, the constituent of reagent 1 and weight percentage:
Magnesium chloride (MgCl
2) 0.2%～2.0%
Disodium ethylene diamine tetraacetate (EDTA) 0.02%～0.2%
Oxidized form of nicotinamide-adenine dinucleotide I (NADP
+) 0.02%～0.2%
Six phosphogluconate trisodiums (GPG) 0.01%～0.1%
Tutofusin tris (Tris) 1.5%～2.0%
Hydrochloric acid (HCl) 0.8%～2.0%
Emulsifying agent (OP) 0.2%～0.5%
Distilled water (H
2O) 97.25%～93%
2, the constituent of reagent 2 and weight percentage:
Magnesium chloride (MgCl
2) 0.2%～2.0%
Disodium ethylene diamine tetraacetate (EDTA) 0.02%～0.2%
Oxidized form of nicotinamide-adenine dinucleotide I (NADP
+) 0.02%～0.2%
Glucose six Di-Sodium Phosphates (G6P) 0.2%～0.5%
Tutofusin tris (Tris) 1.5%～2.0%
Hydrochloric acid (HCl) 0.8%～2.0%
Emulsifying agent (OP) 0.2%～0.5%
Distilled water (H
2O) 97.06%～92.6%
3, reagent 3 is distilled water.
(3) material molecule formula, structural formula, character and the place of production:
(1) magnesium chloride molecular formula MgCl
2, 6H
2O
The deliquescent branch crystal of white has bitter saline taste, soluble in water and ethanol.Strong electrolyte.
The place of production: Tianjin good fortune chemical reagent in morning factory
(2) disodium ethylene diamine tetraacetate
Structural formula:
Character: the colourless crystallization solid, water-soluble, complexing agent.Also as toxinicide.
The place of production: Guangdong, Taishan City, Guangdong Province emigrant's reagent plastics company limited
(3) oxidized form of nicotinamide-adenine dinucleotide I (NADP
+)
Title: nicotinamide adenine dinucleotide phosphoric acid
Structural formula:
Character: white powder, water-soluble
The place of production: the Sigma company U.S. (the precious fine horse in middle mountain company buys on behalf)
(4) six phosphogluconate trisodiums (6PG)
Molecular formula: C
6H
10Na
3O
10P
Structural formula:
Character: white powder, water-soluble
The place of production: the Sigma company U.S. (the precious fine horse in middle mountain company buys on behalf)
(5) Tutofusin tris (Tris)
Structural formula:
Character: the white crystalline solid, water-soluble
The place of production: Finland, chemical company limited before tall building (the precious fine horse in middle mountain company buys on behalf)
(6) hydrochloric acid (HCl)
Character: strong acid
The place of production: Guangzhou Chemical Reagent Factory
(7) emulsifying agent (OP)
Character: colourless to the yellowish brown thick liquid, water-soluble, pure equal solvent.
The place of production: Shantou City, Guangdong Province brilliance laboratory
(8) glucose six Di-Sodium Phosphates (G6P)
Molecular formula: C
6H
11O
9PNa
2
Structural formula:
Character: white powder, water-soluble
The place of production: the Sigma company U.S. (the precious fine horse in middle mountain company buys on behalf)
(4) Pei Zhi technological process:
(1) reagent preparation 1: at first add the Tutofusin tris (Tris) of weighing in the Glass Containers of cleaning, add the ionogen magnesium chloride (MgCl of weighing
2), the complexing agent (EDTA) of adding weighing, continuation adds the oxidized form of nicotinamide-adenine dinucleotide I (NADP) of weighing, add six phosphogluconate trisodiums (6PG) of weighing, add hydrochloric acid (HCl) solution again, make and regulate PH to certain value, agitation and filtration adds distilled water again to a constant volume then;
(2) reagent preparation 2: identical with the technological process of reagent preparation (1), and just do not add six phosphogluconate trisodiums (6PG), and add glucose six Di-Sodium Phosphates (G6P), add distilled water at last to a constant volume.
(3) reagent preparation 3: prepare purified distilled water with distillation plant, cool off standby.
(4) with the above-mentioned reagent can branch of preparing to bottle, use to make medical inspection; Reagent 1 can 45ml * 1 is bottle standby, reagent 2 can 7ml * 1 are bottle standby; Reagent 3 distilled water can 125ml * 2 bottles are standby.
(5) require the operating process of the above-mentioned three kinds of reagent of preparation all in sterile workshop, to carry out; Three kinds of reagent are colourless transparent liquid, 2～8 ℃ of preservations.
(5) advantage of the present invention: (1) various producer reagent performance and reagent performance comparison sheet of the present invention
| Producer | Taiwan unit gives birth to | Britain's Landau | Guangzhou rice base | This reagent |
| Principle | Can't get rid of the interference of 6PG | Can't get rid of the interference of 6PG | Can't get rid of the interference of 6PG | Can get rid of the interference of 6PG |
| Method | Speed A method | Speed A method | The ratio end-point method | The blank method of sample speed |
| Structure | Dry powder | Dry powder | Liquid | Liquid |
| Purposes | The diagnosis favism | The diagnosis favism | The diagnosis favism | The diagnosis favism |
| Advantage | Shelf time is long | Shelf time is long | Do not measure hemoglobin concentration without centrifugation | Shelf time reaches 1 year, and biliquid needn't be prepared direct use, does not measure hemoglobin concentration |
| Shortcoming | Need measure hemoglobin concentration simultaneously | Need measure hemoglobin concentration simultaneously | Manual operations is numerous and diverse fully, and the operating time is long, and the reagent preparation back shelf time is very short, special lower, poor repeatability | Require to use packed red cells |
| Sample pre-treatments | With physiological saline Washed Red Blood Cells three times | With physiological saline Washed Red Blood Cells three times | Need not | Need not |
| The heterozygote recall rate | Low | Low | Than higher | The highest |
| Automatic degree | Can only operate by hand | Relatively good | Relatively poor, the time is long, needs 30 minutes go out the result, can only do in batches, can not be with to doing | Be fit to automatic clinical chemistry analyzer and use, patient is with to doing, and goes out the result behind the suction sample in 10 minutes, can be with to doing |
(2) this reagent be used to measure G6PD have easy and simple to handle, specificity good, highly sensitive, be not subjected to 6PGD to influence, be applicable to that the full-automatic biochemical analyzer uses.
[description of drawings]
Further understand manufacture method of the present invention below in conjunction with description of drawings and embodiment.
Accompanying drawing 1 is a manufacturing process flow diagram of the present invention;
Accompanying drawing 2 is the can assembly drawing of three kinds of reagent of the present invention.
[embodiment]
Adopt three kinds of reagent of the present invention to carry out to glucose six phosphate dehydrogenase detection by quantitative.
It is 1 * 45ml that volume is used in reagent 1 bottling;
It is 1 * 7ml that volume is used in reagent 2 bottlings;
It is 2 * 125ml that volume is used in reagent 3 bottlings.
This reagent can be used for the automatic clinical chemistry analyzer analysis to be used, and reagent 1, reagent 2 can not be mixed into single reagent and use, and adds reagent 1 in preceding 5 minutes and adds reagent 2 in back 5 minutes but divide in same reaction cup; The method that this reagent uses is sample blank rate method or speed B method, and is different fully with speed A method or end-point method that other reagent uses.
It is as follows that this invention reagent detects step to the medical staff:
1, sample is prepared.Extract clinical blood sample, with heparin, ACD Precerving liquid or EDTA anti-freezing, the time to be checked is no more than 24 hours; Get 1ml and draw in the packed red cells layer 20ul adding small test tube with sample injector after centrifugal 5 minutes with 4000r/min through the whole blood of anti-freezing, add R3 reagent 1ml again, mixing treats that red corpuscle dissolves mensuration in back 30 minutes fully.
2, the concrete parameter setting of location parameter is adjusted with reference to its specification sheets by the different model biochemical instruments
| Parameter name | Requirement | Parameter name | Requirement |
| Measure temperature | 37℃ | The R1 consumption | 1.1ml |
| Commplementary wave length/predominant wavelength | 405nm/340nm | The R2 consumption | 0.125ml |
| Colorimetric is calculated the cup optical path | 1.0cm | Amount of samples | 0.05ml |
| Measuring method | Connect speed monitoring method (positive reaction) | Time of lag | 200s |
| Minute | 180s |
3, application of sample step
| Admixture | Blank pipe (B) | Sample hose (s) |
| Reagent 1 (ml) | 1.1 | 1.1 |
| Distilled water (ml) | 0.125 | - |
| Sample (ml) | 0.05 | 0.05 |
| Mixing, 37 ℃ water-soluble 3 minutes | ||
| Reagent 2 (ml) | - | 0.125 |
| Mixing, 37 ℃ postpone to read first absorbance after 20 seconds, read 1 time every 1 minute, read 3 minutes altogether, and calculate average per minute absorbancy velocity of variation Δ A/min. | ||
4, calculation formula
In the formula:
The absorbancy velocity of variation of the average per minute of Δ AS/min sample hose
The absorbancy velocity of variation of the average per minute of Δ AB/min sample blank pipe
TV total reaction volume (ml)
The mmole molecular extinction coefficient of ε NADPH at the 340nm place is 6.22
SV sample volume (ml)
The P cuvette calculates optical path (cm), and exhausted big portion automatic clinical chemistry analyzer is 1
1000 changed factors
51 diluted sample multiples
5, term of reference
Adult's packed red cells G6PD activity＞1300U/L; Newborn infant or Cord blood packed red cells G6PD activity＞2500U/L.
6, specifically detect example
(1) XXX hospital inspection report (biochemical miscellaneous)
Name: the refined levelling of Lin is other: woman's age: 56 catalogue number(Cat.No.)s: 1 prints:
Department: bed label: patient ID: diagnosis:
| Project | The prompting result | Unit | Reference value |
| Sample classification: packed red cells glucose six phosphate dehydrogenases (G6PD) | 1496 | U/L | 1300～5300 |
Receive inspection: the 2006-02-27 censorship: the XXX report: 2006-02-27 00:24 check: XXX audit: XXX
(2) XXX hospital inspection report (biochemical miscellaneous)
The graceful sex of name: Huang Yi: woman's age: 5 catalogue number(Cat.No.)s: 2 print:
Department: bed label: patient ID: judgement:
| Project | The prompting result | Unit | Reference value |
| Sample classification: packed red cells glucose six phosphate dehydrogenases (G6PD) | 3600 | U/L | 1300～5300 |
Receive inspection: the 2006-02-27 censorship: the XXX report: 2006-02-27 00:24 check: XXX audit: XXX
(3) XXX hospital inspection report (biochemical miscellaneous)
Name: yellow Xiao Fang's sex: woman's age: catalogue number(Cat.No.): 3 print:
Department: bed label: patient ID: diagnosis:
| Project | The prompting result | Unit | Reference value |
| Sample classification: packed red cells glucose six phosphate dehydrogenases (G6PD) | 2612 | U/L | 1300～5300 |
Receive inspection: the 2006-02-27 censorship: the XXX report: 2006-02-27 00:24 check: XXX audit: XXX
(4) XXX hospital inspection report (biochemical miscellaneous)
Name: Yang Li duckweed sex: woman's age: 56 catalogue number(Cat.No.)s: 4 print:
Department: bed label: patient ID: diagnosis:
| Project | The prompting result | Unit | Reference value |
| Sample classification: packed red cells glucose six phosphate dehydrogenases (G6PD) | L 1496 | U/L | 1300～5300 |
Receive inspection: the 2006-02-27 censorship: the XXX report: 2006-02-27 00:24 check: XXX audit: XXX
(5) XXX hospital inspection report (biochemical miscellaneous)
Name: the blue or green sex of remy hair: man's age: 56 catalogue number(Cat.No.)s: 1 prints:
Department: bed label: patient ID: diagnosis:
| Project | The prompting result | Unit | Reference value |
| Sample classification: packed red cells glucose six phosphate dehydrogenases (G6PD) | L 56 | U/L | 2500～6600 |
Receive inspection: the 2006-02-27 censorship: the XXX report: 2006-02-27 00:24 check: XXX audit: XXX
Preliminary judge: from above concrete detection example, have three tested persons to think the normal people, a severe lacks G6PD (56U/L), and a patient is G6PD moderate shortage (765U/L).
Can tentatively be judged as " favism " patient.
Conclusion: this reagent system adopts Double bottom thing (G6P and 6PG), measures the activity (this method is not disturbed by the activity of G6PD, detects the activity of real G6PD) of G6PD with blank method of the sample speed of Biochemical Analyzer or speed B method.The at present domestic sale of not seeing active reagent report of the packed red cells G6PD that the mensuration certain volume is arranged and reagent product, therefore, the proposition of this preparation method of reagent thereof is forward-looking.
Claims (3)
1, a kind of glucose six phosphate dehydrogenases (G6PD) are measured the manufacture method of reagent, and it comprises distilled water reagent (3), it is characterized in that being made up of reagent (1) and reagent (2):
(1) composition and the weight percent of reagent (1) are as follows:
Magnesium chloride (MgCl
2) 0.2%～2.0%
Disodium ethylene diamine tetraacetate (EDTA) 0.02%～0.2%
Oxidized form of nicotinamide-adenine dinucleotide I (NADP
+) 0.02%～0.2%
Six phosphogluconate trisodiums (GPG) 0.01%～0.1%
Tutofusin tris (Tris) 1.5%～2.0%
Hydrochloric acid (HCl) 0.8%2.0%
Emulsifying agent (OP) 0.2%～0.5%
Distilled water (H
2O) 97.25%～93%
(2) composition and the weight percent of reagent (2) are as follows:
Magnesium chloride (MgCl
2) 0.2%～2.0%
Disodium ethylene diamine tetraacetate (EDTA) 0.02%～0.2%
Oxidized form of nicotinamide-adenine dinucleotide I (NADP
+) 0.02%～0.2%
Glucose six Di-Sodium Phosphates (G6P) 0.2%～0.5%
Tutofusin tris (Tris) 1.5%～2.0%
Hydrochloric acid (HCl) 0.8%～2.0%
Emulsifying agent (OP) 0.2%～0.5%
Distilled water (H
2O) 97.06%～92.6%
(3) Pei Zhi technological process is as follows:
Reagent preparation (1):
The Tutofusin tris (Tris) that in the Glass Containers of cleaning, at first adds weighing, the ionogen magnesium chloride (MgCl of adding weighing
2), the complexing agent (EDTA) of adding weighing, continuation adds the oxidized form of nicotinamide-adenine dinucleotide I (NADP) of weighing, add six phosphogluconate trisodiums (6PG) of weighing, add hydrochloric acid (HCl) solution again, make and regulate PH to certain value, agitation and filtration adds distilled water again to a constant volume then;
Reagent preparation (2):
Reagent preparation (2) is identical with the technological process of reagent preparation (1), does not just add six phosphogluconate trisodiums (6PG), and adds glucose six Di-Sodium Phosphates (G6P), adds distilled water at last to a constant volume.
Reagent preparation (3)
The distilled water of in the Glass Containers of cleaning, packing into and making.
2, a kind of glucose six phosphate dehydrogenases according to claim 1 (G6PD) are measured the manufacture method of reagent, it is characterized in that reagent (1) can 45ml * 1 bottle standby; Reagent (2) can 7ml * 1 bottle is standby; Reagent (3) distilled water can 125ml * 2 bottles is standby.
3, a kind of glucose six phosphate dehydrogenases according to claim 1 and 2 (G6PD) are measured the manufacture method of reagent, it is characterized in that the reagent preparation process all carries out in sterile workshop; Reagent (1), reagent (2) and reagent (3) are colourless transparent liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610034659 CN1858239A (en) | 2006-03-24 | 2006-03-24 | Method for producing glucose-6-phosphate dehydrogenase detecting reagent |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610034659 CN1858239A (en) | 2006-03-24 | 2006-03-24 | Method for producing glucose-6-phosphate dehydrogenase detecting reagent |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1858239A true CN1858239A (en) | 2006-11-08 |
Family
ID=37297197
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610034659 Pending CN1858239A (en) | 2006-03-24 | 2006-03-24 | Method for producing glucose-6-phosphate dehydrogenase detecting reagent |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1858239A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108893521A (en) * | 2018-07-12 | 2018-11-27 | 广州市米基医疗器械有限公司 | Detect the kit and detection method of Red Blood Cells Glucose -6- phosphate dehydrogenase |
| CN111060503A (en) * | 2020-01-02 | 2020-04-24 | 青岛汉唐生物科技有限公司 | G-6-PD detection kit free from anemia and detection method thereof |
-
2006
- 2006-03-24 CN CN 200610034659 patent/CN1858239A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108893521A (en) * | 2018-07-12 | 2018-11-27 | 广州市米基医疗器械有限公司 | Detect the kit and detection method of Red Blood Cells Glucose -6- phosphate dehydrogenase |
| CN111060503A (en) * | 2020-01-02 | 2020-04-24 | 青岛汉唐生物科技有限公司 | G-6-PD detection kit free from anemia and detection method thereof |
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