CN105866440A - Detection method of glycosylated hemoglobin of blood - Google Patents
Detection method of glycosylated hemoglobin of blood Download PDFInfo
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- CN105866440A CN105866440A CN201610373183.0A CN201610373183A CN105866440A CN 105866440 A CN105866440 A CN 105866440A CN 201610373183 A CN201610373183 A CN 201610373183A CN 105866440 A CN105866440 A CN 105866440A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/72—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood pigments, e.g. haemoglobin, bilirubin or other porphyrins; involving occult blood
- G01N33/721—Haemoglobin
- G01N33/723—Glycosylated haemoglobin
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/795—Porphyrin- or corrin-ring-containing peptides
- G01N2333/805—Haemoglobins; Myoglobins
Abstract
The invention discloses a detection method of glycosylated hemoglobin of blood. The detection method comprises the following steps: (1) preparing an immune detection kit; (2) putting a blood sample to be detected into the immune detection kit, and reading an immune detection result a of the glycosylated hemoglobin of a sample and a corresponding standard contrast product immune detection result b of the glycosylated hemoglobin; (3) putting the same blood sample to be detected into the immune detection kit, and reading an immune detection result c of the hemoglobin of the sample and a corresponding standard contrast product immune detection result d of the hemoglobin; (4) calculating to obtain a specific value A=a/b and a specific value B=c/d; and (5) calculating content=A/B of the glycosylated hemoglobin. The detection method is high in sensitivity and strong in specificity and can be quantified; the problems that operation steps of a traditional quantitative detection method of the glycosylated hemoglobin are complicated and detection time is long are solved; and meanwhile, the problem that the qualitative detection accuracy of the glycosylated hemoglobin is low is solved.
Description
Technical field
The present invention relates to measure Blood glycated haemoglobin method and technology field, a kind of Blood glycated blood
The detection method of Lactoferrin.
Background technology
Glycolated hemoglobin (HbA1c) is that blood-glucose passes through non-enzymatic effect, in cell membrane with erythrocyte
Hemoglobin-chain a word used in person's names propylhomoserin combines the product formed, its synthesis rate and the concentration of sugar in erythrocyte local environment
It is directly proportional, is the product that in blood of human body, endoerythrocytic hemoglobin is combined with blood glucose.
It is irreversible reaction that the combination of blood glucose and hemoglobin generates glycolated hemoglobin, and becomes with blood sugar concentration
Direct ratio, and keep about 120 days.In the blood that blood glucose is the carbohydrate breakdown from food and comes
Monosaccharide, the most only refers to glucose, and blood glucose test result reflection is blood sugar level at once.So HbAle
Albumen relatively blood sugar detection more has clinical meaning, is described as " goldstandard " of diabetic condition monitoring, examines in clinic
Survey carries out the routine testing index as glycemic control.
The method that predominantly detects measuring glycolated hemoglobin the most clinically has high performance liquid chromatography electrophoresis method
Affinity chromatography etc..
Ion-exchange high-performance liquid chromatography analytic process, it is on the basis of classical liquid chromatography, has introduced gas
The theory chromatographed mutually has all advantages of gas chromatography.Need before test to use instrumentation in blood sample
Hemoglobin and glycolated hemoglobin separate, and large-scale chromatogram analysis equipment need to be used during test to detect,
Therefore detection must be carried out by special messenger in hospital, it is difficult to meet the demand that glycolated hemoglobin detects immediately.
Electrophoresis method, compared to both the non-glycated hemoglobin, the total electrical charge changed because of saccharifying and glycolated hemoglobin
Isoelectric point, IP change be agarose gel or pH gradient 5.0～6.5 isoelectrofocusing gel electrophoretic separation
Basis.The hemoglobin subfraction resolution of agarose gel electrophoresis is the least, and isoelectrofocusing can be preferably
Subfraction is made to separate.The automaticity being likely to be due to test is not enough, and importance has declined.
Affinity chromatography, after the hemoglobin in blood sample is added to chromatographic column, all of glycolated hemoglobin
(HbA1 and the hemoglobin of side chain saccharifying, total glycolated hemoglobin) is combined rather than HbAle egg with boric acid
In vain can be measured by chromatographic column.Also the polyhydroxy complex of cis-hydroxyl groups is comprised, such as in addition high concentration
After sorbitol, the combination of glycolated hemoglobin and boric acid is replaced and elutes from pillar.Affinity chromatograph
The method hemoglobin on modifying after translated and pathology hemoglobin affect relative insensitivity.Utilize affine
Chromatography, is only capable of measuring total glycolated hemoglobin.Widely used affinity chromatograph method, is the most only to use experience
Algorithm calculates " HbA1c of standard " from total saccharifying Hemoglobin Value, therefore exists and quantitatively cannot ask accurately
Topic.
Summary of the invention
The technical problem to be solved in the present invention is: overcome the deficiency in prior art, it is provided that a kind of easy and simple to handle,
The detection method of highly sensitive Blood glycated haemoglobin.
The technical solution adopted for the present invention to solve the technical problems is: the inspection of a kind of Blood glycated haemoglobin
Survey method, specifically includes following steps:
(1), immunity detection reagent is prepared, including the preparation of standard comparison product, described standard comparison
Product be not with hemoglobin, anti-hemoglobin antibodies, glycolated hemoglobin and anti-glycolated hemoglobin antibody
There is the material of immunity association reaction；
(2), blood sample to be detected is put into immunity detection reagent, using standard comparison product as reference product,
Carry out immune detection reaction with glycolated hemoglobin, read immune detection result a of sample glycolated hemoglobin
And standard comparison product immune detection result b that glycolated hemoglobin is corresponding；
(3), immunity detection reagent will be put into identical blood sample to be detected in step (2), with step
Suddenly in (2), identical standard comparison product, as reference product, carry out immune detection reaction with hemoglobin, read sample
Immune detection result c of this hemoglobin and standard comparison product immune detection result d corresponding to hemoglobin；
(4), ratio A=a/b, ratio B=c/d are calculated；
(5) content=A/B of glycolated hemoglobin, is calculated.
Further, described standard comparison product are rabbit igg, donkey IgG, horse IgG, sheep IgG, Mus IgG
One or both or two or more.
Further, described anti-hemoglobin antibodies be anti-hemoglobin monoclonal antibody and anti-hemoglobin many
One or both of clonal antibody.
Further, described anti-glycolated hemoglobin antibody is anti-glycolated hemoglobin monoclonal antibody, anti-sugar
Change hemoglobin polyclonal antibody, anti-hemoglobin monoclonal antibody, the one of anti-hemoglobin polyclonal antibody
Plant or two or more.
Further, the application in saccharification hemoglobin immune detection product development of the described detection method.
The invention has the beneficial effects as follows: the present invention is easy and simple to handle, detection highly sensitive, high specificity, and
Can be quantitative, solve traditional glycolated hemoglobin quantitative detecting method complex operation step, detect the time
Long problem, solves the problem that glycolated hemoglobin qualitative detection accuracy is low simultaneously.
Detailed description of the invention
Now preferably the present invention is further illustrated for embodiment.
Embodiment 1, the detection method of colloidal gold method detection glycolated hemoglobin is specifically as follows in step:
(1), the preparation of glycolated hemoglobin gold-immunochromatographyreagent reagent for assay box, glycolated hemoglobin gold colloidal detect
Preparation mainly glycolated hemoglobin test strips and the preparation of hemoglobin test strips of test kit, including colloid
Being coated of gold, the preparation of gold-marking binding pad and nitrocellulose membrane；
(1), the preparation of gold colloidal, comprise the following steps:
A. it is the AuCl of 0.01% by 100ml mass solubility3It is heated to boiling under solution magnetic agitation, accurately adds
Entering 1.5-3ml mass solubility is the citric acid three sodium solution of 1%, continues heating 15 minutes after colour stable,
Add pure water after cooling and be supplemented to 100ml, keep in Dark Place；
B. K is used2CO3Regulation pH to 7-9；
C. K is added2CO3Standing 5 minutes, every milliliter of gold solution adds 10-30ug hemoglobin monoclonal antibody,
It is preferably 20ug, stands 30 minutes；
D. adding quality solubility is the bovine serum albumen solution of 10%, and be added to bovine serum albumin ultimate density is always
0.1%, stand 20 minutes；
E. above-mentioned solution 12000rpm is centrifuged 50min, abandons supernatant, with redissolving the redissolution of liquid equal-volume；
(2), the preparation of gold-marking binding pad, wide including the solution after above-mentioned redissolution is uniformly added drop-wise to 6mm
On pad, 37 DEG C are dried 12 hours, finally prepare gold-marking binding pad；
(3), being coated of nitrocellulose membrane, be coated including glycolated hemoglobin ELISA test strip line, comparison line
Be coated, hemoglobin ELISA test strip line is coated and being coated of comparison line；Wherein glycolated hemoglobin examination
Paper slip detection line is coated glycolated hemoglobin monoclonal antibody, and comparison line is coated rabbit igg；Hemoglobin test strips
Detection line is coated hemoglobin polyclonal antibody, and comparison line is coated rabbit igg；Comprise the following steps:
A. hemoglobin polyclonal antibody, glycolated hemoglobin monoclonal antibody and rabbit igg antibody pH7.4
PBS dilutes, glycolated hemoglobin monoclonal, and hemoglobin monoclonal antibody concentration is 1mg/ml, and rabbit igg resists
Bulk concentration is 0.8mg/ml；
B. it is coated detection line and comparison line according to the speed of 1ul/cm；For preventing diffusion, the PVC board being coated
It is put into 37 DEG C immediately to be dried 12 hours；
(4), after above-mentioned steps all completes, respectively by the sample pad of described glycolated hemoglobin test strips,
Pad, NC film and adsorptive pads are pasted in PVC board the most successively, are cut into 4mm bar, pack sealing, room
Temperature saves backup.
(2), the detection method of glycolated hemoglobin gold-immunochromatographyreagent reagent for assay box, comprise the following steps:
(1), take blood sample to be measured, add at above-mentioned prepared glycolated hemoglobin gold-immunochromatographyreagent reagent for assay box
Sample hole adds 3～4, after 20min, puts into gold colloidal detector；Using rabbit igg as reference product, with
Glycolated hemoglobin carries out immune detection reaction, reads the immune detection line signal value a of sample glycolated hemoglobin
And the rabbit igg immunity comparison line signal value b that glycolated hemoglobin is corresponding；
(2), identical blood sample to be measured is taken, at the glycolated hemoglobin gold colloidal identical with step (1)
Detection kit well adds 3～4, after 20min, puts into gold colloidal detector；Make with rabbit igg
For reference product, carry out immune detection reaction with hemoglobin, read the immune detection line letter of sample hemoglobin
Number value c and rabbit igg immunity comparison line signal value d corresponding to hemoglobin；
(3), ratio A=a/b, ratio B=c/d are calculated；
(4) content=A/B of glycolated hemoglobin, is calculated.
The accuracy testing of colloidal gold method detection glycolated hemoglobin and replica test.
(1), accuracy testing
Take patient's anticoagulated whole blood specimen that glycolated hemoglobin is 16%, with a batch of test kit duplicate detection
3 times, calculating meansigma methods M is 15.33%, and relative deviation B value is-0.04%, meets product design requirement,
Such as table 1.
(2), replica test
Take patient's anticoagulated whole blood specimen that glycolated hemoglobin is 16%, detect with a batch of test kit simultaneously
10 times, calculating meansigma methods M is 16.40%, and standard deviation SD is 0.018, and coefficient of variation CV value is 11.21%,
Meet product design requirement, such as table 2.
Table 1 gold colloidal-accuracy testing
| Sequence number | Result |
| 1 | 15% |
| 2 | 16% |
| 3 | 15% |
| M | 15.33% |
| B | -0.04% |
Table 2 gold colloidals-replica test
| Sequence number | Result |
| 1 | 19% |
| 2 | 16% |
| 3 | 14% |
| 4 | 15% |
| 5 | 16% |
| 6 | 15% |
| 7 | 17% |
| 8 | 20% |
| 9 | 16% |
| 10 | 16% |
| M | 16.40% |
| SD | 0.018 |
| CV | 11.21% |
Embodiment 2, the detection method of fluorescent immune method detection glycolated hemoglobin is specifically as follows in step:
(1), the preparation of glycolated hemoglobin fluorescence immunoassay detection kit, comprise the following steps:
(1), the coupling of fluorescent dye and antibody, by the fluorescent dyes rhodamine of a length of for transmitted wave 665nm with
The hemoglobin monoclonal antibody mixing of l mg/ml, room temperature reaction 3h, add 1mol/L oxammonium hydrochloride. and terminate
Reaction, and isolated and purified by chromatographic column or chromatographic column, obtain the hemoglobin monoclonal anti that fluorescent dye is modified
Body, fluorescence emission wavelengths is 665nm；
(2), the structure of glycolated hemoglobin fluorescence immunoassay detection kit, comprise the steps:
A. take fluorochrome label thing, add bovine serum albumin that quality solubility is 1%, quality solubility is 10%
Sucrose and surfactant triton x-100 that quality solubility is 0.8%, even application is at labelling subsequently
On pad, 40 DEG C of dried sealings, preserve under room temperature；
B. glycolated hemoglobin fluorescence immunoassay detectable bar and hemoglobin fluorescence immunoassay detectable are assembled
Bar, is made up of sample pad, label pad, chromatographic film, adsorptive pads, is sequentially pasted on white base plate, wherein,
Sample pad is poroid barrier film, selects glass fibre, for testing sample collecting region；Pad contaminates containing fluorescence
Material DCLHb monoclonal antibody；Fix in glycolated hemoglobin fluorescence immunoassay detectable bar chromatographic film
There are quantitative glycolated hemoglobin detection line and comparison line, in hemoglobin fluorescence immunoassay detectable bar chromatographic film
Be fixed with hemoglobin quantitation detection line and comparison line, detection line and comparison line is spaced apart 5mm, and saccharifying blood
Lactoferrin detection line is fixed with glycolated hemoglobin monoclonal antibody, and hemoglobin detection line is fixed with blood red egg
White polyclonal antibody, comparison line is fixed with rabbit igg；After assembling, it is cut into required width as requested；
C. glycolated hemoglobin reagent strip is placed in buckle, adds desiccant encapsulation, with Wash buffer
Jointly it is configured to glycolated hemoglobin fluorescence immunoassay detection kit.
(2), the detection method of glycolated hemoglobin fluorescence immunoassay detection kit, comprise the following steps:
(1), blood sample to be measured is taken, in above-mentioned prepared glycolated hemoglobin fluorescence immunoassay detection kit
Well drips 80ul, forward chromatography reaction 15min respectively；
(2), preparing Wash buffer, pH value is 7.5, and buffer system is the phosphate of 20mM, adds
Quality solubility be 1% bovine serum albumin, quality solubility be 10% sucrose and quality solubility be 0.8%
Surfactant triton x-100, respectively adds 50 μ L at 2 sample wells, stands 5min；
(3) acquisition fluorescence signal intensity, it is placed in fluorescent quantitation instrument；
(4), the rabbit igg that sample glycolated hemoglobin examines side line signal value a, glycolated hemoglobin is corresponding is read
Comparison line signal value b, sample hemoglobin inspection side line signal value c and rabbit igg comparison corresponding to hemoglobin
Line signal value d；
(5), ratio A=a/b, ratio B=c/d are calculated；
(6) content=A/B of glycolated hemoglobin, is calculated.
The accuracy testing of fluorescent immune method detection glycolated hemoglobin and replica test.
(1), accuracy testing
Take patient's anticoagulated whole blood specimen that glycolated hemoglobin is 16%, with a batch of test kit duplicate detection
3 times, calculating meansigma methods M is 16.33%, and relative deviation B value is 0.02%, meets product design requirement,
Such as table 3.
(2), replica test
Take patient's anticoagulated whole blood specimen that glycolated hemoglobin is 16%, detect with a batch of test kit simultaneously
10 times, calculating meansigma methods M is 16.50%, and standard deviation SD is 0.020, and coefficient of variation CV value is 11.87%,
Meet product design requirement, such as table 4.
Table 3 fluorescence immunoassays-accuracy testing
| Sequence number | Result |
| 1 | 17% |
| 2 | 15% |
| 3 | 17% |
| M | 16.33% |
| B | 0.02% |
Table 4 fluorescence immunoassays-replica test
| Sequence number | Result |
| 1 | 17% |
| 2 | 18% |
| 3 | 14% |
| 4 | 19% |
| 5 | 17% |
| 6 | 17% |
| 7 | 14% |
| 8 | 19% |
| 9 | 14% |
| 10 | 16% |
| M | 16.50% |
| SD | 0.020 |
| CV | 11.87% |
Embodiment 3, the detection method of euzymelinked immunosorbent assay (ELISA) detection glycolated hemoglobin is specifically as follows in step:
(1), the preparation of glycolated hemoglobin enzyme-linked immunologic detecting kit, comprise the following steps:
(1), preparing the carrier being coated with standard comparison product, carrier is microwell plate, is coated carrier by following
Prepared by method: standard comparison product such as rabbit igg, with being coated buffer dilution, takes carrier to be coated, will be through dilute
The rabbit igg released is carried on carrier, after being coated end, sucks and is coated buffer, adds Block buffer,
It is placed in 37 DEG C and closes closing in 2 hours or 4 DEG C overnight；Discard confining liquid, dry 3-4 hour of room temperature 18-25 DEG C
Rear addition desiccant packs, and obtains rabbit igg and is coated carrier；Wherein be coated buffer be pH7.2-7.4,
10mmol/L phosphate solution, the diluted concentration of rabbit igg is 1ug/mL；The rabbit igg of dilution is on carrier
Package amount can be every hole 100ul；Coated condition is placed in 37 DEG C and is coated and is coated overnight for 2 hours or 4 DEG C；Envelope
Closing buffer is pH7.2-7.4,10mmol/L phosphate-Tween buffer, and including mass concentration is 1% N
Serum albumin solution and mass concentration are 1～10% sucrose；The addition of Block buffer can be every hole 300ul；
The dry environment relative humidity of carrier it is coated less than 30% after closing；
(2), preparing the carrier being coated with hemoglobin polyclonal antibody, carrier is microwell plate, is coated carrier
It is prepared by the following method: hemoglobin polyclonal antibody, with being coated buffer dilution, takes carrier to be coated,
By being carried on carrier through the hemoglobin polyclonal antibody of dilution, after being coated end, suck and be coated buffering
Liquid, adds Block buffer, is placed in 37 DEG C and closes closing in 2 hours or 4 DEG C overnight；Discard confining liquid,
Room temperature 18-25 DEG C adds desiccant and packs after dry 3-4 hour, obtain hemoglobin monoclonal antibody bag
Loaded body；Wherein being coated buffer is pH7.2-7.4,10mmol/L phosphate solution, many grams of hemoglobin
The diluted concentration of grand antibody is 1ug/mL；The hemoglobin polyclonal antibody of dilution package amount on carrier can
For every hole 100ul；Coated condition is placed in 37 DEG C and is coated and is coated overnight for 2 hours or 4 DEG C；Block buffer
For pH7.2-7.4,10mmol/L phosphate-Tween buffer, including mass concentration is 1% bovine serum albumin
Solution and mass concentration are 1～10% sucrose；The addition of Block buffer can be every hole 300ul；After closing
It is coated the dry environment relative humidity of carrier less than 30%；
(3), preparing the carrier being coated with glycolated hemoglobin monoclonal antibody, carrier is microwell plate, is coated
Carrier is prepared by the following method: glycolated hemoglobin monoclonal antibody, with being coated buffer dilution, takes and waits to wrap
Loaded body, will be carried on carrier through the glycolated hemoglobin monoclonal antibody of dilution, after being coated end,
Suck and be coated buffer, add Block buffer, be placed in 37 DEG C and close closing in 2 hours or 4 DEG C overnight；
Discarding confining liquid, room temperature 18-25 DEG C adds desiccant and packs after dry 3-4 hour, obtain HbAle
Protein monoclonal antibody is coated carrier；Wherein being coated buffer is that pH7.2-7.4,10mmol/L phosphate is molten
Liquid, the diluted concentration of glycolated hemoglobin monoclonal antibody is 1ug/mL；The glycolated hemoglobin Dan Ke of dilution
Grand antibody package amount on carrier can be every hole 100ul；Coated condition is placed in 37 DEG C and is coated 2 hours
Or 4 DEG C be coated overnight；Block buffer is pH7.2-7.4,10mmol/L phosphate-Tween buffer, interior
It is 1% bovine serum albumen solution containing mass concentration and mass concentration is 1～10% sucrose；Adding of Block buffer
Entering amount can be every hole 300ul；The dry environment relative humidity of carrier it is coated less than 30% after closing；
(4), preparation hemoglobin monoclonal antibody enzyme marker, for the enzyme of labelling be alkali phosphatase time,
The preparation method of enzymic-labelled antibody is: use Euplotes woodruffi by hemoglobin monoclonal antibody and alkalescence phosphorus
Acid enzyme coupling, fully dialyses with pH7.2,10mmol/L phosphate-Tween buffer, the combination after dialysis
Thing solution adds isopyknic glycerol, less than-20 DEG C preservations；The hemoglobin monoclonal antibody enzyme that labelling is good
Conjugate can be placed in 4 DEG C preserve to effect duration, tire by 20% hyclone diluted to working concentration
Ox blood serum diluent is pH7.4,20mmol/L phosphate-Tween buffer, and including mass concentration is 20% tire
Ox blood serum, mass concentration be 1～10% sucrose and mass concentration be 0.01% Food Red.
(2), the detection method of glycolated hemoglobin enzyme-linked immunologic detecting kit, comprise the following steps:
(1), sample process, when sample to be tested is blood plasma, take blood 2mL, 1500r/min with EDTA anticoagulant tube
Centrifugal 10 minutes, collect supernatant；When sample to be tested is serum, take blood 2mL with common tube or coagulant pipe, 4 DEG C
After lower placement 30 minutes, 1500r/min is centrifuged 10 minutes, collects supernatant；
(2), pattern detection, by sample, analysis buffer is added separately to solid phase rabbit igg, and hemoglobin is many
In the microwell plate of clonal antibody and glycolated hemoglobin monoclonal antibody, after reaction 30min, the blood in sample
Red eggs Pseudobulbus Bletillae (Rhizoma Bletillae) glycolated hemoglobin is specific binding with insolubilized antibody respectively, washes away free composition, adds enzyme mark
Note thing lucifuge reaction 30min, is formed " folder by the hemoglobin monoclonal antibody of enzyme labelling with antigen, insolubilized antibody
The heart " complex, wash away free composition, add nitrite ion, after lucifuge reaction 5min, measure each hole light absorption value；
(4), the rabbit igg that sample glycolated hemoglobin detects hole light absorption value a, glycolated hemoglobin is corresponding is read
Comparison hole light absorption value b, sample hemoglobin detection hole light absorption value c and rabbit igg comparison corresponding to hemoglobin
Hole light absorption value d；
(5), ratio A=a/b, ratio B=c/d are calculated；
(6) content=A/B of glycolated hemoglobin, is calculated.
The accuracy testing of euzymelinked immunosorbent assay (ELISA) detection glycolated hemoglobin and replica test.
(1), accuracy testing
Take patient's anticoagulated whole blood specimen that glycolated hemoglobin is 16%, with a batch of test kit duplicate detection
3 times, calculating meansigma methods M is 16.33%, and relative deviation B value is 0.02%, meets product design requirement,
Such as table 5.
(2), replica test
Take patient's anticoagulated whole blood specimen that glycolated hemoglobin is 16%, detect with a batch of test kit simultaneously
10 times, calculating meansigma methods M is 15.30%, and standard deviation SD is 0.017, and coefficient of variation CV value is 11.13%,
Meet product design requirement, such as table 6.
Table 5 enzyme linked immunologicals-accuracy testing
| Sequence number | Result |
| 1 | 18% |
| 2 | 15% |
| 3 | 16% |
| M | 16.33% |
| B | 0.02% |
Table 6 enzyme linked immunologicals-replica test
| Sequence number | Result |
| 1 | 15% |
| 2 | 14% |
| 3 | 13% |
| 4 | 15% |
| 5 | 14% |
| 6 | 16% |
| 7 | 14% |
| 8 | 18% |
| 9 | 18% |
| 10 | 16% |
| M | 15.30% |
| SD | 0.017 |
| CV | 11.13% |
Above-described embodiment only for technology design and the feature of the present invention are described, its object is to allow and is familiar with this skill
The personage of art will appreciate that present disclosure and is carried out, and can not limit the protection model of the present invention with this
Enclose, all equivalence changes made according to spirit of the invention or modification, all should contain the protection in the present invention
In the range of.
Claims (5)
1. the detection method of a Blood glycated haemoglobin, it is characterised in that: comprise the following steps:
(1), immunity detection reagent is prepared, including the preparation of standard comparison product, described standard comparison
Product be not with hemoglobin, anti-hemoglobin antibodies, glycolated hemoglobin and anti-glycolated hemoglobin antibody
There is the material of immunity association reaction；
(2), blood sample to be detected is put into immunity detection reagent, using standard comparison product as reference product,
Carry out immune detection reaction with glycolated hemoglobin, read immune detection result a of sample glycolated hemoglobin
And standard comparison product immune detection result b that glycolated hemoglobin is corresponding；
(3), immunity detection reagent will be put into identical blood sample to be detected in step (2), with step
Suddenly in (2), identical standard comparison product, as reference product, carry out immune detection reaction with hemoglobin, read sample
Immune detection result c of this hemoglobin and standard comparison product immune detection result d corresponding to hemoglobin；
(4), ratio A=a/b, ratio B=c/d are calculated；
(5) content=A/B of glycolated hemoglobin, is calculated.
The detection method of Blood glycated haemoglobin the most according to claim 1, it is characterised in that:
Described standard comparison product be rabbit igg, donkey IgG, horse IgG, sheep IgG, Mus IgG one or both or two
More than Zhong.
The detection method of Blood glycated haemoglobin the most according to claim 1, it is characterised in that:
Described anti-hemoglobin antibodies is anti-hemoglobin monoclonal antibody and the one of anti-hemoglobin polyclonal antibody
Plant or two kinds.
The detection method of Blood glycated haemoglobin the most according to claim 1, it is characterised in that:
Described anti-glycolated hemoglobin antibody is many grams of anti-glycolated hemoglobin monoclonal antibody, anti-glycolated hemoglobin
Grand antibody, anti-hemoglobin monoclonal antibody, one or both or two kinds of anti-hemoglobin polyclonal antibody
Above.
5. the detection method according to any one of claim 1-4 is opened at saccharification hemoglobin immune detection product
Application in Faing.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107490684A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin collaurum detection method |
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| CN107490684A (en) * | 2016-06-09 | 2017-12-19 | 常州博闻迪医药科技有限公司 | A kind of Blood glycated haemoglobin collaurum detection method |
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