CN107490680A - A kind of Blood glycated haemoglobin enzyme-linked immune detection method - Google Patents
A kind of Blood glycated haemoglobin enzyme-linked immune detection method Download PDFInfo
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Abstract
The invention discloses a kind of Blood glycated haemoglobin enzyme-linked immune detection method.This detection method, including：（1）The preparation of enzyme-linked immunologic detecting kit；（2）Blood sample to be detected is put into enzyme-linked immunologic detecting kit, reads standard comparison product immune detection result b corresponding to the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin；（3）Identical blood sample to be detected is put into enzyme-linked immunologic detecting kit, reads standard comparison product immune detection result d corresponding to the immune detection result c and hemoglobin of sample hemoglobin；（4）Ratio A=a/b, ratio B=c/d is calculated；（5）Calculate content=A/B of glycosylated hemoglobin.The high sensitivity that the present invention detects, high specificity, and can quantify, solve the problem of traditional glycosylated hemoglobin quantitative detecting method is complex for operation step, and detection time is long, while solve the problems, such as that glycosylated hemoglobin qualitative detection accuracy is low.
Description
Technical field
The present invention relates to technical field of immune assay, more particularly to a kind of Blood glycated haemoglobin enzyme-linked immune detection method.
Background technology
Glycosylated hemoglobin（HbA1c）It is that blood-glucose passes through non-enzyme effect, the product combined to form through hemoglobin-chain a word used in person's names propylhomoserin in cell membrane and red blood cell, its synthesis rate is directly proportional to concentration sugared in red blood cell local environment, is the product that endoerythrocytic hemoglobin is combined with blood glucose in blood of human body.
The combination generation glycosylated hemoglobin of blood glucose and hemoglobin is irreversible reaction, and directly proportional to blood sugar concentration, and is kept for 120 days or so.Blood glucose is the monose in the blood come from the carbohydrate breakdown in food, generally only refers to glucose, and what blood glucose test result reflected is blood sugar level at once.So glycosylated hemoglobin more has clinical meaning compared with blood sugar detection, " goldstandard " of diabetic condition monitoring is described as, index of the routine testing as glycemic control is carried out in clinical detection.
The method that predominantly detects for clinically determining glycosylated hemoglobin at present has Gao Xiao Ye Xiang Se Pu ﹑ electricity Yong Fa ﹑ affinity chromatographies etc..
Ion-exchange high-performance liquid chromatography analytic approach, it is on the basis of classical liquid chromatography, and having introduced the theory of gas chromatography has all advantages of gas chromatography.Need to separate the hemoglobin in blood sample and glycosylated hemoglobin using instrumentation before test, it need to be detected during test using large-scale chromatogram analysis equipment, therefore detection must be carried out in hospital by special messenger, it is difficult to meet the needs of glycosylated hemoglobin detects immediately.
Electrophoresis, compared to both the non-glycated hemoglobin, the isoelectric point change of the total electrical charge changed by saccharification and glycosylated hemoglobin is the basis of the isoelectrofocusing gel electrophoretic separation of Ago-Gel or pH gradient 5.0~6.5.The hemoglobin subfraction resolution ratio very little of agarose gel electrophoresis, and isoelectric focusing preferably can separate subfraction.It may decline due to the automaticity deficiency of experiment, importance.
Affinity chromatography, after the hemoglobin in blood sample is added into chromatographic column, all glycosylated hemoglobins（HbA1 and the hemoglobin of side chain saccharification；Total glycosylated hemoglobin）Combined with boric acid rather than glycosylated hemoglobin can be measured by chromatographic column.The polyhydroxy base complex of cis-hydroxyl groups is also included in addition high concentration, such as after sorbierite, the combination of glycosylated hemoglobin and boric acid is replaced and eluted from pillar.Affinity chromatography is to the translated influence relative insensitivity of the hemoglobin of modification and pathology hemoglobin later.Using affinity chromatography, it is only capable of determining total glycosylated hemoglobin.Widely used affinity chromatography method, also only it is to calculate " the HbA1c " of standard, therefore quantitatively can not be accurate the problem of be present from total saccharification Hemoglobin Value with empirical algorithms.
The content of the invention
In order to solve the above technical problems, the present invention uses Enzyme-multiplied immune technique, specific technical scheme is：
A kind of glycosylated hemoglobin enzyme-linked immune detection method, comprises the following steps：
（1）Enzyme-linked immunologic detecting kit is prepared, including the preparation of standard comparison product, the standard comparison product are the not material with hemoglobin, anti-hemoglobin antibodies, glycosylated hemoglobin and anti-glycosylated hemoglobin antibody generation immunity association reaction；
（2）Blood sample to be detected is put into enzyme-linked immunologic detecting kit, reference product is used as using standard comparison product, immune detection reaction is carried out with glycosylated hemoglobin, reads standard comparison product immune detection result b corresponding to the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin；
（3）Will be with step（2）In same blood sample to be detected be put into enzyme-linked immunologic detecting kit, with step（2）In same standard comparison product as reference product, carry out immune detection reaction with hemoglobin, read standard comparison product immune detection result d corresponding to the immune detection result c and hemoglobin of sample hemoglobin；
（4）Ratio A=a/b, ratio B=c/d is calculated；
（5）Calculate content=A/B of glycosylated hemoglobin.
Further, the detection method is raw materials used including standard comparison product, anti-hemoglobin antibodies, anti-glycosylated hemoglobin antibody, coating carrier, enzyme marker, nitrite ion, analysis buffer and concentrated cleaning solution.
Further, the standard comparison product be rabbit igg, donkey IgG, horse IgG, sheep IgG, mouse IgG it is one or two kinds of or two or more.
Further, the anti-hemoglobin antibodies are anti-hemoglobin monoclonal antibody and the one or two of anti-hemoglobin polyclonal antibody.
Further, the anti-glycosylated hemoglobin antibody be anti-glycosylated hemoglobin monoclonal antibody, anti-glycosylated hemoglobin polyclonal antibody, anti-hemoglobin monoclonal antibody, anti-hemoglobin polyclonal antibody it is one or two kinds of or two or more.
Further, the carrier is microwell plate.
Embodiment
Embodiment 1：The preparation method of glycosylated hemoglobin enzyme-linked immunologic detecting kit
The glycosylated hemoglobin enzyme-linked immunologic detecting kit preparation method comprises the following steps：
Step 1, prepare the carrier for being coated with standard comparison product
Carrier is microwell plate, and coating carrier is prepared by the following method：Standard comparison product select rabbit igg, are diluted with coating buffer solution, take carrier to be coated with, the standard comparison product by dilution are carried on carrier, after coating terminates, sucked coating buffer solution, add Block buffer, are placed in 37 DEG C of closings closing in 2 hours or 4 DEG C overnight；Confining liquid is discarded, drier is added after 18~25 DEG C of room temperature dry 3-4 hours and is packed, obtains standard comparison product coating carrier, the coating buffer solution is pH7.2~7.4,10mmol/L phosphate solutions, and the diluted concentration of standard comparison product is 1ug/mL；Package amount of the standard comparison product of dilution on carrier can be per hole 100ul；Coated condition can be placed in 37 DEG C of coatings, 2 hours or 4 DEG C coatings overnight；Block buffer is pH7.2~7.4,10mmol/L PBST solution, includes 1%BSA and 1~10% sucrose；The addition of Block buffer can be per hole 300ul；The dry environment relative humidity that carrier is coated with after closing is less than 30%；
Step 2, prepare the carrier for being coated with hemoglobin polyclonal antibody：
Carrier is microwell plate, and coating carrier is prepared by the following method：Hemoglobin polyclonal antibody is diluted with coating buffer solution, takes carrier to be coated with, and the hemoglobin polyclonal antibody by dilution is carried on carrier, after coating terminates, coating buffer solution is sucked, adds Block buffer, is placed in 37 DEG C of closings closing in 2 hours or 4 DEG C overnight；Discard confining liquid, drier is added after the drying 3~4 hours of 18-25 DEG C of room temperature to pack, hemoglobin polyclonal antibody coating carrier is obtained, the coating buffer solution is pH7.2~7.4,10mmol/L phosphate solutions, and hemoglobin polyclonal antibody I diluted concentration is 1ug/mL；Package amount of the hemoglobin polyclonal antibody of dilution on carrier can be per hole 100ul；Coated condition can be placed in 37 DEG C of coatings, 2 hours or 4 DEG C coatings overnight；Block buffer is pH7.2~7.4,10mmol/L PBST solution, includes 1%BSA and 1~10% sucrose；The addition of Block buffer can be per hole 300ul；The dry environment relative humidity that carrier is coated with after closing is less than 30%；
Step 3, prepare the carrier for being coated with glycosylated hemoglobin monoclonal antibody：
Carrier is microwell plate, and coating carrier is prepared by the following method：Glycosylated hemoglobin monoclonal antibody is diluted with coating buffer solution, takes carrier to be coated with, and the glycosylated hemoglobin monoclonal antibody by dilution is carried on carrier, after coating terminates, coating buffer solution is sucked, adds Block buffer, is placed in 37 DEG C of closings closing in 2 hours or 4 DEG C overnight；Discard confining liquid, drier is added after the drying 3~4 hours of 18-25 DEG C of room temperature to pack, glycosylated hemoglobin monoclonal antibody coating carrier is obtained, the coating buffer solution is pH7.2~7.4,10mmol/L phosphate solutions, and the diluted concentration of glycosylated hemoglobin monoclonal antibody is 1ug/mL；Package amount of the glycosylated hemoglobin monoclonal antibody of dilution on carrier can be per hole 100ul；Coated condition can be placed in 37 DEG C of coatings, 2 hours or 4 DEG C coatings overnight；Block buffer is pH7.2~7.4,10mmol/L PBST solution, includes 1%BSA and 1~10% sucrose；The addition of Block buffer can be per hole 300ul；The dry environment relative humidity that carrier is coated with after closing is less than 30%；
Step 4, prepare hemoglobin monoclonal antibody enzyme marker：
Enzyme for mark is alkaline phosphatase, and the preparation method of enzymic-labelled antibody is：Hemoglobin monoclonal antibody and alkaline phosphatase are coupled using Euplotes woodruffi, fully dialysed with pH7.2,10mmol/L PBS, isometric glycerine, less than -20 DEG C preservations are added in the conjugate solution after dialysis；The hemoglobin monoclonal antibody enzyme conjugates marked can use 20% hyclone diluted to working concentration, 4 DEG C are placed in preserve to the term of validity, hyclone dilution is pH7.4,20mmol/L PBST solution, includes 20% hyclone, 1~10% sucrose, 0.01% Food Red；
Step 5, analysis buffer is prepared, analysis buffer pH7.4,10mmol/L PBST buffer solutions, includes 1%BSA；
Step 6, concentrated cleaning solution is prepared, concentrated cleaning solution pH7.2-7.4,20mmol/L PBS, includes 1% tween；
Step 7, glycosylated hemoglobin enzyme-linked immunologic detecting kit is assembled, coating carrier, enzyme marker, nitrite ion, analysis buffer and concentrated cleaning solution are assembled into glycosylated hemoglobin enzyme-linked immunologic detecting kit.
Glycosylated hemoglobin enzyme-linked immunologic detecting kit provided by the invention and preparation method, using double-antibody sandwich reaction pattern, single-minded quantitative detection can be carried out to the glycosylated hemoglobin in human blood sample sheet.Have the advantages that high sensitivity, detection range are wide, stability is good, easy to operate quick pollution-free.The present invention is combined with other open type full automatic enzyme linked immunosorbent detection platforms, can effectively shorten detection time, reduces testing cost and manpower is spent.The product wide market of project development, economic and social benefit are notable；Compared with the methods of immunoturbidimetry, this invention simplifies operating procedure, shortens detection time, the sensitivity for substantially increasing measure and accuracy.
The hemoglobin monoclonal antibody that the present invention uses, hemoglobin polyclonal antibody and glycosylated hemoglobin monoclonal antibody have high degree of specificity, and obtained glycosylated hemoglobin enzyme-linked immunologic detecting kit and preparation method can substitute other kits to carry out the quantitative detection of glycosylated hemoglobin.
Embodiment 2：The detection mode of glycosylated hemoglobin enzyme-linked immunologic detecting kit
The concrete operation method of glycosylated hemoglobin in the detection clinical sample given below using the enzyme-linked immunologic detecting kit prepared in embodiment：
（1）, sample process, when sample to be tested is blood plasma, takes blood 2mL, 1500r/min to centrifuge 10 minutes with EDTA anticoagulant tubes, collect supernatant；When sample to be tested is serum, with common tube or promotees solidifying pipe and take blood 2mL, after being placed 30 minutes at 4 DEG C, 1500r/min is centrifuged 10 minutes, collection supernatant；
（2）, pattern detection, by sample, analysis buffer is added separately to solid phase rabbit igg, in hemoglobin polyclonal antibody and the microwell plate of glycosylated hemoglobin monoclonal antibody, after reacting 30min, hemoglobin and glycosylated hemoglobin in sample are specifically bound with insolubilized antibody respectively, wash away free composition, add enzyme marker lucifuge reaction 30min, free composition is washed away, nitrite ion is added, each hole light absorption value is determined after lucifuge reaction 5min；
（4）, read sample glycosylated hemoglobin detection hole light absorption value a, rabbit igg corresponding to glycosylated hemoglobin than rabbit igg corresponding to device to hole light absorption value b, sample hemoglobin detection hole light absorption value c and hemoglobin than device to hole light absorption value d；
（5）, ratio A=a/b, ratio B=c/d is calculated；
（6）, calculate glycosylated hemoglobin content=A/B.
Embodiment 3：Kit Performance Evaluation is tested
1. accuracy testing
Same patient's anticoagulated whole blood sample is taken, repeats detection 3 times with a batch of kit, it is 0.02% to calculate relative deviation B values, meets product design requirement；
2. replica test
Same patient's anticoagulated whole blood sample is taken, is detected 20 times simultaneously with a batch of kit, it is 11.13% to calculate coefficient of variation CV values, meets product design requirement.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, and within the spirit and principles of the invention, any modification, equivalent substitution and improvements made etc., should be included in the scope of the protection.
Claims (6)
1. a kind of glycosylated hemoglobin enzyme-linked immune detection method, comprises the following steps：
（1）Enzyme-linked immunologic detecting kit is prepared, including the preparation of standard comparison product, the standard comparison product are the not material with hemoglobin, anti-hemoglobin antibodies, glycosylated hemoglobin and anti-glycosylated hemoglobin antibody generation immunity association reaction；
（2）Blood sample to be detected is put into enzyme-linked immunologic detecting kit, reference product is used as using standard comparison product, immune detection reaction is carried out with glycosylated hemoglobin, reads standard comparison product immune detection result b corresponding to the immune detection result a and glycosylated hemoglobin of sample glycosylated hemoglobin；
（3）Will be with step（2）In same blood sample to be detected be put into enzyme-linked immunologic detecting kit, with step（2）In same standard comparison product as reference product, carry out immune detection reaction with hemoglobin, read standard comparison product immune detection result d corresponding to the immune detection result c and hemoglobin of sample hemoglobin；
（4）Ratio A=a/b, ratio B=c/d is calculated；
（5）Calculate content=A/B of glycosylated hemoglobin.
2. detection method as claimed in claim 1, it is characterised in that：The detection method is raw materials used including standard comparison product, the antibody of anti-hemoglobin, the antibody of anti-glycosylated hemoglobin, coating carrier, enzyme marker, nitrite ion, analysis buffer and concentrated cleaning solution.
3. detection method as claimed in claim 1, it is characterised in that：The standard comparison product be rabbit igg, donkey IgG, horse IgG, sheep IgG, mouse IgG it is one or two kinds of or two or more.
4. the detection method of Blood glycated haemoglobin as claimed in claim 1, it is characterised in that：The anti-hemoglobin antibodies are anti-hemoglobin monoclonal antibody and the one or two of anti-hemoglobin polyclonal antibody.
5. the detection method of Blood glycated haemoglobin as claimed in claim 1, it is characterised in that：The anti-glycosylated hemoglobin antibody be anti-glycosylated hemoglobin monoclonal antibody, anti-glycosylated hemoglobin polyclonal antibody, anti-hemoglobin monoclonal antibody, anti-hemoglobin polyclonal antibody it is one or two kinds of or two or more.
6. detection method as claimed in claim 1, it is characterised in that：The carrier is microwell plate.
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