CN110441463A - A kind of reagent of ion exchange chromatography detection Glycohemoglobin HbA1c - Google Patents

Abstract

The invention discloses a kind of reagents of ion exchange chromatography detection Glycohemoglobin HbA1c, including following solution: the main ingredient of A eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, the potassium chloride of 0.2~1g/L and the preservative of 0.02~0.4g/L, pH range is 6.1~7.1, the main ingredient of B eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, the potassium chloride of 0.2~1g/L, the sodium chloride of 1~5g/L and the preservative of 0.02~0.4g/L, and pH is 6.1~7.1.Reagent raw material sources of the present invention are easy to get extensively, and harmless, preparation process is simple, without complicated manufacture craft, finished product reagent can store under normal temperature conditions, and detecting step is convenient, reagent need to be only placed on necessary instrument, with the use of test result can be gone out.

Description

A kind of reagent of ion exchange chromatography detection Glycohemoglobin HbA1c

Technical field

The present invention relates to glycosylated hemoglobin detection technique field in medical test, specially a kind of ion exchange chromatography Detect the reagent of Glycohemoglobin HbA1c.

Background technique

Glycohemoglobin HbA1c be glucose in hemoglobin and blood in red blood cell slowly, continue and can not The product of back reaction, its concentration is relative constant, is able to reflect the nearly 2-3 monthly average blood glucose level of body currently, as international sugar The American Diabetes Association (ADA) of one of urine disease research authoritative institution is as the goldstandard of blood glucose control.It is to sugar The screening, diagnosis, observation of curative effect for urinating disease have a very important significance.

Glycosylated hemoglobin measuring method up to more than 60, is broadly divided into two major classes: the detection side 1. based on charge differences Method, including ion-exchange chromatography, efficient liquid phase chromatographic analysis and electrophoresis etc.；2. the detection method based on architectural difference, including Affinity chromatography and immunization etc..

Affinity chromatography achievees the purpose that separation determination in conjunction with the glucose specificity in HbA1c residue using it, and It is minimum by glycosylated hemoglobin variant and derivative interference, as long as but have glycosylation structure on haemoglobin molecule, can It is captured by affinity column, therefore the testing result of this method is glycosylated hemoglobin total amount, it is more higher than other methods measured value.

Immunization inhibits principle to quantitative determine HbA1c using antibody-mediated latex agglutination, but vulnerable to many homologous sugar Change the interference of haemoglobin variant, and corresponding reagent raw material needs to use the antibody antigen of animality, price is partially expensive, need to also be low It is saved under the conditions of temperature, transportation cost is also higher.

Electrophoresis is separated, electrophoresis according to the glycosylated hemoglobin difference electrically charged with both the non-glycated hemoglobin After, Hb H b each component is HbA1a, HbA1b, HbA1c, HbA respectively from cathode to anode₀.It is swept by absorbance It retouches, the percentage composition of HbA1c can be calculated.But the shortcomings that the method is need to carry out sample analysis in batch, and speed is slow, It can not be measured in real time, the degree of automation is low, and the functions such as emergency treatment insertion, automatic maintenance compare shortcoming.

Ion-exchange high-performance liquid chromatography analytic approach is to detect the goldstandard of Glycohemoglobin HbA1c, it is based on blood The electrically charged difference of institute after the saccharification of hemoglobin beta chain N-terminal valine is different from the adhesive force of cation exchange resin and establish.Cause This can be distinguished different component by HPLC method, be separated, and with detector to the various hemoglobin components after separation Absorbance detected, after analysis, various hemoglobin component results expressed as a percentage.

Reagent and its principle in the present invention is eluent and hemolytic agent with Ion-exchange high-performance liquid chromatography analytic approach Composition, wherein processing product of the hemolytic agent as sample makes the membranolysis in whole blood sample and release whole blood sample In hemoglobin, play the role of rupture of membranes, do not occur chemically react and generate new chemical substance.Cation in chromatographic column Exchanger resin is because having certain cationic polar after its activation, from glycosylated hemoglobin, the sugar of charges different in whole blood sample Change haemoglobin variant and both the non-glycated hemoglobin forms the ionic bond of certain strong and weak difference, when the elution of specific pH intensity When liquid is by the chromatographic column that is adsorbed with whole blood sample, then the glycosylated hemoglobin that ionic bond can be made weaker takes the lead in washing from chromatographic column It is de- to separate, and glycosylated hemoglobin variant and both the non-glycated hemoglobin be then because binding force is stronger, can only by pH intensity compared with High eluent elutes by several times, and therefore, the eluent in the present invention does not occur biological or chemical with whole blood sample and reacts, Only a kind of absorption, displacement, the process being detached from.

Summary of the invention

(1) the technical issues of solving

In view of the deficiencies of the prior art, the present invention provides a kind of ion exchange chromatographies to detect glycosylated hemoglobin The reagent of HbA1c solves the problems in above-mentioned background technique.

(2) technical solution

To achieve the above object, the invention provides the following technical scheme: a kind of ion exchange chromatography detects HbAle The reagent of albumen HbA1c, including following solution:

The main ingredient of A eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 0.2 The potassium chloride of~1g/L and the preservative of 0.02~0.4g/L, pH range are 6.1~7.1.

The main ingredient of B eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 0.2 The preservative of the potassium chloride of~1g/L, the sodium chloride of 1~5g/L and 0.02~0.4g/L, pH are 6.1~7.1.

The main ingredient of C eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 0.2 The potassium chloride of~1g/L, the sodium chloride of 2~10g/L, 0.02~0.4g/L preservative, pH be 5.9~6.9.

The main ingredient of D eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 10 The sodium chloride of~20g/L and the preservative of 0.02~0.4g/L, pH are 6.1~7.1.

The main component of hemolytic agent is the surfactant of the boric acid of 15~30g/L, the borax of 5~20g/L, 1~5g/L With the preservative of 0.02~0.4g/L, pH is 7.0~8.0.

It is anti-corrosion agent addition agent of the present invention that wherein present invention selection content, which is 0.01% to 99.9% methylisothiazolinone,.

The technical program is advanced optimized, the substitution ingredient of the surfactant has Triton-X100, Brj-35, gathers Ethylene glycol.

(3) beneficial effect

Compared with prior art, the present invention provides a kind of ion exchange chromatography detection Glycohemoglobin HbA1cs Reagent, have it is following the utility model has the advantages that

1, the reagent of ion exchange chromatography detection Glycohemoglobin HbA1c, reagent raw material sources are easy to get extensively, right Human body is harmless, and preparation process is simple, and without complicated manufacture craft, finished product reagent can store under normal temperature conditions.

2, the reagent of ion exchange chromatography detection Glycohemoglobin HbA1c, detecting step is convenient, only need to be by reagent It is placed on necessary instrument, with the use of test result can be gone out.

Detailed description of the invention

Fig. 1 is invention test effect figure.

Specific embodiment

Below in conjunction with the embodiment of the present invention, present invention is further described in detail:

Embodiment 1

Note: "-", which represents, is free of this ingredient；

Embodiment 2

Note: "-", which represents, is free of this ingredient；

Embodiment 3

Note: "-", which represents, is free of this ingredient；

Simultaneously based in the present embodiment 1,2,3 respectively at being grouped as and according to People's Republic of China's pharmaceuticals industry standard Repeatability, linear and accuracy clause should be met the following requirements in YY/T1246-2014 " glycolated hemoglobin analysis ":

Repeatability: the sample that measurement concentration is 4.0%~6.5%, METHOD FOR CONTINUOUS DETERMINATION 20 times, the repeated measuring results coefficient of variation CV should be not more than 3.0%；

Linear: in detection interval, the linearly dependent coefficient r of testing result should be not less than 0.9900；

Accuracy: it is measured with nominal reference substance, the abnormal reference material that can trace to the source to IFCC or NGSP, relatively partially Difference should be in ± 8% section；

Test data:

1, repeated

Embodiment 1:

Embodiment 2:

Embodiment 3:

2, linear

Embodiment 1:

Embodiment 2:

Embodiment 3:

3, accuracy

Embodiment 1:

Embodiment 2:

Embodiment 3:

As can be seen from the above results, agent prescription of the present invention is matched with existing instrument, and testing result meets Industry code requirements.

From Figure of description 1 as can be seen that agent prescription of the invention can isolate HbA1a, HbA1b, HbA1c, HbA0 component, the testing result of Glycohemoglobin HbA1c have 3 kinds of representation methods, American National glycosylated hemoglobin standard meter Draw (NGSP) unit (%) and international clinical chemistry association (IFCC) unit (mmol/mol) and average blood sugar (eAG) unit (mmol/L)。

Meanwhile being found after a large amount of selection test, existing preservative is being used for liquid chromatography color on the market at present It all can be to microballoon filler inside liquid chromatography chromatography column and chromatographic column when composing the elution reagent or other matched reagents of column Service life and performance have an immense impact on, and performance is to make the filler nigrescence of liquid chromatography chromatographic column and block microballoon inside chromatographic column Gap between filler, causes that the pressure of entire liquid chromatography chromatographic system quickly increases or filler is displaced to form chromatographic column gap Shape, so that original service life of chromatographic column and the decline of filling adsorption ability, and use methylisothiazolinone as liquid chromatogram layer Problems are just not present in the eluent or matched reagent for analysing column, similar with the antiseptic property of Sodium azide, select new preservative The main reason for be because Sodium azide belongs to and meets water and generate extremely toxic substance, and in strenuous vibration and 40 DEG C under dry powder Above environment is easy to happen explosion, be easy to cause environmental pollution and personal injury.

Technical characteristic of the present invention without description can realize that details are not described herein by or using the prior art, certainly, Above-mentioned specific embodiment is not limitation of the present invention, and the present invention is also not limited to above-mentioned specific embodiment, this skill The variations, modifications, additions or substitutions that the those of ordinary skill in art field is made within the essential scope of the present invention should also be fallen into In protection scope of the present invention.

Claims (2)

1. a kind of reagent of ion exchange chromatography detection Glycohemoglobin HbA1c, it is characterised in that: including following solution:

The main ingredient of A eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 0.2~1g/ The potassium chloride of L and the preservative of 0.02~0.4g/L, pH range are 6.1~7.1；

The main ingredient of B eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 0.2~1g/ The preservative of the potassium chloride of L, the sodium chloride of 1~5g/L and 0.02~0.4g/L, pH are 6.1~7.1；

The main ingredient of C eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 0.2~1g/ The potassium chloride of L, the sodium chloride of 2~10g/L, 0.02~0.4g/L preservative, pH be 5.9~6.9；

The main ingredient of D eluent is the disodium hydrogen phosphate of 0.2~1g/L, the sodium dihydrogen phosphate of 0.5~1.5g/L, 10~20g/ The sodium chloride of L and the preservative of 0.02~0.4g/L, pH are 6.1~7.1；

The main component of hemolytic agent be the boric acid of 15~30g/L, the borax of 5~20g/L, 1~5g/L surfactant and The preservative of 0.02~0.4g/L, pH are 7.0~8.0.

It is anti-corrosion agent addition agent of the present invention that wherein present invention selection content, which is 0.01% to 99.9% methylisothiazolinone,.

2. a kind of reagent of ion exchange chromatography detection Glycohemoglobin HbA1c according to claim 1, feature Be: the substitution ingredient of the surfactant has Triton-X100, Brj-35, polyethylene glycol.