The affinity sorbent for the determination of glycated hemoglobin

 

(57) Abstract:

The affinity sorbent for the determination of glycated hemoglobin contains porous silica gel modified with glyceryltrinitrate brand SP-500, activated with epichlorohydrin, the General formula

The invention enables the obtaining of a sorbent with high selectivity.

The invention relates to the chemistry of polymers and can be used in medicine for the diagnosis of diabetes, namely for affinity determination of glycosylated hemoglobin.

Known sorbent similar purpose - agarose with immobilized meta-aminophenylarsonic acid (m-AFBC), which is used for determination of glycated hemoglobin (Catalogue company Glico-Gel Pierce Chemical Company, 1986, p.3-7). The capacity of agarose on the ligand is 5-15 μm in 1 ml of sorbent.

The disadvantage of the sorbent on the basis of agarose is that the rate of use of one portion of the sorbent is limited to 3-6 cycles of reliable quantitative determination of glycated hemoglobin. It's an expensive sorbent.

Known sorbent similar purpose - linked polyvinyl alcohol (medium) with immobility is the 2093525, IPC 08 F 30/06, publ. 20.10.97 year). The capacity of agarose on the ligand is 5-15 μm in 1 ml of sorbent.

The disadvantage of the sorbent on the basis of polyvinyl alcohol should also be considered insufficient number of reliable definitions of ghb on one portion of the sorbent (20-30).

Closest to the proposed chemical composition of the sorbent is silica containing grafted group m-aminophenylarsonic acid having the structural formula

(EN 2040963, IPC B 01 J 20/10 from 08.09.1995 year).

The disadvantage of this sorbent is a high value for nonspecific binding of proteins and glycoproteins that make it inefficient when used as a sorbent for affinity determination of glycosylated hemoglobin.

The objective of the invention is improving the efficiency of the sorbent by increasing the selectivity of binding of glycoproteins.

The problem is solved by affinity sorbent for the determination of glycated hemoglobin based on the silica containing immobilized meta-aminophenylarsonic acid, which in contrast to the prototype as the carrier is used porous silica gel, modified g CLASS="ptx2">

Introduction in accordance with the declared entity as spacer (bridge) between the silica gel and m-AFBC of glycerylphosphorylcholine and epichlorohydrin increases the hydrophilicity of the sorbent, resulting in reduced non-specific adsorption of glycoproteins that increases the selectivity of the determination of glycosylated hemoglobin.

The affinity sorbent for the determination of glycated hemoglobin was obtained as follows.

1) Activation of the epichlorohydrin porous silica gel modified by glycerylphosphorylcholine

2) the Reaction of binding of the activated carrier with m-AFBC

Confirmation of the occurrence of a chemical reaction between the ligand (m-ABFC) and a carrier, activated epichlorohydrin, is the iodometric titration of the aldehyde groups of the carrier, resulting after its oxidation by periodate sodium before and after planting m-AFBC: the number of aldehyde groups after binding m-AFBC 14, 2±0,6% less than the number obtained for the unmodified media.

According to the claimed to receive sorbent used silica gel firm "Serva" brand SP-500, modified glycerylphosphorylcholine silica gel with the General formula SiO2·H2O, permeated by pores with an average diameter of 500 angstroms. The surface of the particles modified by glycerylphosphorylcholine due to the reaction between the silanol groups and the surface silanol groups of the silica gel. The result of this moditication the surface of the carrier loses sorption capacity and acquires a surface hydroxyl group.

The specified connection is activated with epichlorohydrin under alkaline conditions for the introduction of the surface active oxirane groups. Then there was the binding of m-AFBC under alkaline conditions due to the reaction of the amino group m-AFBC with oxirane groups associated with the surface of the sorbent. Unreacted oxirane group was removed by reaction with monoethanolamine.

Elemental analysis of 30 samples obtained sorbent showed that the boron content in it is 0,18±0,04%, which confirms its high specific capacity.

The essence of the claimed invention is confirmed by the examples.

Example 1

To 50 ml of silica gel firm "Serva" brand SP-500-modified glycerylphosphorylcholine, suspended in 50 ml of distilled water was added to 10 ml of epichlorohydrin and 20 ml of 2M NaOH was dissolved in the tech and 100 volumes of distilled water and 0.1 M carbonate buffer binding pH of 9.5, then was added m-AFBC rate of 20 mg/ml of medium and shaken at room temperature for 18 hours. Washed from excess m-AFBC sorbent used for the quantitative determination of ghb. According to the elemental analysis of the sorbent contains 0.18±0.6% of boron. In the prototype, the content of boron on the results of the elemental analysis is 0.12%.

Example 2

For determination of glycated hemoglobin obtained sorbent was placed in a column (1 ml) and was washed with the starting buffer (0.025 M sodium phosphate buffer, pH 8.5). The hemolysate was prepared by adding one volume of washed erythrocytes three volumes of 0.02% sodium azide. The column was applied 0.1 ml of hemolysate. To associate a sample current buffer was stopped for 15 minutes and washed the column with the starting buffer for collecting replicationmanager hemoglobin. Glycosylated hemoglobin was suirable buffer containing 200 mm sorbitol, pH 8.5. The obtained fractions 1 and 2 were photometrically at 414 nm. The content of glycated hemoglobin was determined by the formula

where OP(1) and OP(2) is the optical density at 414 nm of fractions 1 and 2, respectively;

V1 and V2 is the volume of fractions 1 and 2 ml.

The multiplicity accurate definition wide-angle is) proved the reduction in the number of bound hemoglobin in samples probably due to the partial destruction of the sorbent and the associated capacity decrease.

Example 3

For determination of glycated hemoglobin obtained sorbent was placed in a column (1 ml) and was washed with the starting buffer (0.025 M sodium phosphate buffer, pH 8.5). The hemolysate was prepared by adding one volume of washed erythrocytes three volumes of 0.02% sodium azide. In the hemolysate was performed determining the amount of glycated hemoglobin in the chromatograph AUTO A1 STMA-8110 (Kyoto, Called Co, Japan). The result of this definition is considered the true value of the glycosylated forms in the sample. The column with the test sorbent was applied 0.1 ml of hemolysate. To associate a sample current buffer was stopped for 15 minutes and washed the column with the starting buffer for collecting replicationmanager hemoglobin. Glycosylated hemoglobin was suirable buffer containing 200 mm sorbitol, pH 8.5. The obtained fractions 1 and 2 were photometrically at 414 nm. The content of glycated hemoglobin was determined by the formula

The multiplicity of reliable definitions (which gives a result different from the result obtained using chromatograph, not more than 5%) on the received surrealist linked hemoglobin in samples probably due to the partial destruction of the sorbent and the associated capacity decrease.

Example 4

Determination of ghb was carried out similarly to example 3 with the difference that instead of the proposed sorbent used sorbent prepared according to the prototype. The amount of hemoglobin determined in the samples at 90±15% exceeded the amount of glycosylated hemoglobin defined on the chromatograph AUTO A1 STMA-8110 (Kyoto, Called Co, Japan), which suggests that the use of this sorbent for the quantitative determination of glycated form of hemoglobin leads to unreliable results.

Thus, the claimed sorbent based on porous silica gel modified by glycerylphosphorylcholine and epichlorohydrin, with immobilized meta-aminophenylarsonic acid provides a 45-fold determination of ghb with high accuracy far exceeding the accuracy of its setting when using sorbent of the prototype.

The affinity sorbent for the determination of glycated hemoglobin based on the silica containing immobilized meaningfulbeauty acid, characterized in that as the carrier is used porous silica gel, modified g is

 

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