Method for measuring glycated hemoglobin

Abstract: A method for measuring glycated hemoglobin is disclosed, which comprises contacting a sample with an anti-hemoglobin antibody being bound to a solid phase, adding thereto an anti-glycated hemoglobin antibody, and then measuring the anti-glycated hemoglobin antibody bound to the solid phase via glycated hemoglobin and the anti-hemoglobin antibody. A method for measuring glycated hemoglobin is further disclosed, which comprises mixing a sample with particles on which an anti-glycated hemoglobin antibody is bound to cause immunoagglutination reaction, and then measuring agglutinations formed by the immunoagglutination reaction. A method for measuring glycated hemoglobin is furthermore disclosed, which comprises mixing a sample with a solution containing a monoclonal antibody specific for glycated hemoglobin, and then measuring the change in the turbidity of the resulting mixture. There is still disclosed a method for measuring glycated hemoglobin which comprises mixing a sample with unsensitized latex particles and an anti-glycated hemoglobin antibody, or with unsensitized latex particles and subsequently with an anti-glycated hemoglobin antibody, to cause immunoagglutination reaction, and then measuring agglutinations formed by the immunoagglutination reaction. There is still further disclosed a method for measuring glycated hemoglobin which comprises mixing a sample with unsensitized latex particles and particles on which an anti-glycated hemoglobin antibody is bound, or with unsensitized latex particles and subsequently with particles on which an anti-glycated hemoglobin antibody is bound, to cause immunoagglutination reaction, and then measuring agglutinations formed by the immunoagglutination reaction. Still furthermore, an anti-glycated hemoglobin monoclonal antibody having a high specificity for human glycated hemoglobin is disclosed.

Description: FIELD OF THE INVENTION This invention relates to a method for measuring glycated hemoglobin. BACKGROUND OF THE INVENTION Since it has been known that the concentration of glycated hemoglobin (A 1c ) in blood increases in the case of diabetic patients, measurement of glycated hemoglobin is used for the diagnosis of diabetes and examination of the progress of the disease, together with the measurement of blood sugar level. In the prior art method, measurement of glycated hemoglobin is carried out by binding glycated hemoglobin in a sample (erythrocyte extract) directly to a solid phase, washing the solid phase, allowing the bound hemoglobin to react with a labeled anti-glycated hemoglobin antibody, washing the solid phase again and then measuring levels of the label. This method, however, requires a time-consuming pretreatment for the direct binding of glycated hemoglobin in each sample to the solid phase (for example, an overnight incubation at 4°C), thus rendering quick measurement of a large number of samples impossible. EP 201 187 discloses an assay in which an anti-glycated haemoglobin antibody is used to detect haemoglobin A 1c . EP 444 787 discloses an assay for measuring non-A 1c glycated haemoglobin. In view of the above, it therefore becomes an object of the present invention to provide a method for the simple and quick measurement of glycated hemoglobin. SUMMARY OF THE INVENTION The inventors of the present invention have conducted intensive studies and found that the time-consuming step involved in the prior art method, namely a step for the binding of glycated hemoglobin in each sample to a solid phase, can be eliminated and therefore glycated hemoglobin can be measured quickly, when an anti-hemoglobin antibody bound to a solid phase in advance is allowed to react with a sample and the reaction product is subsequently allowed to react with an anti-glycated hemoglobin antibody. The present invention has been accomplished on the basis this finding. Particularly, in accordance with the present invention, there is provided a method for measuring glycated hemoglobin which comprises contacting a sample with an anti-hemoglobin antibody being bound to a solid phase, adding thereto an anti-glycated hemoglobin antibody, and then measuring the anti-glycated hemoglobin antibody bound to the solid phase via glycated hemoglobin and the anti-hemoglobin antibody. Other objects and advantages of the present invention will be made apparent as the description progresses. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing effects of the anti-human glycated hemoglobin monoclonal antibody prepared in Example 1 and a commercially available anti-human glycated hemoglobin antibody (Chemicon International Inc. (U.S.A.)) on the measurement of samples containing human glycated hemoglobin at various concentrations. The concentration of the crude peroxidase-labeled glycated hemoglobin solution is referred to as 100 (▪ : Example 2 ; ▴ : Comparative Example 1). DETAILED DESCRIPTION OF THE INVENTION The present invention is described below in further detail. According to the present invention, there is provided a method for measuring the ratio of glycated hemoglobin Δ 1c to total hemoglobin in a sample which comprises contacting a sample, which contains or does not contain glycated hemoglobin A 1c , with an anti-hemoglobin antibody being bound to a solid phase to react the glycated hemoglobin A 1c with the anti-hemoglobin antibody, adding thereto an anti-glycated hemoglobin A 1c antibody to react the anti-glycated hemoglobin A 1c antibody with the glycated hemoglobin A 1c , and then measuring said anti-glycated hemoglobin A 1c antibody being bound to said solid phase via said glycated hemoglobin A 1c and said anti-hemoglobin antibody and determining said ratio. According to the present invention, when a large quantity of a solid phase material to which an anti-hemoglobin antibody is bound is prepared in advance, glycated hemoglobin in a sample can be bound to the solid phase via the anti-hemoglobin antibody in each measurement, by allowing the sample and the anti-hemoglobin antibody being bound to the solid phase to undergo antigen-antibody reaction. In comparison with the prior art process in which glycated hemoglobin is directly bound to a solid phase, this method can be completed within a considerably shortened period of time by a factor of 1/10 or less. The solid phase to be used in the method of the present invention may be any of those used in conventional art immunoassays. For example, wells of a plastic plate may preferably be used. The anti-hemoglobin antibody is well known in the art, which may be either a polyclonal antibody or a monoclonal antibody. Binding of the anti-hemoglobin antibody to a solid phase may be effected by adding a solution of the antibody having a concentration of about 1 µg/ml to the solid phase and allowing the resulting solid phase to stand an overnight at 4°C. After the binding treatment, blocking with a protein such as BSA (bovine serum albumin) is carried out in the usual way in order to block non-specific protein adsorption sites. When human glycated hemoglobin is measured, it is preferred to bind an anti-human hemoglobin antibody to the solid phase. Next, the anti-hemoglobin antibody thus bound to the solid phase is allowed to contact with a sample to effect binding of glycated hemoglobin in the sample to the solid phase via the solid phase-bound anti-hemoglobin antibody by antigen-antibody reaction. The sample may be either erythrocyte extracts or total blood. The antigen-antibody reaction may be carried out, for example, at room temperature for about 2 hours. After washing, the glycated hemoglobin bound to the solid phase via the anti-hemoglobin antibody is allowed to undergo antigen-antibody reaction with an anti-glycated hemoglobin antibody. The anti-glycated hemoglobin antibody may be either a polyclonal antibody or a monoclonal antibody, but preferably a monoclonal antibody form the viewpoint of high measuring accuracy. The present inventors have succeeded in preparing a monoclonal antibody which is capable of undergoing specific reaction with glycated hemoglobin but not substantially with normal hemoglobin (monoclonal antibody 3F10; a hybridoma capable of producing monoclonal antibody 3F10 has been deposited on June 10, 1992 at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry of Japan, 1-3, Higashi 1-chome, Tsukubashi, Ibaraki, 305 Japan under accession No. FERM P-12998 (FERM BP-4311 under the Budapest Treaty), in accordance with a procedure which will be described later in detail in Example. This monoclonal antibody may be used preferably in the methods of the present invention. The antigen-antibody reaction of this case may be carried out under the same conditions as described above. After subsequent washing, the anti-glycated hemoglobin antibody thus bound to the solid phase via the glycated hemoglobin and the anti-hemoglobin antibody is measured in accordance with various prior art techniques known in the field of immunoassays. For example, the measurement may be effected by labeling the anti-glycated hemoglobin antibody with an enzyme, a fluorescence dye, a radioactive material or the like and then measuring the label. It may be effected also by binding biotin to the anti-glycated hemoglobin antibody, allowing the bound biotin to react with labeled avidin and then measuring the label. The measurement may also be effected by allowing the anti-glycated hemoglobin antibody to react with a labeled antibody which can undergo specific reaction with the anti-glycated hemoglobin antibody and then measuring the label. Specific examples of the animal erythrocytes include ones obtained from animals such as fowls, ducks, goats, sheep, cattle and horses. The following examples are provided to further illustrate the present invention. It is to be understood, however, that the examples are for purpose of illustration only and are not intended as a definition of the limits of the present invention. EXAMPLE 1 (1) Preparation of monoclonal antibody specific for glycated hemoglobin: Glycated hemoglobin was thoroughly dispersed in Freund's complete adjuvant, and mice of Balb/c line were immunized with 100 µl of the suspension four times at intervals of two weeks. The spleen was excised from each of the immunized mice to obtain 10 6 spleen cells which were subsequently fused with mouse myeloma cells in the presence of PEG. After culturing the fused cells, the resulting culture supernatant was checked for the presence of an anti-human glycated hemoglobin antibody by ELISA method. Thereafter, positive cells for the antibody were checked by the limiting dilution method to isolate anti-human glycated hemoglobin antibody-producing cells. The thus obtained cells were used as anti-human glycated hemoglobin mouse monoclonal antibody-producing cells (hybridoma cells) and cultured in a large scale, and the cultured cells were administered to mice by intraperitoneal injection. After 2 weeks of the administration, the ascitic fluid was collected at intervals of 3 days to obtain the anti-human glycated hemoglobin monoclonal antibody of interest. The thus obtained anti-human glycated hemoglobin monoclonal antibody was named 3F10, and a hybridoma capable of producing 3F10 has been deposited at National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, Ministry of International Trade and Industry of Japan under accession No. FERM P-12998 (FERM BP-4311 under the Budapest Treaty). It was confirmed that the monoclonal antibody 3F10 is IgG. (2) Measurement of human glycated hemoglobin by ELISA: A 10 µg/ml solution of an anti-human hemoglobin antibody (rabbit antibody) was dispensed in 50 µl portions into wells of a 96 well plate for ELISA use (available from Nunc) and allowed to stand overnight at 4°C. The resulting plate was washed five times with a phosphate-buffered physiological saline containing 2 % by weight BSA and 0.02 % by weight Tween® 20 and then stored at 4°C after distributing the same buffer in 200 µl portions. After removing the supernatant fluid, 150 µl of a sample containing glycated hemoglobin was added dropwise to each well of the plate and allowed to stand for 2 hours at room temperature. The thus treated plate was then washed four times with physiological saline containing 0.02 % by weight Tween® 20. Next, a peroxidase-labeled anti-human glycated hemoglobin-specific mouse monoclonal antibody solution (500 ng/ml) was distributed in 150 µl portions into wells of the above plate and allowed to stand for 2 hours at room temperature. After washing the plate four times with the physiological saline containing 0.02 % by weight Tween® 20, a 0.05 % by weight H 2 O 2 solution containing 0.1 % by weight ABTS (2,2'-azobis(3-ethylbenzothiazoline-6-sulfonic acid)) was distributed in 200 µl portions into wells of the thus washed plate and allowed to stand for 30 minutes at room temperature to effect color development. Thereafter, the thus developed color was measured colorimetrically by measuring absorbances at 415 nm and 492 nm. The results are shown in Table 1. Ratio of glycated hemoglobin to total hemoglobin (%) Ratio of Absorbance at 415 to Absorbance at 492 0 0.06 1 0.10 3 0.18 6 0.29 9 0.47 12 0.61 15 0.80 18 1.00 COMPARATIVE EXAMPLE 2 Using the anti-glycated human hemoglobin monoclonal antibody prepared in Example 1 and a commercially available anti-human glycated hemoglobin monoclonal antibody, glycated human hemoglobin contained in samples in varied amounts of was quantitatively determined in the following manner. The anti-glycated human hemoglobin monoclonal antibody prepared in Example 1 or a commercially available anti-human glycated hemoglobin monoclonal antibody (Chemicon International Inc. (U.S.A.)) was subjected to sensitization onto a 96 well microplate. To the resulting microplate was added peroxidase (POD)-labeled glycated hemoglobin antigen by varying the added amount (concentration). After carrying out the immune reaction at room temperature for 2 hours, the plate was washed with PBS-Tween®. Thereafter, ABTS/H 2 O 2 (substrate) was distributed in 100 µl portions into wells of the thus treated plate, and the reaction was proceeded out at room temperature for 1 hour. After adding 50 µl of a reaction termination solution, absorbance at a wave length of 415 nm was measured, with the results shown in Fig. 1. As is evident from Fig. 1, the commercially available anti-human glycated hemoglobin antibody can hardly respond to the increasing amount of the antigen (Comparative Example 1), while the absorbance increases in proportion to the amount of the antigen when the anti-glycated human hemoglobin monoclonal antibody prepared in Example 1 is used (Example 2). In consequence, it was confirmed that glycated hemoglobin can be measured over a broad range of concentration by the use of the anti-glycated human hemoglobin monoclonal antibody prepared in Example 1. Thus, it is apparent that there has been provided, in accordance with the present invention, a simple and quick measuring method of glycated hemoglobin.

Claims: A method for measuring the ratio of glycated hemoglobin A 1c to total hemoglobin in a sample which comprises contacting a sample with an anti-hemoglobin antibody being bound to a solid phase, adding thereto a solution of anti-glycated hemoglobin A 1c antibody, and then measuring said anti-glycated hemoglobin A 1c antibody bound to said solid phase via said glycated hemoglobin A 1c and said anti-hemoglobin antibody and determining said ratio. A method according to Claim 1, wherein said anti-glycated hemoglobin A 1c antibody is a labeled anti-glycated hemoglobin A 1c antibody. A method according to Claim 2, wherein the label of said labeled anti-glycated hemoglobin A 1c antibody is an enzyme.

Inventors: KURANO YOSHIHIRO, SUGIYAMA MASAMI, TANAKA AIKO, TANIMOTO TETSUJI, UCHIDA YOSHIAKI, KURANO, YOSHIHIRO, SUGIYAMA, MASAMI, TANAKA, AIKO, TANIMOTO, TETSUJI, UCHIDA, YOSHIAKI
Applicants: FUJIREBIO KK, FUJIREBIO INC.
Application year: 1993
Main classification: G01N33
IPC codes: C07K16