Human Serum Albumin에 대한 단세포군 항체의 생성, 특성분석 및 microalbuminuria 검색을 위한 sandwich ELISA의 개발에 관한 연구 = Production and Characterization of Monoclonal Antibodies against Human Serum Albumin and Development of Sandwich ELISA for Detection of Microalbuminuria in Diabetes Mellitus

다국어 초록 (Multilingual Abstract)

Alumin of a variety of species has been the subject of extensive studies by protein chemists and immunologists for over 3 deades. Human serum albumin (HSA) is the most abundant protein in plasma. It is a nonglycosylated protein consisting of a single polypeptide chain of 585 amino acids with 34 cystive residues. The molecule contains 9 serial double loops formed by 17 disulfide bridges arranged as 3 repeat units or domains.
Since polyclonal antibodies bind to almost all antigenic determinants of antigen, the experiments base4d on polyclonal antibodies yielded frequently low-titre, of broad specificity, and are not completely reproducible, although the heterogeneity of fine specificity could be minimized by affinity chromatography on fragments of the immunogens. Recently, the development of hybridoma techniques has made it possible to produce a large amount of homogenous and specific anti-protein antibody which reacts with a single epitope of antigen.
Diabetic nephropathy is the major cause of the increased mortality in insulin-dependent diabetes mellitus (IDDM), affecting over 40% of these patients.
Recent studies have shown that an increased urinary albumin excretion rate (microalbuminuria) in IDDM patients is a good predictor of the development of diabetic nephropathy. The possibility that therapeutic intervention instituted at this early stage of the disease might reverse, arrest or at least slow its progression to late and irreversible stages promotes the necessity to develop a screening test for microalbuminuria.
Microalbuminuria can be measured by radioimmunoassay(RIA), nephelometric laser, immunoturbidimetry and enzyme linked immunosorbent assay(ELISA). However, RIA has some disadvanges such as isotope-related health hazards, and expense of equipment used to measure gamma-emitting isotope.
The aims in this study were to produce monoclonal antibodies against HSA and to develop a simple, rapid, and sensitive sandwich ELISA using anti-human albumin monoclonal antibodies for detecting microalbuminuria.
Four monoclonal antibodies(MHA-1, MHA-2, MHA-3, and MHA-4) which reacted selectively against HSA were produced by hybridoma technology. Isotype of each monoclonal antibody was found to be IgG₁.
All monoclonal antibodies except MHA-1 were species-specific which were demonstrated on ELISA and immunoblotting. Since MHA-4 reacted with albumin in only nonreducting condition, it was suggested that this antibody recognized the conformational epitope of the albumin. Utilizing ingibition ELISA, it was speculated that these four antibodies recognized respectively 4 different epitopes of albumin and among those epitopes, epitopes of MHA-2 and MHA-3 same or located very approximately.
Sandwich ELISA using two monoclonal antibodies(MHA-4 as capture antibody and MHA-2 as detector antibody), which reacted with different epitopes, were developed to detect microalbuminuria. Dynamic range of this assay was from 30 to 5000㎍/1 and its lowere limit of detection was 30㎍/1, corresponding to 1.5ng of albumin per well. The intra-assay and the inter-assay coefficient of variation were 3.3-4.0% and 6.8-8.0%, respectively. Analytical recovery ranged from 92 to 103%. Concentrations of 84 urine samples were measured by the sandwich ELISA and competitive RIA. Correlation between two assays was good(r=0.983, p<0.005) over a range of albumin concentration tested.
Albumon excretions in 24 hour urine samples from non-insulin-dependent diabetes mellitus(NIDDM) patients (mean 16.7mg/day, n=39, 8yr duration) were significantly increased (t=2.53, p=0.015) as compared with those from healthy subjects(mean 5.6mg/day).
However, 24 hour urine albumin excretions in IDDM patient(mean 11.1mg/day, n=25, 6.8yr duration) werer not significantly different from those in normal control(t=1.16, p=0.26). The 24 hour urine albumin(mg/day) correlated well with the urine albumin/creatinine ratio(r=0.816, p<0.005) on log data.
This study indicates that this newly established sandwich ELISA is found to have good analytical characteristics, outstanding in its specificty, sensitivity, accuracy, and precision, and is suitable and easily accessable for detection of microalbuminuria in clinical medicine.