Human Serum Albumin에 대한 단세포군 항체의 생성, 특성분석 및 microalbuminuria 검색을 위한 sandwich ELISA의 개발에 관한 연구

구자욱,고광욱,고재경 한양대학교 의과대학 1992 한양의대 학술지 Vol.12 No.1

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.

Structural insights into the binding behavior of isoflavonoid glabridin with human serum albumin

Razzak, Md. Abdur,Lee, Ji Eun,Choi, Shin Sik Elsevier 2019 Food hydrocolloids Vol.91 No.-

<P><B>Abstract</B></P> <P>Glabridin (GB), a prenylated isoflavonoid in licorice root has been used as foods and medicines with beneficial biological activities. Human serum albumin (HSA) is the most abundant blood plasma protein binding and transporting ligands such as GB. The present study investigated the interaction between GB and HSA using multi-spectroscopic and molecular docking techniques. Fluorescence spectroscopy demonstrated that GB strongly quenched the tryptophan fluorescence emission of HSA, suggesting that GB-HSA interaction followed a static mode of quenching with a binding constant (K, 10<SUP>5</SUP>-10<SUP>4</SUP> M<SUP>−1</SUP>, 25-37 °C) reflecting strong-to-moderate affinity of GB to HSA. Molecular displacement and protein-ligand docking simulations showed that the probable GB-binding positions of HSA exist near the site I of HSA. The hydrophobic interaction is a dominant binding force for forming GB-HSA complexes, and the binding resulted in conformational changes of HSA protein with more β-sheet structures. Furthermore, the complex formation of GB with HSA changed the hydrodynamic size and zeta-potential of HSA with enhanced solubility of GB. The elucidation of GB-HSA binding behavior will provide insights into the application of GB in food and pharmaceutical industries.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Binding of glabridin to human serum albumin was demonstrated by spectroscopy and molecular docking simulation. </LI> <LI> Interaction of glabridin with human serum albumin belongs to static quenching. </LI> <LI> Glabridin binds to the site I of human serum albumin through hydrophobic interactions. </LI> <LI> Glabridin binding changes the structure of human serum albumin with more β-sheets. </LI> <LI> Aqueous solubility of glabridin was enhanced by complexation with human serum albumin. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Albumin nanoscience: homing nanotechnology enabling targeted drug delivery and therapy

Shrawani Lamichhane,이상길 대한약학회 2020 Archives of Pharmacal Research Vol.43 No.1

Albumin is a biocompatible, non-immunogenicand versatile drug carrier system. It has been widely usedto extend the half-life, enhance stability, provide protectionfrom degradation and allow specifi c targeting of therapeuticagents to various disease states. Understanding the roleof albumin as a drug delivery and distribution system hasincreased remarkably in the recent years from the developmentof albumin-binding prodrugs to albumin as a drugcarrier system. The extraordinary surface property of albuminmakes it possible to bind various endogenous and exogenousmolecules. This review succinctly deals with severalalbumin-drug conjugates and nanoparticles along with theirpreparation techniques and focuses on surface-modifi edalbumin and targeting of albumin formulation to specifi corgans and tissues. It also summarizes research eff orts onalbumin nanoparticles used for delivering drugs to tumorcells and describes their role in permeation through tumorvasculature and in receptor mediated endocytosis, which isalso described in this review. The versatility of albumin andease of preparation makes it a suitable drug carrier system,swhich is the major objective of this review.

Albumin: An Emerging Opportunity in Drug Delivery

Parastou Rahimizadeh,양성태,임성인 한국생물공학회 2020 Biotechnology and Bioprocess Engineering Vol.25 No.6

Albumin, the most abundant and long-lived serum protein, exhibits novel features as a carrier that can greatly enhance the pharmacological action of therapeutic payloads. Besides passive trafficking by enhanced permeability and retention effect, albumin has been shown to accumulate within the tumor environment or inflamed tissues by receptor-mediated active transport, lending itself to being a promising scaffold for targeted drug delivery. Albumin has recently been found to be a scavenger for amyloid-β with the potential to treat neurodegenerative diseases. The hydrophobic binding pockets, conjugatable thiol residue, and surface-exposed N- and C-termini in albumin inherently serve as useful spots for carrying various kinds of peptidyl and non-peptidyl drugs. Beyond its long-standing role as a half-life extender, albumin is emerging as a versatile drug carrier to aid numerous therapeutic agents that have poor pharmacokinetics, targetability, solubility, and instability in vivo.

PEGylated human serum albumin nanocarrier for chemotherapy

김동민,정재백,임수연,김다훤,조희주,정지훈 한국공업화학회 2020 한국공업화학회 연구논문 초록집 Vol.2020 No.-

Paclitaxel(PTX) is representative chemo-drug which has high effect and low solubility. PTX needs oil/detergent-based solvent to be dissolved due to its poor solubility in an aqueous environment. However, those formulations often cause undesirable complications including hypersensitivity reactions and limited tumor distribution, resulting in a lower dose than recommended dose. Now, we introduce a facile and oil free method to prepare albumin-based PTX nanoparticles for efficient systemic cancer therapy using a conjugate of human serum albumin (HSA) and poly(ethylene glycol) (PEG). PTX were efficiently incorporated in the self-assembled PEGyl-HSA nanoparticles (PEGyl-HSA/PTX) using a simple film casting and re-hydration procedure without additional processes such as application of high pressure/shear or chemical crosslinking. The spherical PEGyl-HSA nanoparticle mediates efficient cellular delivery, leading to high cytotoxicity in various breast cancer cells.

유방암 환자에서 99mTc-Antimony Trisulfide Colloid, 99mTc-Tin Colloid, 99mTc-Human Serum Albumin을 이용한 감시림프절 매핑 성적의 비교

장성준 ( Sung June Jang ),문승환 ( Seung Hwan Moon ),김석기 ( Seok Ki Kim ),김범산 ( Bom Sahn Kim ),김석원 ( Seok Won Kim ),정기욱 ( Ki Wook Chung ),강건욱 ( Keon Wook Kang ),이은숙 ( Eun Sook Lee ) 대한핵의학회 2007 핵의학 분자영상 Vol.41 No.6

목적: 유방암 환자에게 불필요한 액와림프절 전절제술을 예방하기 위해서는 감시림프절을 절제하여 전이 여부를 평가하는 것이 중요하다. 감시림프절 매핑을 위한 방사성교질 중 antimony trisulfide colloid (ASC), tin colloid (TC), human serum albumin (HSA) 이상의 3가지 교질에 99mTc을 표지하여 각각에서 림포신티그라피, 감시림프절 매핑의 성적을 비교하였다. 대상 및 방법: 2001년 10월부터 2006년 12월까지 임상적으로 액와림프절 전이가 없는 조기 유방암 환자 총 397명에게 시행한 림포신티그라피와 수술 중 감시림프절 절제 동결절편 검사, 및 수술 후 병리 소견을 후향적으로 평가하였다. 림포신티그라피 영상 소견을 분석하고, 감시림프절의 발견율(identification rate)과 위음성율(false negative rate), 그리고 음성예측도(negative predictive value)를 구하여 각 군의 자료에 대해 Fisher 직접확률법으로 비교하였다. 결과: 202명에게는 99mTc ASC를, 120명에게는 99mTc-TC를, 75명에게는 99mTc-HSA를 사용하였으며 평균 연령, 병기, 원발 종양의 크기 등에서는 각 군별 환자들 사이에 유의한 차이가 없었다. ASC는 59명에게는 유륜부 피내 혹은 피하주사법을 사용했으며, 136명에게는 종양주위 주사를, 그리고 7명의 환자에게는 두 가지를 병용하였다. TC와 HSA를 사용한 환자들에게는 모두 피내 혹은 피하주사하였다. 액와림프절 전이는 ASC 사용 군에서 33.2%, TC 사용군에서 31.7%, HSA 사용 군에서 22.7%였으며 통계적으로 유의한 차이는 없었다. 감시 림프절 발견율(IR)과 위음성율(FNR) 그리고 음성예측도(NPV)는 사용한 교질 ASC/TC/HSA 각각에 대해 99.0%, 21.5%, 90.5% / 96.7%, 20.5%, 90.7% / 94.7%, 17.7%, 94.7%로 사용한 교질의 종류에 따라 통계적으로 유의한 차이가 없었다. 감시림프절 매핑 성적은 방사성교질의 주사방법에 따라서 유의한 차이를 보이지 않았다. 림포신티그라피의 영상에서 감시림프절이 명확히 구분된 경우는 ASC에서 79.6%, TC에서 92.5%, HSA에서 88.6%였다. 림프관이 관찰된 비율은 ASC에서 43.6%, TC에서 0.8%, HSA 에서 96.8%이었다. 림포신티그라피에서 관찰된 감시림프절의 개수는 HSA가 가장 많았지만 통계적으로 유의한 차이는 없었다. 결론: ASC, TC 및 HSA등의 방사성교질을 이용한 감시림프절 매핑 성적은 서로 유의한 차이를 보이지 않았다. Purpose: In the breast cancer patient, lymphatic mapping and sentinel lymph node biopsy are the most important procedure for axillary lymph node staging. We aimed to compare the three radiocolloids [99mTc-antimony trisulfide colloid (ASC), 99mTc-tin colloid (TC), and 99mTc-human serum albumin (HSA)] for sentinel lymph node mapping. Subjects and Methods: Totally, 397 patients with clinically N0 stage were enrolled. 99mTc-ASC was injected in 202 out of 397 patients, 99mTc-TC was injected in 120 patients, and 99mTc-HSA was injected in the remaining 75 patients. The sentinel lymph nodes were localized by lymphoscintigraphy and selected using intraoperative gamma probe. All sentinel lymph nodes were investigated by intraoperative pathologic consultation. The axillary lymph nodes which were harvested by the lymph node dissection were also investigated. Results: The patients of each group showed similar clinical characteristics. There were no significant differences (p>0.05) in the identification rate of sentinel lymph nodes (IR), false negative rate (FNR), and negative predictive value (NPV). The axillary lymphadenectomy revealed axillary lymph node metastases in those three groups (ASC-33.2%, TC-31.7%, HSA-22.7%). The IR, FNR, and NPV were not significantly different among those groups. Conclusion: Those three 99mTc-labeled radiocolloids showed equivalent results in sentinel lymph node mapping of breast cancer. (Nucl Med Mol Imaging 2007;41(6):546-552)

Drug-biomacromolecule interaction V

Kim, Chong-Kook,Ahn, Hae-Young,Han, Byung-Hoon,Hong, Soon-Keun The Pharmaceutical Society of Korea 1983 Archives of Pharmacal Research Vol.6 No.1

The binding properties of three ginsenosides, Rb$_{1}$, Rc and Re, to bovine and human serum albumins have been examined by fluorescence probe technique. 1-anilinonphathalene-8-sulfonate (ANS) was used as the fluorescence probe. Protopanaxatriol glycoside, Re, did not quench the fluorscence of ANS to the bovine serum albumin. Competitive bindings between protopanaxadiol glycosides, Rb$_{1}$ and Rc are both 3.3 . The binding constants for Rb$_{1}$ and Rc with bovine serum albumin were 1.91 * 10$_{4}$M$_{-1}$ AND 1.04 * 10$^{[-994]}$ M$^{-1}$ , respectively. The ginsenosides, Rb$_{1}$, Rc and Re did not quench the fluorescence of ANS bound to human serum albumin.

PEGylated Albumin-based nanoplatforms for cancer chemotherapy

김병덕,임수연,김다훤,박채은,황위보,( Lyu Siyan ),정지훈 한국공업화학회 2021 한국공업화학회 연구논문 초록집 Vol.2021 No.-

Human serum albumin-based calpain 2 sensor is useful for detection of calpain 2 enriched NSCLC

Min Soo Kang,Seung-Hae Kwon,Taemin Wi,Yong Il Park,Minwoo Kim,Ruda Lee 한국실험동물학회 2021 한국실험동물학회 학술발표대회 논문집 Vol.2021 No.7

Calpain 2 (CAPN2) is well known as one of the major calpain isoforms. Aberrant expression of CAPN2 is linked to angiogenesis, cell proliferation, cell migration and tumorigenesis in various cancer organs such as lung, prostate, mammary and nervous system. Non-small cell lung cancer is the leading cause of cancer-related deaths worldwide because that symptoms of lung cancer do not appear until the disease is already at an advanced stage. In present study, we devised human serum albumin bounded calpain 2 (HSA-CAPN2) by conjugating HSA and CAPN2 sensitive peptide substrate attached NIR dye and BHQ-3 to investigate potency of the HSA-CAPN2 as a sensor for detecting CAPN2 aberrant tumor. Characteristics and enzyme specificity of the HSA-CAPN2 were analyzed by transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy (FTIR), high speed liquid chromatography (HPLC) and chromatography. In vitro experiments were performed to investigate intercellular capability and signal recovery of the HSA-CAPN2 using A-549 cells. Furthermore, target desired distribution and signal recovery of HSA-CAPN2 were evaluated using non-small cell lung cancer xenograft mouse model in vivo and ex vivo. The target specific transferring ability and its function as a sensor on CAPN2 expressed tumor tissue were confirmed by NIR fluorescence analysis. The present data demonstrated the HSA-CAPN2 is a potential sensor for detection of CAPN2 enriched tumor.

사람치아 단백질을 분리 흡착한 PVDF막의 생체반응에 관한 연구

강나라(Nara Kang),홍종락(Jong-Rak Hong),정필훈(Pill-Hoon Choung) 대한구강악안면외과학회 2004 대한구강악안면외과학회지 Vol.30 No.3

Purpose: Human tooth proteins are highly heterogeneous, comprising diverse proteins derived from a number of genes. The attempts to identify protein for activity of tooth matrix proteins have been defied by several factors. First, the amount of proteins within teeth is very small relative to many extracellular matrix proteins of other tissues. Second, the bioassay system is tedious and needed for long time. Therefore we tried to find easy techniques, which increase the product rate, and an assay of small proteins, with which amino acid sequence is possible without additional procedures. Materials and Methods: Total protein were extracted from 300 g enamel removed teeth and 600 g teeth with 4 mol/L guanidine HCl and purified by gel chromatography. Aliquot of proteins was implanted into muscle pouches in Sprague-Dawley rats for bioassay. By SDS-PAGE and membrane blotting, molecular weight of each protein was estimated and a partial amino acid sequence was obtained. Each fraction blotted on the membrane was cut out and inserted in rat ectopic model. Results: In dissociative method, total tooth proteins were obtained 1mg/ml from enamel removed teeth and 3.5 mg/ml from teeth. In SDS-PAGE, four clear bands at the sites corresponding to 66, 40, 20 and 18 kD. Especially The 66 kD band was clearly exhibited. Amino acid sequencing from tooth could be possible using PVDF membrane blotting technique. In amino acid sequencing, 66 kD protein was identified as albumin. Conclusion: Compared with conventional method for extraction of teeth protein and bioassay of proteins, the methods in this study were easy, time-saving and more productive technique. The matured tooth proteins omitting additional procedure of mechanical removal of enamel were simply analyzed using blotted PVDF membrane. This method seems to make a contribution as a technique for bioassay and amino acid sequencing of protein.