OBJECTIVE CONCLUSIONS ABSTRACT Establishment of ELISA for AgI/II protein detection using monoclonal anti-AgI/II antibody Jae-Gon Kim, jeong-Yeol Park,

Slides:

Advertisements

Similar presentations

General Microbiology Lecture Twelve Identification of Bacteria

Advertisements

Enzyme Linked Immunosorbent Assay

Monoclonal Antibodies and Cancer Therapy. Definition: Mono: One Clone: A strain of cells descended form a single cell Antibody: A molecule of animal.

Western Analysis Laboratory procedure that allows you to:

One of the most useful ways to test a humoral (antibody) response is to inoculate (immunize) an animal with an antigen (foreign substance) and then measure.

Altogen Labs, 4020 S Industrial Dr, Suite 130, Austin TX 78744, USA  ELIS A Enzyme-Linked Immunosorbent Assay ELISA.

Enzyme-Linked Immunosorbent Assay ELISA 1Dr. Nikhat Siddiq.

(Enzyme Linked Immunosorbent Assay)

Enzyme-Linked Immunosorbent Assay

Expression, Purification and Isolation of the MinE protein By Arsalan Wasim and Nicholas Wong.

Chapter 20 Experimental Systems Dr. Capers.  In vivo ○ Involve whole animal  In vitro ○ Defined populations of immune cells are studied under controlled.

WHAT IS A WESTERN BLOT?.

Expression and Localization of a Hypothetical Protein of Trypanosoma brucei Savannah Mwesigwa 1, Monica Namayanja 1, Phillip Magambo 1, Sylvester Ochwo.

Results 1.Miller JR, Siripurkpong P, Hawes J, Majdalawieh A, Ro HS, McLeod RS. The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin.

CHOIRUNIL CHOTIMAH INTRODUCTION Leishmaniasis Leishmania Parasites promastigotes amastigotes Difficult to treat Need effective vaccine.

Western Blot Megan Sova Stacie Huffmon Beth Meyer.

Basic Parts Update Made 7 more parts since last week: cel9A, gliadin scFV, TevC, cel3A, OprF AtD, cl02365 AtD, Hia AtD Available passengers –Gliadin scFv,

TREATMENT TRAINING RESEARCH DEVELOPMENT OF CHIKEN YOLK IMMUNOGLOBULIN (IGY) TO SOME BACTERIAL AND BIOTOXIN AGENTS Colonel A/Prof. Le Van Dong et al., Vietnam.

Lecturer: David. * Reverse transcription PCR * Used to detect RNA levels * RNA is converted to cDNA by reverse transcriptase * Then it is amplified.

Dengue fever caused by dengue virus (DENV), a member of Flaviviridae leads to large global disease burden. Detection of immunoglobulin M (IgM) and nucleic.

Kaitlyn Zakuciya; Dr. Chad Sethman PhD.; Waynesburg University Department of Biology, Waynesburg PA The Effect of Artificial Sweeteners on Streptococcus.

Figure S1 A MVNFVSAGLFRCLPVSCPEDLLVEELVDGLLSLEEELKDKEEEEAVLDGL LSLEEESRGRLRRGPPGEKAPPRGETHRDRQRRAEEKRKRKKEREKE EEKQTAEYLKRKEEEKARRRRRAEKKAADVARRKQEEQERRERKWRQ.

Telephone    Provider of Global Contract Research Services Accelerating Preclinical Research, Drug Discovery.

The ELISA Enzyme-Linked ImmunoSorbent Assay A highly specific and sensitive procedure allowing evaluation of multiple agents for a single serum dilution.

Figure S1. Production of recombinant NS1 protein

Enzyme-linked immunosorbent assay (ELISA)

GENE EXPRESSION STUDY ON PROTEIN LEVEL

Enzyme Linked Immunosorbent Assay

Haemagglutination assay

Development of Med28 Specific Monoclonal Antibodies

Volume 63, Issue 2, Pages (February 2003)

Figure 3. Crude N-acetylneuraminic acid obtained from glycomacropeptide (G-NANA) inhibits the growth curve of H. felis. H. felis was grown overnight in.

Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor Runping Gao, David R. Brigstock

Bid, a Bcl2 Interacting Protein, Mediates Cytochrome c Release from Mitochondria in Response to Activation of Cell Surface Death Receptors Xu Luo, Imawati.

Volume 66, Issue 4, Pages (October 2004)

Macrophage-Derived Metalloelastase Is Responsible for the Generation of Angiostatin in Lewis Lung Carcinoma Zhongyun Dong, Rakesh Kumar, Xiulan Yang,

Regulation of monocyte migration by amphoterin (HMGB1)‏

Purusharth Rajyaguru, Meipei She, Roy Parker Molecular Cell

Sphingosylphosphorylcholine is a Potent Inducer of Intercellular Adhesion Molecule-1 Expression in Human Keratinocytes Genji Imokawa, Yutaka Takagi,

Overexpression of Laminin-8 in Human Dermal Microvascular Endothelial Cells Promotes Angiogenesis-Related Functions Jie Li, Lisa Zhou, Hoang T. Tran,

by Quanzhi Li, Joy L. Frestedt, and John H. Kersey

Specific Lysis of Melanoma Cells by Receptor Grafted T Cells is Enhanced by Anti- Idiotypic Monoclonal Antibodies Directed to the scFv Domain of the Receptor

Generation and Characterization of a Polyclonal Antipeptide Antibody to Human Glycodelin Archna S Poddar, Jong G Kim, Kiran P Gill, Barry N Bates, Nalini.

Human Keratinocytes Express Functional CD14 and Toll-Like Receptor 4

Aquaporin 3 Colocates with Phospholipase D2 in Caveolin-Rich Membrane Microdomains and Is Downregulated Upon Keratinocyte Differentiation Xiangjian Zheng,

17β-estradiol, Progesterone, and Dihydrotestosterone Suppress the Growth of Human Melanoma by Inhibiting Interleukin-8 Production Naoko Kanda, Shinichi.

Volume 63, Issue 2, Pages (February 2003)

Cell-Density-Dependent Regulation of Expression and Glycosylation of Dopachrome Tautomerase/Tyrosinase-Related Protein-2 Thomas J. Hornyak, Daniel J.

Gangliosides GD1b, GT1b, and GQ1b Suppress the Growth of Human Melanoma by Inhibiting Interleukin-8 Production: the Inhibition of Adenylate Cyclase1

Volume 70, Issue 7, Pages (October 2006)

1,25-dihydroxyvitamin D3 inhibits renal interstitial myofibroblast activation by inducing hepatocyte growth factor expression Yingjian Li, Bradley C.

Volume 62, Issue 3, Pages (September 2002)

Yang Shen, Monica Naujokas, Morag Park, Keith Ireton Cell

Makiko Iguchi, Setsuya Aiba, Yumiko Yoshino, Hachiro Tagami

Volume 56, Issue 1, Pages (July 1999)

Connective Tissue Growth Factor (CCN2) in Rat Pancreatic Stellate Cell Function: Integrin α5β1 as a Novel CCN2 Receptor Runping Gao, David R. Brigstock

The PbGAPDH G6 fragment interacts with recombinant CD68.

Volume 86, Issue 1, Pages (July 1996)

Signaling Events During Induction of Plasminogen Activator Inhibitor-1 Expression by Sphingosylphosphorylcholine in Cultured Human Dermal Fibroblasts

Transforming Growth Factor β1 Induces Apoptosis in Normal Melanocytes but not in Nevus Cells Grown in Type I Collagen Gel Tuomo Alanko Journal of Investigative.

Differential Roles of Insulin Receptor and Insulin-Like Growth Factor-1 Receptor in Differentiation of Murine Skin Keratinocytes Efrat Wertheimer, Meirav.

IL-12 affects Dermatophagoides farinae–induced IL-4 production by T cells from pediatric patients with mite-sensitive asthma Takeshi Noma, MD, PhD, Izumi.

Severity and Phenotype of Bullous Pemphigoid Relate to Autoantibody Profile Against the NH2- and COOH-Terminal Regions of the BP180 Ectodomain Silke.

Volume 10, Issue 12, Pages (June 2000)

Relationship Between Cell-Associated Matrix Metalloproteinase 9 and Psoriatic Keratinocyte Growth Nathalie Buisson-Legendre, Hervé Emonard, Philippe.

Sequential E2s Drive Polyubiquitin Chain Assembly on APC Targets

Envoplakin and Periplakin, the Paraneoplastic Pemphigus Antigens, are also Recognized by Pemphigus Foliaceus Autoantibodies Shideh Kazerounian, Mỹ G.

Histone H4 Is a Major Component of the Antimicrobial Action of Human Sebocytes Dong-Youn Lee, Chun-Ming Huang, Teruaki Nakatsuji, Diane Thiboutot, Sun-Ah.

Nitrogen Regulates AMPK to Control TORC1 Signaling

Presentation transcript:

OBJECTIVE CONCLUSIONS ABSTRACT Establishment of ELISA for AgI/II protein detection using monoclonal anti-AgI/II antibody Jae-Gon Kim, jeong-Yeol Park, Yeon-Mi Yang, Byeong-Ju Baik School of Dentistry, Chonbuk National University, JeonJu, Korea MATERIALS AND METHODS RESULTS Bacterial strains and culture conditions Serotype C S. mutans GS-5 was grown in BHI medium at 37 o C in an anaerobic chamber for 24 h then transferred into 5 ml of a fresh BHI broth. The optical density was measured at 540nm.. Streptococcus mutans has been known as a causative agent for dental caries. As well as acid production yielding the deminerali- zation of tooth enamel, adherence and colonization of S. mutans to the teeth are also important for their virulence. S. mutans attaches the dental pellicle by an adhesion from surface protein called as Antigen I/II. A secretory IgA for intact AgI/II or its saliva- ry-protein-binding segment has been found to block adherence of S. mutans. Therefore, it will be of most importance to detect the level of AgI/II molecules in saliva regarding dental caries and other systemic disease. Through the expression pattern of AgI/II of S. mutans GS-5, we suggest that AgI/II of S. mutans GS-5 file up in the culture media time dependently. We tested the reaction of between recombinant AgI/II-N, or native AgI/II and monoclonal anti-AgI/II antibody by ELISA. As a result, our monoclonal anti- AgI/II antibody can detect 1ng recombinant AgI/II-N and predict amount of the native AgI/II. In conclusion, through ELISA against AgI/II using our monoclonal anti-AgI/II antibody, we can detect the AgI/II in native condition. Also, we thought that analysis of secretory AgI/II can be utilized for prediction of disease associated S. mutans. The aim of this study was to evaluate the establishment of secretory AgI/II by ELISA using monoclonal anti-AgI/II antibody for prediction of dental caries. For the purpose, the expression pattern of AgI/II protein during the growth of S. mutans GS-5 was investigated, the optimal condition for AgI/II detection was searched using recombinant AgI/II protein and this ELISA condition was tested whether AgI/II protein in the supernatant of the bacterial culture was detected. We confirmed the presence of native AgI/II piling up in culture media. We established ELISA condition against recombinant AgI/II and native AgI/II We detected the 1 ng of recombinant AgI/II at 5 min. We detected native AgI/II in culture media containing others. Generation of monoclonal anti-AgI/II antibodies ELISA The 96 well plate were coated with 1, 10, 50, or 100 ng recombinant AgI/II-N, culture supernatant with or without concentrated by 50% ammonium sulfate at 4 o C, and blocked with 1% skim milk. Monoclonal anti-AgI/II-N antibody (1:100, 1:1000, 1:10000, 1:50000) was added in duplicate. After washing with PBS, AgI/II-N-specific antibody was detected using anti-mouse IgG conjugated alkaline phosphatase, and the absorbance was measured at 405nm. Expression and Purification of recombinant AgI/II Fig 1. SDS-PAGE analysis of S. mutans GS-5 culture supernatant. S. mutnas GS-5 culture supernatant (20ul) were collected at various time points during bacterial growth and separated in 8% SDS-PAGE gel. Fig 2. Western blot analysis of S. mutans GS-5 culture supernatant. S. mutans GS-5 culture supernatants (20ul) were collected at various time points of the bacterial growth and analyzed in Western blot using monoclonal anti-AgI/II antibodies. Fig 3. Time dependent ELISA against 1ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 4. Time dependent ELISA against 10ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 5. Time dependent ELISA against 50ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 6. Time dependent ELISA against 100ng of recombinant AgI/II. Monoclonal anti-AgI/II antibody 1:100 (closed circle), 1:1000 (open circle), 1:10000 (closed triangle), and 1:50000 (open triangle) dilution was used to detect the 1 ng recombinant AgI/II protein. The results represent the mean values ± S.E.M. from two separate experiments. Mean antibody titers are given as –log 2 dilution. Fig 7. Comparison of ELISA titer by 1:1000 dilution of monoclonal anti AgI/II antibody. Titer of recombinant AgI/II 1, 10, 50, and 100 ng was compared with time dependently color density by monoclonal anti AgI/II antibody (1:1000). Fig 9 Time dependent ELISA against concentrated protein of culture supernatant. Concentrated protein S. mutans GS-5 culture supernatant (100 ng) was detected using monoclonal anti-AgI/II antibody 1:100 (circle) and 1:1000 (triangle) dilution. Fig 8. Comparison of ELISA titer by 1:100 dilution of monoclonal anti AgI/II antibody. Titer of recombinant AgI/II 1, 10, 50, and 100 ng was compared with time dependently color density by monoclonal anti AgI/II antibody (1:100). To obtain recombinant AgI/II-N protein, induced its expression using isopropyl-  -D-thiogalactopyranoside(IPTG) and purified the protein with nickel chelated agarose column. To quantify purified AgI/II-N, carried out SDS-PAGE and transferred it to nitrocellulose paper for western blot analysis using monoclonal anti-AgI/II-N. Figure 10. Comparison of ELISA titer between concentrated protein and culture supernatant. Titer of concentrated protein of S. mutans GS-5 culture supernatant, 50 ng and 100 ng, and culture supernatant of S. mutans GS-5, 100 ul was compared by 1: 100 dilution of monoclonal anti AgI/II antibody.