scholarly journals Screening Antibody Libraries with Colony Assay Using scFv-Alkaline Phosphatase Fusion Proteins

Molecules ◽  
2020 ◽  
Vol 25 (12) ◽  
pp. 2905
Author(s):  
Yoshiro Hanyu ◽  
Mieko Kato
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Proteins ◽  
Direct Detection ◽  
Screening Methods ◽  
Single Chain Variable Fragment ◽  
Antibody Library ◽  
Single Chain ◽  
Library Size ◽  
Colony Assay ◽  
Antibody Libraries

Screening antibody libraries is an important step in establishing recombinant monoclonal antibodies. The colony assay can identify positive clones without almost any false-positives; however, its antibody library is smaller than those used in other recombinant screening methods such as phage display. Thus, to improve the efficiency of colony assays, it is necessary to increase library size per screening. Here, we report developing a colony assay with single-chain variable fragment (scFv) fused to the N-terminus of bacterial alkaline phosphatase (scFv-PhoA). The scFv-PhoA library was constructed in an expression vector specifically designed for this study. Use of this library allowed the successful and direct detection of positive clones exhibiting PhoA activity, without the need for a secondary antibody. Colony assay screening with scFv-PhoA is simple, rapid, offers a higher success rate than previous methods based on scFv libraries, and—most importantly—it enables high-throughput procedures.

scholarly journals Selection of Single-Chain Variable Fragment Antibodies to Black Currant Reversion Associated Virus from a Synthetic Phage Display Library

1998 ◽  
Vol 88 (3) ◽  
pp. 230-233 ◽  
Author(s):  
Petri Susi ◽  
Angelika Ziegler ◽  
Lesley Torrance
Keyword(s):  
Alkaline Phosphatase ◽  
Phage Display ◽  
Plant Extracts ◽  
Fusion Proteins ◽  
Phage Display Library ◽  
Antibody Fragments ◽  
Healthy Plant ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Black Currant

Single-chain variable fragment (scFv) antibodies that bind to black currant reversion associated virus (BRAV) were obtained from a synthetic phage display antibody gene library without recourse to animal immunizations. Several different BRAV-specific phage scFv were obtained quickly, after only three rounds of selection against immobilized virus antigen. The phage scFv gave enzyme-linked immunosorbent assay (ELISA) absorbance values that were greater than seven times the control healthy plant extracts. In contrast, comparative tests using a rabbit antiserum failed, because unacceptably high background values were obtained with healthy plant extracts. Two of the scFv were subcloned into the pDAP2 vector for the rapid and efficient production of scFv-alkaline phosphatase fusion proteins. Functional fusion proteins were obtained after expression in Escherichia coli, and preparations from periplasmic extracts detected BRAV in ELISA. The results demonstrate that antibody fragments obtained from a synthetic phage display library are useful research tools, and they proved to be a viable practical alternative when traditional antisera failed to detect BRAV, a weak immunogen. Furthermore, the genetic fusion of antibody fragments to alkaline phosphatase obviates the need for further chemical coupling procedures, and the fusion proteins can be obtained cheaply.

scholarly journals Lactobacillus helveticus MIMLh5-Specific Antibodies for Detection of S-Layer Protein in Grana Padano Protected-Designation-of-Origin Cheese

2013 ◽  
Vol 80 (2) ◽  
pp. 694-703 ◽  
Author(s):  
Milda Stuknytė ◽  
Eeva-Christine Brockmann ◽  
Tuomas Huovinen ◽  
Simone Guglielmetti ◽  
Diego Mora ◽  
...  
Keyword(s):  
Western Blotting ◽  
Lactobacillus Helveticus ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
Content Type ◽  
Bacterial Surface ◽  
Protected Designation Of Origin ◽  
Antibody Libraries ◽  
Grana Padano ◽  
Detection And Localization

ABSTRACTSingle-chain variable-fragment antibodies (scFvs) have considerable potential in immunological detection and localization of bacterial surface structures. In this study, synthetic phage-displayed antibody libraries were used to select scFvs against immunologically active S-layer protein ofLactobacillus helveticusMIMLh5. After three rounds of panning, five relevant phage clones were obtained, of which four were specific for the S-layer protein ofL. helveticusMIMLh5 and one was also capable of binding to the S-layer protein ofL. helveticusATCC 15009. All five anti-S-layer scFvs were expressed inEscherichia coliXL1-Blue, and their specificity profiles were characterized by Western blotting. The anti-S-layer scFv PolyH4, with the highest specificity for the S-layer protein ofL. helveticusMIMLh5, was used to detect the S-layer protein in Grana Padano protected-designation-of-origin (PDO) cheese extracts by Western blotting. These results showed promising applications of this monoclonal antibody for the detection of immunomodulatory S-layer protein in dairy (and dairy-based) foods.

Construction of Single Chain Variable Fragment (ScFv) and BiscFv-Alkaline Phosphatase Fusion Protein for Detection ofBacillusAnthracis

2006 ◽  
Vol 78 (4) ◽  
pp. 997-1004 ◽  
Author(s):  
Shi-Hua Wang ◽  
Ji-Bin Zhang ◽  
Zhi-Ping Zhang ◽  
Ya-Feng Zhou ◽  
Rui-Fu Yang ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Fusion Protein ◽  
Single Chain Variable Fragment ◽  
Single Chain

Generation of a single-chain variable fragment phage display antibody library from naïve mice panned against Fasciola hepatica antigens

2019 ◽  
Vol 205 ◽  
pp. 107737
Author(s):  
Luke J. Norbury ◽  
Katarzyna Basałaj ◽  
Piotr Bąska ◽  
Anna Zawistowska-Deniziak ◽  
Alicja Kalinowska ◽  
...  
Keyword(s):  
Phage Display ◽  
Fasciola Hepatica ◽  
Single Chain Variable Fragment ◽  
Antibody Library ◽  
Single Chain ◽  
Phage Display Antibody

scholarly journals Clonning, Expression and Identification by Immunohistochemistry of Humanized Single-Chain Variable Fragment Antibody against Hepatitis C Virus Core Protein

2011 ◽  
Vol 60 (1) ◽  
pp. 13-17 ◽  
Author(s):  
YONG-ZHI LUN ◽  
JUN CHENG ◽  
YAN-WEI ZHONG ◽  
ZENG-GUO YU ◽  
QI WANG ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Microtiter Plate ◽  
Single Chain Variable Fragment ◽  
Antibody Library ◽  
Single Chain ◽  
Virus Core ◽  
Phage Antibody Library ◽  
Healthy Persons

Expression of single-chain variable fragment (scFv) antibodies on the surface of bacteriophage is widely used to prepare antibodies with pre-defined specificities. A phage antibody library containing the gene for scFv antibody against Hepatitis C virus core protein was panned with core protein immobilized on microtiter plate wells. After five rounds of panning 60 phage clones specific to core protein were obtained and one selected clone was sequenced. It was found that the specifically detected antigen consists of 774bp and is capable of encoding 257 amino acids in the patients but not in healthy persons.

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