The human immune response to red blood cell antigens as revealed by repertoire cloning

1998 ◽  
Vol 17 (1-2) ◽  
pp. 239-251 ◽  
Author(s):  
Don L. Siegel
Keyword(s):  
Immune Response ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Human Immune Response ◽  
Human Immune

scholarly journals Expression and characterization of recombinant anti-Rh(D) antibodies on filamentous phage: a model system for isolating human red blood cell antibodies by repertoire cloning

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2334-2344 ◽  
Author(s):  
DL Siegel ◽  
LE Silberstein
Keyword(s):  
Immune Response ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Expression Vectors ◽  
Filamentous Phage ◽  
Soluble Antigen ◽  
Fab Fragments ◽  
Human Immune Response ◽  
Membrane Bound ◽  
Human Immune

Abstract The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents. Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation. By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces. Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments. Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens. To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig. After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)- expressing phage per 10(4) antitetanus toxoid-expressing phage. A method was developed for screening the library with intact Rh(D)- positive RBCs. After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100%. Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography. The authenticity of the Fabs was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively. Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay. Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.

scholarly journals Expression and characterization of recombinant anti-Rh(D) antibodies on filamentous phage: a model system for isolating human red blood cell antibodies by repertoire cloning

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2334-2344
Author(s):  
DL Siegel ◽  
LE Silberstein
Keyword(s):  
Immune Response ◽  
Blood Cell ◽  
Red Blood Cell ◽  
Expression Vectors ◽  
Filamentous Phage ◽  
Soluble Antigen ◽  
Fab Fragments ◽  
Human Immune Response ◽  
Membrane Bound ◽  
Human Immune

The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents. Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation. By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces. Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments. Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens. To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig. After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)- expressing phage per 10(4) antitetanus toxoid-expressing phage. A method was developed for screening the library with intact Rh(D)- positive RBCs. After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100%. Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography. The authenticity of the Fabs was confirmed by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively. Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay. Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.

scholarly journals Assessing the human immune response to SARS-CoV-2 variants

2021 ◽  
Vol 27 (4) ◽  
pp. 571-572 ◽  
Author(s):  
Roberto Burioni ◽  
Eric J. Topol
Keyword(s):  
Immune Response ◽  
Human Immune Response ◽  
Human Immune

scholarly journals Approaches to Modelling the Human Immune Response in Transition of Candidates from Research to Development

2014 ◽  
Vol 2014 ◽  
pp. 1-6 ◽  
Author(s):  
Diane Williamson
Keyword(s):  
Immune Response ◽  
Human Immune Response ◽  
Human Immune ◽  
Animal Rule

This review considers the steps required to evaluate a candidate biodefense vaccine or therapy as it emerges from the research phase, in order to transition it to development. The options for preclinical modelling of efficacy are considered in the context of the FDA’s Animal Rule.

The effects of anabolic steroids and strength training on the human immune response

1989 ◽  
Vol 21 (4) ◽  
pp. 386???392 ◽  
Author(s):  
LEONARD H. CALABRESE ◽  
SUSAN M. KLEINER ◽  
BARBARA P. BARNA ◽  
CHRISTINE I. SKIBINSKI ◽  
DONALD T. KIRKENDALL ◽  
...  
Keyword(s):  
Immune Response ◽  
Strength Training ◽  
Anabolic Steroids ◽  
Human Immune Response ◽  
Human Immune

Development of A First-Responder Fluorescence Reader for Microarray Cytokine Assay of Human Immune Response to Disease

2007 ◽  
Vol 1004 ◽  
Author(s):  
David B. Fenner ◽  
David I Rosen ◽  
Anthony A Ferrante ◽  
Amy E Stevens ◽  
Chad E Bigelow ◽  
...  
Keyword(s):  
Immune Response ◽  
Image Analysis ◽  
Optical Design ◽  
Human Saliva ◽  
Minimally Processed ◽  
Portable Apparatus ◽  
New Apparatus ◽  
Human Immune Response ◽  
Human Immune ◽  
Cytokine Assay

AbstractDevelopment of field portable apparatus and methods for cytokine assay of human saliva by fluorescent-reporting microarray plates is described. Multiplexed assay of 12 cytokines for minimally-processed saliva is read with a CCD-based imager under LED excitation. Immune responsive cytokines are measured at levels significant for indication of human disease state. The motivational context for the new apparatus development, the general optical design issues, saliva protocol, and image analysis are described.

Human Immune Response to a Peptide Mimic ofNeisseria meningitidisSerogroup C in hu-PBMC-SCID Mice

Hybridoma ◽  
1999 ◽  
Vol 18 (2) ◽  
pp. 121-129 ◽  
Author(s):  
WENDY A. HUTCHINS ◽  
THOMAS KIEBER-EMMONS ◽  
GEORGE M. CARLONE ◽  
M.A. JULIE WESTERINK
Keyword(s):  
Immune Response ◽  
Scid Mice ◽  
Peptide Mimic ◽  
Human Immune Response ◽  
Human Immune
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