High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies.
Defining human B cell repertoires to viral pathogens is critical for design of vaccines that induce broadly protective antibodies to infections such as HIV-1 and influenza. Single B cell sorting and cloning of immunoglobulin (Ig) heavy- and light-chain variable regions (V(H) and V(L)) is a powerful technology for defining anti-viral B cell repertoires. However, the Ig-cloning step is time-consuming and prevents high-throughput analysis of the B cell repertoire. Novel linear Ig heavy- and light-chain gene expression cassettes were designed to express Ig V(H) and V(L) genes isolated from sorted single B cells as IgG1 antibody without a cloning step. The cassettes contain all essential elements for transcriptional and translational regulation, including CMV promoter, Ig leader sequences, constant region of IgG1 heavy- or Ig light-chain, poly(A) tail and substitutable V(H) or V(L) genes. The utility of these Ig gene expression cassettes was established using synthetic V(H) or V(L) genes from an anti-HIV-1 gp41 mAb 2F5 as a model system, and validated further using V(H) and V(L) genes isolated from cloned EBV-transformed antibody-producing cell lines. Finally, this strategy was successfully used for rapid production of recombinant influenza mAbs from sorted single human plasmablasts after influenza vaccination. These Ig gene expression cassettes constitute a highly efficient strategy for rapid expression of Ig genes for high-throughput screening and analysis without cloning.
Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, 27710, United States.
Hua-Xin
HX
Liao
Marc C
MC
Levesque
Ashleigh
A
Nagel
Ashlyn
A
Dixon
Ruijun
R
Zhang
Emmanuel
E
Walter
Robert
R
Parks
John
J
Whitesides
Dawn J
DJ
Marshall
Kwan-Ki
KK
Hwang
Yi
Y
Yang
Xi
X
Chen
Feng
F
Gao
Supriya
S
Munshaw
Thomas B
TB
Kepler
Thomas
T
Denny
M Anthony
MA
Moody
Barton F
BF
Haynes
D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000914:Antibodies, Viral / Q000235:genetics / Q000276:immunology; D001402:B-Lymphocytes / Q000276:immunology*; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D005803:Genes, Immunoglobulin; D015497:HIV-1 / Q000276:immunology; D004854:Herpesvirus 4, Human / Q000276:immunology; D006801:Humans; D008967:Molecular Biology / Q000379:methods*
D000911:Antibodies, Monoclonal; D000914:Antibodies, Viral; D004247:DNA