PPubMed26 Dec 2012Cited 31×

A Novel Rabbit Immunospot Array Assay on a Chip Allows for the Rapid Generation of Rabbit Monoclonal Antibodies with High Affinity Rabbit Immunospot Array Assay on a Chip

Tatsuhiko Ozawa, Xiuhong Piao, Eiji Kobayashi, Yue Zhou, Hiroaki Sakurai, Tsugunobu Andoh, Aishun Jin, Hiroyuki Kishi, Atsushi Muraguchi, Srinivas Kaveri

Abstract

'Hiroaki Sakurai' 'Tsugunobu Andoh' 'Aishun Jin' 'Hiroyuki Kishi' 'Atsushi Muraguchi' 'Srinivas Kaveri'] Antigen-specific rabbit monoclonal antibodies (RaMoAbs) are useful due to their high specificity and high affinity, and the establishment of a comprehensive and rapid RaMoAb generation system has been highly anticipated. Here, we present a novel system using immunospot array assay on a chip (ISAAC) technology in which we detect and retrieve antigen-specific antibody-secreting cells from the peripheral blood lymphocytes of antigen-immunized rabbits and produce antigen-specific RaMoAbs with 10-12 M affinity within a time period of only 7 days. We have used this system to efficiently generate RaMoAbs that are specific to a phosphorylated signal-transducing molecule. Our system provides a new method for the comprehensive and rapid production of RaMoAbs, which may contribute to laboratory research and clinical applications.

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A chip‑based ISAAC system enables rapid (7‑day) generation of rabbit monoclonal antibodies with picomolar (10‑12 M) affinity.

What’s new

i.Developed a novel rabbit immunospot array assay on a chip (ISAAC) to detect and retrieve antigen‑specific antibody‑secreting cells
ii.Demonstrated rapid generation of rabbit monoclonal antibodies with 10‑12 M affinity within 7 days
iii.Showed that high‑affinity antibodies produce smaller immunospots (D1/2 < 60), enabling affinity‑based screening
iv.Applied the system to generate phospho‑peptide‑specific antibodies (pTAK1) and validated their specificity in western blots

How they did it

Rabbits were immunized, peripheral‑blood IgG⁺ lymphocytes were isolated and arrayed on microwell chips coated with capture antibodies. Antigen‑specific secreted antibodies were visualized by fluorescence, single cells were retrieved with a micromanipulator, their antibody genes amplified and expressed in CHO cells, and affinities were measured by ELISA/Scatchard analysis.

What they found

RaMoAbs with 10‑12 M affinity were produced within 7 days
Affinity range of HEL‑specific antibodies was 1×10⁻⁷ to 1×10⁻¹² M; 56% (14/24) had KD 10⁻¹⁰‑10⁻¹² M
Limit of detection of a 10‑12 M RaMoAb was ~0.4 pM, 25‑fold lower than a mouse antibody (~10 pM)
Immunospots with D1/2 < 60 had average KD 2.56×10⁻¹¹ M versus 7.34×10⁻¹⁰ M for D1/2 > 60
64 pTAK1‑peptide‑specific monoclonal antibodies were obtained; 59 cross‑reacted with non‑phosphorylated peptide
RaMoAbs detected Thr‑187‑phosphorylated TAK1 in HeLa cells 2‑5 min after TNF‑α stimulation, whereas a commercial antibody did not

Caveats

✕The D1/2 cutoff may vary with different antigens and antibodies
✕Only IgG‑secreting cells were screened with ISAAC
✕A majority of pTAK1‑specific antibodies (59/64) also bound the non‑phosphorylated peptide, indicating cross‑reactivity

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