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Example 1 Production of Clones R 3F10, R 3A12 and R 6D12 HA peptide preparation

The following peptides were synthesized:

Bio-C-HA (acetyl-YPYDVPDYAGSGSK (ε-biotinoyl)-amide) (SEQ ID NO: 2) Bio-N-HA (biotinoyl-ε-Aca-SGSGYPYDVPDYA-amide) (SEQ ID NO: 3) KLH-MPS-CUZU-HA-C KLH-MPS-CUSU-HA-N Immunization of small mammals

Lou/C rats were immunized intraperitoneally with KLH-coupled HA peptide according to the following scheme:

For the primary immunization the animals were injected with 50 μl KLH-coupled HA peptide in complete Freund's adjuvant.

Further immunizations were carried out with 50 μg KLH-coupled HA peptide in incomplete Freund's adjuvant.

Fusion

The subsequent fusion of the spleen cells of the Lou/C rats was carried out using mouse P3x63-Ag8,653 in the presence of PEG according to Krammer et al (1990) Hybridoma 9, 309-317.

Selecting the clones

Screening scheme:

1st Screen

An SA-coated microtitre plate (MTP) was coated with 1 μg/ml Bio-C-HA or Bio-N-HA, hybridoma supernatants were used undiluted and added to the coated MTP, the bound antibodies were detected with the aid of anti-rat-POD conjugate/TMB substrate. 2nd Screen

A maxisorb MTP was coated with HA-tagged GST (1 μl/ml in carbonate buffer), the hybridoma supernatants selected in the 1st screen were again used undiluted and added to the coated MTP, the bound antibodies were detected with the aid of anti-rat-POD conjugate/TMB substrate. 3rd/4th Screen

BIACORE® measurements were carried out with an analogous coating. Result

5 clones with the highest affinity and the longest half-time of dissociation were selected with the aid of plasmon resonance in the BIACORE® system. They were named R 3F10, R 3A12, R 6D12, R 4H10 and M5B9. The affinity was only slightly different depending on the biotinoylated position of the peptides (C-terminus of N-terminus cf. FIG. 1).

The clones R 3F10, R 3A12 and R 6D12 were established as cell lines. They exhibit a good growth and a stable antibody production, and the antibodies that are produced have a 10-fold to 100-fold higher affinity than the monoclonal antibodies of the prior art 12CA5 and anti-HA BabCo.

The monoclonal antibody produced by hybridoma R 3A12 deposited at the “Deutsche Sammlung für Mikroorganismen und Zellkulturen” under Accession No. DSM ACC2286 (08.10.1996).
[[Category:Antibodies]]

HA antibody - Revision history

Paul.xu: Created page with "Example 1 Production of Clones R 3F10, R 3A12 and R 6D12 HA peptide preparation The following peptides were synthesized: Bio-C-HA (acetyl-YPYDVPDYAGSGSK (ε-biotinoyl)-amide..."