J Vet Sci.  2006 Sep;7(3):293-295. 10.4142/jvs.2006.7.3.293.

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf)

• Shin G
• Lee H
• Palaksha K
• Kim Y
• Lee E
• Shin Y
• Lee E
• Park K
• Jung T

• KMID: 1089916
• DOI: http://doi.org/10.4142/jvs.2006.7.3.293

Abstract

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.

Keyword

black rockfish; immunoglobulin; monoclonal antibodies; Sebastes schlegeli Higendorf

MeSH Terms

Animals
Antibodies, Monoclonal/*biosynthesis/immunology
Chromatography, Affinity/veterinary
Electrophoresis, Gel, Two-Dimensional/veterinary
Immunoblotting/veterinary
Immunoglobulin Heavy Chains/immunology
Immunoglobulin Light Chains/immunology
Immunoglobulins/blood/*immunology
Perciformes/blood/*immunology

Figure

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  Fig. 1 SDS-PAGE analysis of black rockfish immunoglobulin (Ig)s purified using protein A, mannan binding protein (MBP), and goat IgG affinity columns. MBP purification of Ig was achieved with the ImmunoPure IgM Purification Kit (Pierce, USA). The purified Igs were resolved on 10% SDS-PAGE gels and stained with Coomassie brilliant blue G. lane 1; black rockfish whole serum, lane 2; MBP-positive Ig, lane 3; protein A-positive Ig, lane 4; goat IgG-positive Ig.

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  Fig. 2 Cross-reactivity of R7B4-8 (panel A) and R7B4-9 (panel B) MAbs to immunoglobulin (Ig)s purified by three different affinity columns (immunoblot analysis). lanes 1 and 5; Igs purified by MBP column, lanes 2 and 6; Ig purified by protein A column, lanes 3 and 4; Igs purified by IgG column.

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  Fig. 3 Identification of Immunoglobulin heavy chain on 2-DE immunoblot profile using R7B4-9 MAb. 2-DE was performed by isoelectric focusing using IPG dry strip (pH 4-7, 7 cm), followed by 12.5% SDS-PAGE. After 2-DE, one gel was stained with CBG (A) and the other gel was used for immunoblot assays with R7B4-9 MAb (B).

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