Production of Monoclonal Antibody Against IgM of Common Carp (Cyprinus carpio)

Abstract

Introduction: Monoclonal antibodies widely used in different immunological studies,
immunohistochemistry, diagnosis and treatment of aquatic diseases. This study was aimed to
product anti-heavy chain immunoglobulin M of common carp, Due to its inaccessibility in
Iran.
Materials & Methods: In order to produce anti-IgM monoclonal antibody of carp, initially
serum IgM was partially purified through ammonium sulfate sedimentation method.
Prepared semi-purified IgM, was injected to the Balb/C mice with six to eight weeks age.
Afterwards, the spleen cells of one of the high immunity mice was removed to produce
monoclonal antibody and merged with myeloma cells. After about 10 days of incubation in
CO2 incubator, fluids of produced hybridoma cells, serum of MBP-immunized fish and
anti-immunoglobulin conjugated peroxidase in mice were screened by ELISA method. The
hybridomas that antibodies in their supernatant were able to detected fish MBP-linked IgM,
considered positive for the production of anti-IgM monoclonal antibodies and cloned again
three times. Subsequently, SDS-page and Western blotting were used in denatured condition
and non-denatured conditions to detect the antibodies produced in response to IgM heavy
chain. Moreover, produced anti-carp IgM Monoclonal antibodies capability to detect
anti-albumin and anti- sheep RBC antibodies in serum of immunized fish with albumin and
sheep RBC were verified and conformed.
Results: The results of SDS-page gel showed that common carp monoclonal antibody
anti-chain IgM monomer chain well seen in the range of 75 kDa and tetramer chain in the
760 kDa range. Moreover, the monoclonal antibody has the ability to separate the group of
immunized RBC sheep and bovine serum albumin from the control group.
Conclusion: The results of the production of IgM anti-chain monoclonal antibody,
commonly produced by ammonium sulfate, have been successfully performed by
semi-purification of the serum.