Development and characterization of a monoclonal antibody against the putative T cells of Labeo rohita

Abstract

In this study, we have described the development and characterization of monoclonal antibodies (MAbs) directed against thymocytes of rohu, Labeo rohita. MAbs were obtained by immunizing BALB/c mice with freshly isolated and nylon wool column enriched mononuclear cells of thymus. Positive clones against thymocytes were screened by cellular ELISA. The hybridoma showing strong reactivity with nylon wool enriched mononuclear cells, and non-reactivity with a rohu thymus macrophage cell line and rohu serum was selected and subjected to single cell cloning by limiting dilution. The MAbs secreted by a positive clone were designated as E6 MAb. Western blotting of reduced protein from enriched thymocytes showed that E6 reacted with a 166.2 kDa polypeptide and belongs to the IgG1 subclass. Flow cytometric analysis of gated lymphocytes, revealed that the percentage of E6 positive (E6?) cells in thymus (n = 5, 720.4 ± 79.70 g) was 89.7 %. Similarly, the percentage of E6? cells in kidney, spleen and blood (n = 5) was 6.71, 1.71 and 1.88 %, respectively. In indirect immunoperoxidase test, E6? cells appeared to be lymphoid cells with a high nucleus to cytoplasmic ratio and were densely packed in the central region of thymus whereas, a few cells were found to be positive in kidney and spleen sections. E6 MAb also reacted with a small population of lymphocytes in blood smear. This MAb appears to be a suitable marker for T lymphocytes and can be a valuable tool in studying immune response and ontogeny of L. rohita immune system.

CD4: a vital player in the teleost fish immune system

“…Accordingly, it was suggested that the presence of CD4-positive T cells in the ginbuna crucian carp is the equivalent of the helper T-lymphocyte in mammals. Additionally, mAbs directed against thymocytes of rohu, ( Labeo rohita ) was developed and characterized [32]. This mAbs emerged to be a suitable marker for T-lymphocytes and could be a valuable tool in studying the immune response and ontogeny of the rohu immune system.…”

Sleeping With the Enemy? The Current Knowledge of Piscine Orthoreovirus (PRV) Immune Response Elicited to Counteract Infection

“…In addition, cd4-1 , the gene encoding the T cell co-receptor, was also upregulated in infected salmon ( 34 ), all indicating that PRV elicited the adaptive immune response in Atlantic salmon, which probably involves T cell proliferation. CD4 + CD3 + T cells (T helper) have been in fact identified, isolated and characterized in some fish species, including Japanese Pufferfish ( Takifugu rubripes ), ginbuna crucian carp ( Carassius auratus langsdorfii ), zebrafish ( Danio rerio ), rainbow trout ( Oncorhynchus mykiss ) ( 42 – 47 ); and rohu ( Labeo rohita ) ( 48 ). Although in Atlantic salmon, T cell isolation and characterization awaits further studies, transcriptional data and the studies in rainbow trout ( 44 , 49 – 52 ) support the presence of T lymphocytes in this fish species and its role in response to the pathogens or model antigens.…”

Production and characterisation of monoclonal antibodies against immunoglobulins of Cirrhinus mrigala (Hamilton 1822)

“…To date, MAbs have been established for a variety of freshwater and marine fish species. These include Ictalurus punctatus (Edholm et al, 2010), Paralichthys olivaceus (Li et al, 2007), Takifugu rubripes (Miyadai et al, 2004), Labeo rohita (Rathore et al, 2008), Channa striata (Sood et al, 2011), Clupea pallasii (Purcell et al, 2012), Clarias batrachus (Sood et al, 2012), Labeo rohita (Rebello et al, 2016), Lateolabrax japonicas (Yang et al, 2018), Oncorhynchus mykiss (Hayashi et al, 2019) and Carassius auratus gibelio (Wu et al, 2020). All of these MAbs developed have numerous diagnostic applications in fish health management.…”