Analysis of the expression of IBMR3 antigen in cells and tissues from rodents
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Date
2009
Authors
Alrawi, Qutaiba K. Jassim
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Abstract
Monoclonal antibodies (Mab) and their fragments have been widely used for
diagnostic and therapeutic purposes. IBMR3 hybridoma cells were produced in a previous
study. The cell culture supernatant of the hybridoma cells was collected and stored at -20
°C freezer. The aim of this research is to investigate the expression profile of the IBMR3
antigens in different tissue of mouse, rat and cell lines HT29, 3T3 by using immunoblotting
technique and immunohistochemical staining using the peroxidase technique. In this study
the expression of IBMR3 antigen was evaluated in eighteen different organs taken from
Balb/c mouse and Sprague Dawley rat, established cell lines, human colorectal HT29
cancer cell line and mouse fibroblast 3T3. Various mouse and rat tissues were snap- frozen
and stored in liquid nitrogen. For immunoblotting six micron frozen sections of the various
tissues were prepared and collected in Eppendorf tubes. The sections were then lysed in
lysis buffer, and equal concentrations of lysates were run on 12% SDS-PAGE. The
separated protein bands were transferred to PVDF/ polyvinylidene difluoride membrane for
immunoblotting. Immunoblotting were subsequently subjected to densitometric analysis to
get the value of molecular weight, peak height and raw volume of the protein band
(Burnette, 1981). IBMR3Ag was expressed in mouse tissues with the highest MW (98.22)
kDa and highest number (nine) of bands detected in muscle and the lowest MW in heart
(75.00) kDa and liver (78.68) kDa with lowest number (2) of bands. In rat, more bands
(eight) were detected in brain and kidney. While in spleen only four protein bands were
detected. The highest MW was also indicated in brain (120.83 kDa), kidney (85.48 kDa)
and 73.42 kDa in spleen. In both mouse and rat, highest expression of IBMR3 antigen was
found in the lung and lowest in the brain. The immunohistochemical staining method was
used in peroxidase technique for the investigation of the expression profile of the IBMR3
antigens in different frozen tissues from mouse and rat. The positive IBMR3 antigen were
seen under light microscope as brown color, the results between weak, moderate and
intense. Expressed IBMR3 antigens help to analyze the profile for molecular weight, peak
height and raw volume. The different results in immunoblotting and immunohistochemistry
staining gave positive expression for IBMR3 Ag in different part of the tissue sections, this
shed more light on, whether the level of IBMR3 antigens are associated with pathological
parameters. The results from this study suggest that the IBMR3 antigens were differentially
expressed in mouse, rat tissue and cell lines. It was recommended that further research be
conducted to identify and investigate the nature of the IBMR3 antigen and its potential role
in tissue development.
Description
Master
Keywords
Biological Science , Antigen , Cells , Tissues , Rodents