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Updated: Aug 8, 2026
Generation of Murine Monoclonal Antibodies by Hybridoma Technology
Published on: January 2, 2017
Influence of the decrease of intracellular antigenic content on morphometric analysis: effect of the type and
1Department of Pathology, University of Louvain, Faculty of Medicine, Brussels, Belgium.
Insights
B cell degranulation significantly impacts immunohistochemical detection, leading to underestimation of B cell area. Higher antibody concentrations are crucial for accurate B cell detection in pancreatic islets.
Area of Science:
- Endocrinology
- Immunohistochemistry
- Cell Biology
Background:
- Pancreatic B cells are vital for insulin production.
- Degranulation, a process of vesicle release, can alter cellular antigenicity.
- Accurate B cell quantification is essential for understanding diabetes and related research.
Purpose of the Study:
- To investigate how B cell degranulation affects immunohistochemical detection.
- To determine if detection method influences the impact of degranulation.
- To optimize B cell detection techniques in the pancreas.
Main Methods:
- Stimulated B cell degranulation in rat pancreas using glibenclamide.
- Quantified insulin levels via radioimmunoassay (RIA).
- Performed ultrastructural analysis and immunocytochemistry with varying antibody types and concentrations.
Main Results:
- Glibenclamide treatment reduced pancreatic insulin by 83% and B cell granules by 72%.
- B cell degranulation caused a 16% underestimation of B cell area under standard conditions.
- Underestimation worsened with monoclonal antibodies and higher serum dilutions; silver amplification did not correct this.
Conclusions:
- B cell degranulation significantly underestimates B cell area in immunohistochemistry.
- Detection accuracy is dependent on antibody concentration and type.
- Using higher antibody concentrations is essential for reliable B cell quantification in degranulated samples.
Abstract:
The aim of the study is to determine the effect degranulation of B cells on their immunohistochemical detection and to evaluate whether this effect depends on the technique of immunological detection. The biological model is the pancreatic insulin-containing B cell. To decrease the insulin content of the pancreas, insulin release was stimulated by five intraperitoneal injections of glibenclamide (2 mg/kg). Specimens of the pancreas were taken for insulin extraction and quantitation by radioimmunoassay (RIA), ultrastructural analyses and immunocytochemistry. The sections were treated either by polyclonal or monoclonal anti-insulin serum at various concentrations and peroxidase-antiperoxidase (PAP) technique eventually followed by silver amplification. The B-cell insular fraction or relative B-cell area (RBA) was measured with an automatic image analyser. The reference value for the relative B-cell area was established in control rats and corresponds to the ratio of the insular area occupied by immunostained B cells to the islet area. Its value was confirmed by calculation of the area occupied by non-B cells. RIA indicates a decrease of 83% of the insulin content in treated rats while the number of B granules decreases by 72% at the ultrastructural level. In usual conditions (polyclonal serum, 1/1500) the degranulation leads to a 16% underestimation of the B-cell insular fraction. This underestimation increases when monoclonal antibodies are used and further increases when higher dilutions are tested. The silver amplification does not prevent this underestimation and, in this particular model, exclusively acts by increasing the contrast. The only means of restoring the correct level of detection is to use the serum at a higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)
