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Updated: Aug 8, 2026
Quantitative Immunofluorescence to Measure Global Localized Translation
Published on: August 22, 2017
Quantitation of antigen in tissue by immunofluorescence image analysis
J M Basgen1, T E Nevins, A F Michael
1Department of Pediatrics, University of Minnesota Medical School 55455.
Insights
This study developed a method to quantify antigens in tissue sections using immunofluorescence and image analysis. The method accurately measures antigen binding in rat kidneys, correlating well with antigen dose.
Area of Science:
- Nephrology
- Immunology
- Biotechnology
Background:
- Quantifying antigen deposition in kidney tissue is crucial for diagnosing and understanding renal diseases.
- Traditional methods may lack precision or require complex sample preparation.
- Developing robust methods for antigen measurement in situ is essential for research and clinical applications.
Purpose of the Study:
- To establish and validate a quantitative immunofluorescence method for measuring antigen(s) in tissue sections.
- To assess the correlation between administered antigen dose and its deposition in rat glomeruli.
- To evaluate the reliability of quantitative immunofluorescence compared to direct antigen quantification.
Main Methods:
- Utilized fluorescein isothiocyanate (FITC)-conjugated antibodies for immunofluorescence staining of kidney tissue sections.
- Administered serially diluted 125I-labeled goat anti-glomerular basement membrane antibody to Sprague-Dawley rats.
- Quantified bound antigen using both quantitative immunofluorescence and direct radioactivity measurement of isolated glomeruli.
Main Results:
- A strong linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to glomeruli.
- A highly significant correlation (r = 0.98) was found between quantitative immunofluorescence (mean brightness) and bound 125I-IgG per glomerulus below 500 pg.
- Above 500 pg IgG/glomerulus, the correlation between immunofluorescence and bound antigen diminished, suggesting potential epitope masking or antibody accessibility issues.
Conclusions:
- Quantitative immunofluorescence, when validated, provides a reliable method for measuring antigen deposition in tissue.
- The method demonstrates high sensitivity and accuracy, particularly at lower antigen concentrations.
- Potential limitations at higher antigen loads highlight the importance of considering epitope availability in immunofluorescence assays.
Abstract:
A method is described to measure antigen(s) in tissue section using an image analysis system to quantitate immunofluorescence following staining with fluorescein isothiocyanate (FITC)-conjugated antibody. The antigen utilized was an 125I-labeled goat anti-glomerular basement membrane antibody (1409 cpm/micrograms) administered intravenously to each of 11 Sprague-Dawley rats in doses ranging from 1.09 to 34.72 mg IgG. 24 h later, both kidneys were obtained for quantitative immunofluorescence following staining of tissue sections with FITC-labeled rabbit anti-goat IgG and for determination of radioactivity which reflects the amount of goat IgG present in isolated glomeruli. A linear correlation (r = 0.97) was observed between the dose of administered goat 125I-IgG and the amount bound to isolated glomeruli over the entire dosage range. A highly significant correlation (r = 0.98) was also seen between the mean brightness per glomerulus as determined by quantitative immunofluorescence and the amount of 125I-IgG bound per glomerulus but only at values less than 500 pg of IgG per glomerulus. Above these levels no correlation was observed, suggesting the presence of hidden epitopes in the bound goat IgG or the lack of availability of the FITC-labeled rabbit antibody.
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