Quantification of TNFa by ELISA

November 23, 2015

Loyola University Chicago
Microbiology and Immunology
Research Associate

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Company:

BioLegend

Product Name:

Human TNFa protein standard (ELISA)

Catalog Number:

570109

To analyze macrophage activation, THP1 cells were differentiated into macrophage cells and treated with fraction 10 or a control overnight. Cells were stimulated with LPS for 4 hours and then cell culture supernatant was collected for detecting TNFa secretion by ELISA. We found fraction treatment inhibited LPS induced TNFa secretion in macrophages. We are able to quantify the concentration of TNFa using the standard control.

Experimental Design and Results Summary

Application

ELISA to quantify the cytokine concentration

Starting Material

Macrophage cell culture supernatant

Protocol Overview

Microplate was coated with capture antibody overnight. After blocking, serially diluted samples and standard protein were added in microplate and incubated for 1 hr at 37 degrees. After 3 times of wash, biotin-anti-human TNFa antibody (1ug/ml) was added in the plate and incubated at 37 degrees for 1 hr, followed by HPR-streptavidin incubation for 1 hr. After 3 washes, TMB was added to develop the signal and the plate was analyzed by measuring OD 450nm.

Tips

Standard dilution can be from 2 to 500 pg/ml

Results Summary

Concentration of TNFa in samples was quantified. Results showed that bacteria fraction treatment inhibited LPS induced TNFa secretion in human macrophages.

Additional Notes

None

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Summary

The Good

We can detect as low as of 1pg/ml of standard protein. It is sensitive and easy to use.

The Bad

None

The Bottom Line

We are satisfied with this protein standard in combination with human TNFa ELISA antibody pair, all from Biolegend. It is sensitive and specific.

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