New ELISA for B cell-activating factor.
BACKGROUND
The B cell-activating factor of the TNF family (BAFF) is upregulated in autoimmune diseases, but a number of conflicting results have cast doubts on the reliability of the ELISA protocols currently used for its quantification. This situation led us to develop a new ELISA for the measurement of BAFF.


METHODS
BAFF was purified for use alongside nonglycosylated recombinant BAFF. Two monoclonal antibodies (mAbs) and two polyclonal antibodies (pAbs) to BAFF were used.


RESULTS
The optimization process showed that the pAb format was preferable to the mAb format as capture antibody, because the pAbs recognized the glycosylated as well as the nonglycosylated forms of BAFF. The most efficient pair of Abs involved using the unconjugated form of a goat pAb to capture BAFF and the same biotinylated goat pAb to detect bound BAFF. This ELISA was not influenced by the presence of rheumatoid factor.


CONCLUSIONS
This new ELISA helped provide insights into why serum concentrations of BAFF vary between studies for a given population of patients. It is a reliable tool for the management of the diseases in which BAFF is an indication of response to therapy.
Le Pottier
Laëtitia
L
EA 2216 Immunologie et Pathologie and IFR 418 ScInBioS, Université de Brest, Brest, France.
Bendaoud
Boutahar
B
Renaudineau
Yves
Y
Youinou
Pierre
P
Pers
Jacques-Olivier
JO
Daridon
Capucine
C
D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D053264:B-Cell Activating Factor / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D001826:Body Fluids / Q000737:chemistry; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D006031:Glycosylation; D006041:Goats; D006801:Humans; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification; D012463:Saliva / Q000737:chemistry
D000906:Antibodies; D000911:Antibodies, Monoclonal; D053264:B-Cell Activating Factor; D011994:Recombinant Proteins