A very sensitive enzyme-linked immunoabsorbent assay to Staphylococcal protein A in the presence of immunoglobulins.
The production of recombinant hepatitis B virus surface antigen (rHBsAg) purified by immunoaffinity chromatography with monoclonal antibodies is used to obtain a vaccine against this virus. Monoclonal antibodies to rHBsAg from mouse ascites have been purified by Staphylococcal Protein A (SpA)--prior coupling to Sepharose CL-4B (Amersham-Bioscences, Uppsala, Sweden). A high sensitivity immunoassay has been developed for the quantification of part-per-million of SpA contaminants likely to co-purify with monoclonal antibodies obtained by Protein A affinity chromatography, in the presence of immunoglobulins. Specific sheep polyclonal Abs against SpA (SpAc1) were used as plate coating and the SpA detection was possible thanks to the conjugates of sheep Ab fragments F(ab)(2) (fSpAc1) and horseradish peroxidase (fSpAc1-peroxidase), reducing the possible unspecific interaction between SpA and Fc fragments. The immunoassay was shown to be specific for SpA contaminants. The quantification limit of the assay was 0.39 ng/ml spreading to the measurement of contamination levels less than 2 ppm of SpA in final preparations of monoclonal antibodies used for the immunopurification of pharmaceutical products, which is quite low for this application.
Valdés Veliz
Rodolfo
R
Monoclonal Antibody Division, Center for Genetic Engineering and Biotechnology, 10600, P.O. Box 6162, Havana, Cuba. biunayki.reyes@cigb.edu.cu
García
Janet
J
Reyes
Biunayki
B
Muñoz
Lázara
L
Alvarez
Tatiana
T
Padilla
Sigifredo
S
Abrahantes
María del Carmen
Mdel C
Zubiaurrez
Jorge
J
Agraz
Alberto
A
Marrero
Aniebris
A
D000818:Animals; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods* / Q000706:statistics & numerical data; D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens / Q000302:isolation & purification; D007136:Immunoglobulins; D051379:Mice; D012680:Sensitivity and Specificity; D013205:Staphylococcal Protein A / Q000032:analysis*
D006510:Hepatitis B Antibodies; D006514:Hepatitis B Surface Antigens; D007136:Immunoglobulins; D013205:Staphylococcal Protein A