Humoral immune responses to peptides derived from the beta-amyloid peptide C-terminal sequence.
There is a continuing interest in the immunochemical quantification of isoforms of amyloid beta-peptide (Abeta) in body fluids of patients with Alzheimer's disease (AD); however, at present there is no general procedure to produce and test the required antibodies. We examined various methods to generate rabbit anti-Abeta; antibodies that are specific for Abeta(38), Abeta(40) and Abeta(42), and we tested their specificity and sensitivity by ELISA and Western blotting. To produce high-affinity antibodies required repeated inoculations of small doses of peptide conjugates over a period of at least 6 months. Antibodies generated to peptides derived from the Abeta(42) sequence showed some cross-reactivity with Abeta(40), but antibodies generated to Abeta4 peptides did not cross-react with Abeta(42). The shortest peptide capable of generating antibodies of moderate affinity possessed the sequence Met(35)-Ala(42); however, antibodies raised to the peptide Gly(33)-Ala(42) possessed the greatest affinity (K(D) = 1 nM) and specificity for Abeta(42). The latter antibodies were over 50,000-fold more reactive with Abeta(42) than with Abeta(40). They can detect Abeta isoforms in extracts of normal brain, where the peptides are present at levels below one part per billion. Our results provide methods to generate and characterize the specificity and affinity of anti-Abeta antibodies. This information is necessary to develop sensitive and specific immunoassays to quantify Abeta isoforms in brain extracts and in body fluids.
Miller
David L
DL
New York State Institute for Basic Research in Developmental Disabilities. Staten Island, NY 10314, USA. davidlm@interport.net
Potempska
Anna
A
Mehta
Pankaj D
PD
D000595:Amino Acid Sequence; D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000276:immunology*; D000818:Animals; D000906:Antibodies / Q000276:immunology; D000917:Antibody Formation / Q000276:immunology*; D000918:Antibody Specificity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D011817:Rabbits; D012680:Sensitivity and Specificity
D016229:Amyloid beta-Peptides; D000906:Antibodies; D000939:Epitopes; D007106:Immune Sera