Quantitative determination of human IgG antibodies to the peptide subunit determinant of peptidoglycan by an enzyme-linked immunosorbent assay.
An enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative measurement of IgG antibodies to the immunodominant R-D-Ala-D-Ala-OH determinant of peptidoglycan. Synthetic peptides R-D-Ala-D-Ala-OH, revealing structural analogy with the C-terminal sequence of the antigenic determinant H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 of peptidoglycan, were coupled covalently to albumin via their amino groups. The resulting peptidyl proteins were employed as an antigen in an ELISA for the specific detection of human IgG antibodies against the C-terminal R-D-Ala-D-Ala-OH moiety of H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2. Antigenic specificity was proved by comparing the high binding to albumin-(D-Ala-D-Ala-D-Ala-OH)9 with a lack of binding to albumin-(L-Ala-L-Ala-L-Ala-OH)13 and by appropriate inhibition studies of the ELISA. IgG, totally free from IgA and IgM, was isolated from reference serum 004, and the particular specificity was entirely found in this fraction. Quantification of the ELISA was effected by affinity chromatography. Isolated IgG was applied to an affinity column of Sepharose-[albumin-(D-Ala-D-Ala-D-Ala-OH)9]n, unbound IgG was eluted with phosphate-buffered saline and specific IgG against the C-terminal R-D-Ala-D-Ala-OH moiety of H-L-Ala-D-Glu(L-Lys-D-Ala-D-Ala-OH)-NH2 was eluted with 6 M guanidinium chloride.
N
N
Franken
P H
PH
Seidl
E
E
Zauner
H J
HJ
Kolb
K H
KH
Schleifer
L
L
Weiss
D001667:Binding, Competitive; D002648:Child; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000276:immunology*; D006801:Humans; D007124:Immunoenzyme Techniques; D007074:Immunoglobulin G / Q000032:analysis*; D009842:Oligopeptides / Q000276:immunology; D010457:Peptidoglycan / Q000276:immunology*; D012709:Serum Albumin / Q000378:metabolism
D000939:Epitopes; D007074:Immunoglobulin G; D009842:Oligopeptides; D010457:Peptidoglycan; D012709:Serum Albumin; C039944:alanyl-alanyl-alanine