Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation.
A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.
Kasprzyk
P G
PG
National Cancer Institute-Navy Medical Oncology Branch, Bethesda, Maryland 20814.
Cuttitta
F
F
Avis
I
I
Nakanishi
Y
Y
Treston
A
A
Wong
H
H
Walsh
J H
JH
Mulshine
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D005404:Fixatives; D019886:Gastrin-Releasing Peptide; D005976:Glutaral; D006801:Humans; D007457:Iodine Radioisotopes; D008969:Molecular Sequence Data; D010455:Peptides / Q000032:analysis*; D011143:Polyvinyl Chloride; D011863:Radioimmunoassay / Q000379:methods*
D000911:Antibodies, Monoclonal; D005404:Fixatives; D007457:Iodine Radioisotopes; D010455:Peptides; D019886:Gastrin-Releasing Peptide; D011143:Polyvinyl Chloride; D005976:Glutaral