A phage antibody to the active site of human placental alkaline phosphatase with higher affinity to the enzyme-substrate complex.
Selection of specific antibodies from large repertoires is of importance in generating antibodies to specific structural determinants and in studying structure-function relationships. Alkaline phosphatase (AP) has several isozymes with various degrees of homology and a range of common synthetic substrates. We have previously reported the generation of isozyme specific anti-enzyme antibodies to an oncofetal antigen, placental alkaline phosphatase (PLAP) by using a specific uncompetitive inhibitor, L-Phe-Gly-Gly along with the substrate para-nitrophenyl phosphate (pNPP), to elute scFvs from a phage-displayed immunoglobulin library. These antibodies were directed to the active site and inhibited enzyme activity. An uncompetitive inhibitor acts by stabilizing the enzyme-substrate (ES) complex. In the present work, we report the characteristics of a clone VE5, selected by the same method. This clone has a higher binding affinity for ES complex than for enzyme alone. This is true for all the three isozymes (placental, bone and intestinal) tested. However, the other synthetic small molecular substrate, disodium phenyl phosphate inhibits phage binding. The clone possibly binds to the conserved structures of the active site of the AP isozymes and the higher affinity binding to AP-pNPP complex reflects the method of selection. Such anti-enzyme antibodies have a possible potential role in dissecting structure-function relationship of enzymatic antigens.
Jain
Vishal
V
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi 110029, India.
Saini
Deepti
D
Goswami
Pooja
P
Sinha
Subrata
S
D000469:Alkaline Phosphatase; D000906:Antibodies / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D001665:Binding Sites / Q000276:immunology; D003001:Cloning, Molecular; D005260:Female; D058851:GPI-Linked Proteins; D006801:Humans; D007527:Isoenzymes / Q000276:immunology*; D009928:Organ Specificity; D019151:Peptide Library; D010920:Placenta / Q000201:enzymology / Q000276:immunology; D011247:Pregnancy; D013379:Substrate Specificity / Q000276:immunology
D000906:Antibodies; D058851:GPI-Linked Proteins; D007527:Isoenzymes; D019151:Peptide Library; D000469:Alkaline Phosphatase; C019839:alkaline phosphatase, placental