Effect of Antibody Orientation on Immunosorbent Performance

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAg) of 42-48% compared with 18-22% for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation. In summary, at low Mab density and a 2:1 molar rhPC to Mab binding stoichiometry, about 80% or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50% of lost Mab functionality upon immobilization.

Original languageEnglish (US)
Pages (from-to)528-535
Number of pages8
JournalJournal of Molecular Recognition
Volume9
Issue number5-6
DOIs
StatePublished - Jan 1 1996

Fingerprint

Immunosorbents
Monoclonal antibodies
Antibodies
Monoclonal Antibodies
Immobilization
Pepsin A
Antigens
Protein C
Proteins
Masks
Recombinant Proteins
Acetic acid
Stoichiometry
Digestion
Catalytic Domain
Esters
Acetates

Keywords

  • Antibody orientation
  • Immunosorbent performance

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

Cite this

Effect of Antibody Orientation on Immunosorbent Performance. / Subramanian, Anuradha; Velander, William H.

In: Journal of Molecular Recognition, Vol. 9, No. 5-6, 01.01.1996, p. 528-535.

Research output: Contribution to journalArticle

@article{72feece15de74279a76d7027fb6e7325,
title = "Effect of Antibody Orientation on Immunosorbent Performance",
abstract = "The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAg) of 42-48{\%} compared with 18-22{\%} for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation. In summary, at low Mab density and a 2:1 molar rhPC to Mab binding stoichiometry, about 80{\%} or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50{\%} of lost Mab functionality upon immobilization.",
keywords = "Antibody orientation, Immunosorbent performance",
author = "Anuradha Subramanian and Velander, {William H.}",
year = "1996",
month = "1",
day = "1",
doi = "10.1002/(SICI)1099-1352(199634/12)9:5/6<528::AID-JMR296>3.0.CO;2-Q",
language = "English (US)",
volume = "9",
pages = "528--535",
journal = "Journal of Molecular Recognition",
issn = "0952-3499",
publisher = "John Wiley and Sons Ltd",
number = "5-6",

}

TY - JOUR

T1 - Effect of Antibody Orientation on Immunosorbent Performance

AU - Subramanian, Anuradha

AU - Velander, William H.

PY - 1996/1/1

Y1 - 1996/1/1

N2 - The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAg) of 42-48% compared with 18-22% for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation. In summary, at low Mab density and a 2:1 molar rhPC to Mab binding stoichiometry, about 80% or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50% of lost Mab functionality upon immobilization.

AB - The impact of antibody orientation on immunosorbent efficiency was quantitatively assessed. A pH-dependent murine monoclonal antibody (Mab) against human protein C (hPC), recombinant hPC (rhPC) and two different immobilization chemistries and matrices were used as model systems. The lysyl groups of the rhPC were covalently modified with an acetic acid ester of N-hydroxysuccinimide and this modified rhPC was used as a Fab masking agent (FMA). The FMA was used to mask the antigen binding regions (Fab) of the Mab prior to and during covalent immobilization. Thereafter, the residual active sites of the support were inactivated and the FMA was removed. Mab was immobilizeed at low bead-averaged densities of about 0.4-1.1 mg Mab/mL matrix to minimize local density effects. Immunosorbents made using masked Mab (oriented coupling) gave antigen binding efficiencies (nAg) of 42-48% compared with 18-22% for those made by random coupling. The amount of (Fab)2 released from pepsin digestion of immunosorbents was about 3-4-fold higher for matrices having been made with FMA-masked Mab relative to unmasked Mab. Thus, the (Fab)2 accessibility to pepsin correlates well with higher functional efficiency (nAg) and serves as a measure of orientation. In summary, at low Mab density and a 2:1 molar rhPC to Mab binding stoichiometry, about 80% or more of the Mab randomly coupled through amino moieties was improperly oriented relative to oriented coupled Mab, which correlated with about 50% of lost Mab functionality upon immobilization.

KW - Antibody orientation

KW - Immunosorbent performance

UR - http://www.scopus.com/inward/record.url?scp=0030224828&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030224828&partnerID=8YFLogxK

U2 - 10.1002/(SICI)1099-1352(199634/12)9:5/6<528::AID-JMR296>3.0.CO;2-Q

DO - 10.1002/(SICI)1099-1352(199634/12)9:5/6<528::AID-JMR296>3.0.CO;2-Q

M3 - Article

C2 - 9174936

AN - SCOPUS:0030224828

VL - 9

SP - 528

EP - 535

JO - Journal of Molecular Recognition

JF - Journal of Molecular Recognition

SN - 0952-3499

IS - 5-6

ER -