Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay

Tarja Kristina Sorsa-Leslie, H D Mason, William Joseph Harris, Paul Alfred Francois Fowler

Research output: Contribution to journalArticle

2 Citations (Scopus)
4 Downloads (Pure)

Abstract

Background: We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF).

Methods: A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping.

Results: Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants.

Conclusion: This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins that defy conventional approaches. In addition, we conclude that these data support suggestions that GnSAF may be structurally related to serum albumin or very tightly bound to serum albumin.

Original languageEnglish
Pages (from-to)49-66
Number of pages16
JournalReproductive Biology and Endocrinology
Volume3
DOIs
Publication statusPublished - 26 Sep 2005

Keywords

  • escherichia-coli
  • inhibiting factor
  • follicular-fluid
  • factor GNSAF
  • polyacrylamide-gels
  • steroid-production
  • factor bioactivity
  • fragments
  • proteins
  • display

Cite this

Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay. / Sorsa-Leslie, Tarja Kristina; Mason, H D ; Harris, William Joseph; Fowler, Paul Alfred Francois.

In: Reproductive Biology and Endocrinology, Vol. 3, 26.09.2005, p. 49-66.

Research output: Contribution to journalArticle

Sorsa-Leslie, Tarja Kristina ; Mason, H D ; Harris, William Joseph ; Fowler, Paul Alfred Francois. / Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay. In: Reproductive Biology and Endocrinology. 2005 ; Vol. 3. pp. 49-66.
@article{1139a0de39f84baf8ee5c0fa016bdb40,
title = "Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay",
abstract = "Background: We aimed to combine the generation of {"}artificial{"} antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF). Methods: A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Results: Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. Conclusion: This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins that defy conventional approaches. In addition, we conclude that these data support suggestions that GnSAF may be structurally related to serum albumin or very tightly bound to serum albumin.",
keywords = "escherichia-coli, inhibiting factor, follicular-fluid, factor GNSAF, polyacrylamide-gels, steroid-production, factor bioactivity, fragments, proteins, display",
author = "Sorsa-Leslie, {Tarja Kristina} and Mason, {H D} and Harris, {William Joseph} and Fowler, {Paul Alfred Francois}",
year = "2005",
month = "9",
day = "26",
doi = "10.1186/1477-7827-3-49",
language = "English",
volume = "3",
pages = "49--66",
journal = "Reproductive Biology and Endocrinology",
issn = "1477-7827",
publisher = "BioMed Central",

}

TY - JOUR

T1 - Selection of gonadotrophin surge attenuating factor phage antibodies by bioassay

AU - Sorsa-Leslie, Tarja Kristina

AU - Mason, H D

AU - Harris, William Joseph

AU - Fowler, Paul Alfred Francois

PY - 2005/9/26

Y1 - 2005/9/26

N2 - Background: We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF). Methods: A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Results: Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. Conclusion: This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins that defy conventional approaches. In addition, we conclude that these data support suggestions that GnSAF may be structurally related to serum albumin or very tightly bound to serum albumin.

AB - Background: We aimed to combine the generation of "artificial" antibodies with a rat pituitary bioassay as a new strategy to overcome 20 years of difficulties in the purification of gonadotrophin surge-attenuating factor (GnSAF). Methods: A synthetic single-chain antibody (Tomlinson J) phage display library was bio-panned with partially purified GnSAF produced by cultured human granulosa/luteal cells. The initial screening with a simple binding immunoassay resulted in 8 clones that were further screened using our in-vitro rat monolayer bioassay for GnSAF. Initially the antibodies were screened as pooled phage forms and subsequently as individual, soluble, single-chain antibody (scAbs) forms. Then, in order to improve the stability of the scAbs for immunopurification purposes, and to widen the range of labelled secondary antibodies available, these were engineered into full-length human immunoglobulins. The immunoglobulin with the highest affinity for GnSAF and a previously described rat anti-GnSAF polyclonal antiserum was then used to immunopurify bioactive GnSAF protein. The two purified preparations were electrophoresed on 1-D gels and on 7 cm 2-D gels (pH 4-7). The candidate GnSAF protein bands and spots were then excised for peptide mass mapping. Results: Three of the scAbs recognised GnSAF bioactivity and subsequently one clone of the purified scAb-derived immunoglobulin demonstrated high affinity for GnSAF bioactivity, also binding the molecule in such as way as to block its bioactivity. When used for repeated immunopurification cycles and then Western blot, this antibody enabled the isolation of a GnSAF-bioactive protein band at around 66 kDa. Similar results were achieved using the rat anti-GnSAF polyclonal antiserum. The main candidate molecules identified from the immunopurified material by excision of 2-D gel protein spots was human serum albumin precursor and variants. Conclusion: This study demonstrates that the combination of bioassay and phage display technologies is a powerful tool in the study of uncharacterised proteins that defy conventional approaches. In addition, we conclude that these data support suggestions that GnSAF may be structurally related to serum albumin or very tightly bound to serum albumin.

KW - escherichia-coli

KW - inhibiting factor

KW - follicular-fluid

KW - factor GNSAF

KW - polyacrylamide-gels

KW - steroid-production

KW - factor bioactivity

KW - fragments

KW - proteins

KW - display

U2 - 10.1186/1477-7827-3-49

DO - 10.1186/1477-7827-3-49

M3 - Article

VL - 3

SP - 49

EP - 66

JO - Reproductive Biology and Endocrinology

JF - Reproductive Biology and Endocrinology

SN - 1477-7827

ER -