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Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase

Fredrikson, G ; Nilsson, S LU ; Olsson, H LU ; Björck, L LU ; Akerström, B LU and Belfrage, P LU (1987) In Journal of Immunological Methods 97(1). p.65-70
Abstract

The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with... (More)

The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.

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organization
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type
Contribution to journal
publication status
published
subject
keywords
Adipose Tissue/enzymology, Animals, Antibodies/analysis, Antigen-Antibody Complex, Bacterial Proteins/immunology, Hormones/pharmacology, Immunoglobulin G/immunology, Rabbits/immunology, Radioimmunoassay/methods, Rats, Sterol Esterase/analysis
in
Journal of Immunological Methods
volume
97
issue
1
pages
65 - 70
publisher
Elsevier
external identifiers
  • scopus:0023124216
  • pmid:3819438
ISSN
0022-1759
DOI
10.1016/0022-1759(87)90106-2
language
English
LU publication?
yes
id
5c140892-f243-43d3-a3a0-ba60f6ece816
date added to LUP
2019-05-22 10:32:12
date last changed
2020-01-13 01:51:59
@article{5c140892-f243-43d3-a3a0-ba60f6ece816,
  abstract     = {<p>The newly described immunoglobulin G-binding streptococcal surface protein, protein G, was used to prepare and characterize rabbit antibodies. The antibodies were directed against rat hormone-sensitive lipase, the rate-limiting enzyme in the hydrolysis of the triacylglycerols stored in adipose tissue. Antiserum was obtained after two injections with 20 micrograms enzyme protein, and the immunoglobulin fraction was obtained using a protein G-based solid-phase radioimmunoassay. The hydrolysis of acylglycerols by the enzyme was inhibited by the antibodies, and the enzyme could be efficiently removed from a solution using the antibodies and heat-killed streptococci expressing surface protein G. By Western blot and detection with 125I-protein G, the antibodies were found to selectively bind to hormone-sensitive lipase and to a smaller extent to two minor contaminants, possibly proteolytic fragments of the lipase. The amount of 125I-labelled protein G bound to the lipase on the blot was quantitatively related to the amount of enzyme protein down to the detection limit 10 ng.</p>},
  author       = {Fredrikson, G and Nilsson, S and Olsson, H and Björck, L and Akerström, B and Belfrage, P},
  issn         = {0022-1759},
  language     = {eng},
  month        = {02},
  number       = {1},
  pages        = {65--70},
  publisher    = {Elsevier},
  series       = {Journal of Immunological Methods},
  title        = {Use of protein G for preparation and characterization of rabbit antibodies against rat adipose tissue hormone-sensitive lipase},
  url          = {http://dx.doi.org/10.1016/0022-1759(87)90106-2},
  doi          = {10.1016/0022-1759(87)90106-2},
  volume       = {97},
  year         = {1987},
}