Generation of murine monoclonal antihuman milk fat globule membrane antibodies using immunoprecipitation and BIAcore analyses.
A selection of monoclonal antibodies was developed against deoxycholine-solubilized human milk fat globule membranes (HMFG). The antibodies were selected for their ability to immunoprecipitate 125I-labeled HMFG and then further analyzed by surface plasmon resonance on the BIAcore for their reactivity with HMFG and with a fusion protein containing a 4-mer of the muc-1 tandem repeat. Both the HMFG and the fusion protein proved to be robust surfaces for the analysis of crude supernatants. The BIAcore evaluation was useful in identifying true positives. BIAcore analyses of purified antibody preparations were used to determine binding characteristics such as affinity and intensity. The latter proved useful in selecting a panel for evaluation by immunohistochemistry for breast tissue reactivity. Four of 6 antibodies appeared to react more intensely with tumor compared with normal breast tissues. One of those antibodies reacted with the fusion protein 4-mer of the muc-1 tandem repeat.
Bynum
J
J
Cell Biology Division, Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709, USA.
Hutchins
J T
JT
Kull
F C
FC
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis*; D000915:Antibody Affinity; D015374:Biosensing Techniques; D005260:Female; D006801:Humans; D006825:Hybridomas / Q000737:chemistry / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008895:Milk, Human / Q000276:immunology*; D018396:Mucin-1 / Q000276:immunology*; D011233:Precipitin Tests
D000911:Antibodies, Monoclonal; D008562:Membrane Glycoproteins; D018396:Mucin-1