Synthesis and Characterization of Site-Selective Orbitide-BSA Conjugate to Produce Antibodies.
Bioactive flax cyclic peptides (orbitides and linusorbs) were site-specifically ligated through methionine with bovine serum albumin (BSA) to produce immunogenic compounds. In this study, modified flaxseed immunosuppressant orbitides (linusorbs or LOs) containing hydroxyl (OH) groups were synthesized for use as haptens. These compounds were extensively characterized by1H nuclear magnetic resonance (NMR),13C NMR, high-performance liquid chromatography-tandem mass spectrometry, and Fourier transform infrared spectroscopy. The haptens were conjugated to BSA, and the extent of hapten incorporation was determined by matrix-assisted laser desorption and ionization, liquid chromatography-electrospray ionization-mass spectrometry, and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The BSA hapten complexes were used to elicit polyclonal antibody (pAbs) production in rabbits. A competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) was developed that used orbitide-specific pAbs and horseradish peroxidase (HRP) conjugates. The LO assay detection limit was approximately 0.01 μg/mL (ppm), and thus, ELISA can be used for the detection of LOs in tissue and plant samples. The pAbs can be used to detect and quantify LOs in flax and flaxseed samples, to verify the presence of LOs in flaxseed containing foods, and for the detection of LOs in tissue samples, wastes, and body fluids of animals fed flaxseed.
Department of Plant Sciences, University of Saskatchewan , 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada.
Pramodkumar D
PD
Jadhav
Department of Plant Sciences, University of Saskatchewan , 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada.|Prairie Tide Chemicals Inc. , 102 Melville Street, Saskatoon, Saskatchewan S7J 0R1, Canada.
Youn Young
YY
Shim
Department of Plant Sciences, University of Saskatchewan , 51 Campus Drive, Saskatoon, Saskatchewan S7N 5A8, Canada.|Prairie Tide Chemicals Inc. , 102 Melville Street, Saskatoon, Saskatchewan S7J 0R1, Canada.|Guangdong Saskatchewan Oilseed Joint Laboratory, Department of Food Science and Engineering, Jinan University , 601 Huangpu Avenue West, Guangzhou, Guangdong 510632, China.
Martin J T
MJ
Reaney
D000818:Animals; D000906:Antibodies / Q000737:chemistry*; D060326:Chemistry Techniques, Synthetic; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006241:Haptens / Q000737:chemistry*; D018796:Immunoconjugates / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010456:Peptides, Cyclic / Q000737:chemistry / Q000276:immunology; D011817:Rabbits; D012710:Serum Albumin, Bovine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D017550:Spectroscopy, Fourier Transform Infrared
D000906:Antibodies; D006241:Haptens; D018796:Immunoconjugates; D010456:Peptides, Cyclic; D012710:Serum Albumin, Bovine