Use of polystyrene-supported 2-isobutoxy-1-isobutoxycarbonyl-1,2-dihydroquinoline for the preparation of a hapten-protein conjugate for antibody development

Bioorganic and Medicinal Chemistry Letters

Volumn 20, Issue 5, 2010, Pages 1792-1795

  Sathe, Manisha ^a   Derveni, Mariliza ^a   Allen, Marjorie ^a   Cullen, David C ^a  

^a CRANFIELD UNIVERSITY   (United Kingdom)

Author keywords

Bioconjugation; ELISA; Immunogen; Phytanic acid; Polystyrene supported 2 isobutoxy 1 isobutoxycarbonyl 1,2 dihydroquinoline; PS IIDQ

Indexed keywords

2 ISOBUTOXY 1 ISOBUTOXYCARBONYL 1,2 DIHYDROQUINOLINE; ANTIGEN; DIHYDROQUINOLINE DERIVATIVE; PHYTANIC ACID; POLYSTYRENE; UNCLASSIFIED DRUG;

ARTICLE; ENZYME LINKED IMMUNOSORBENT ASSAY; HAPTEN CARRIER COMPLEX; IMMUNOGENICITY; MATRIX ASSISTED LASER DESORPTION IONIZATION TIME OF FLIGHT MASS SPECTROMETRY; MOLECULAR WEIGHT;

ANIMALS; ANTIBODIES; CATTLE; ENZYME-LINKED IMMUNOSORBENT ASSAY; HAPTENS; PHYTANIC ACID; POLYSTYRENES; QUINOLINES; SERUM ALBUMIN, BOVINE; SPECTROMETRY, MASS, MATRIX-ASSISTED LASER DESORPTION-IONIZATION;

BOVINAE;

EID: 76649103021     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2009.12.078     Document Type: Article

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23. The phytanic acid derivative, as an example hapten derivative, was chosen for convenience as it is one of the potential molecular targets for immunoassay development within an associated project within the authors' laboratory-specifically the development of the immunoassay-based Life Marker Chip instrument for detecting biomarkers of Life on Mars.
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25. Conjugation of the phytanic acid derivative to BSA using PS-IIDQ.PS-IIDQ (1.87 mmol/g, 66.6 mg, 124.6 μmol) was added to a stock solution of the phytanic acid derivative (21.25 mg, 50 μmol) in DMF (1 mL). Conjugates of the phytanic acid derivative with the protein (BSA) were prepared at four different protein to hapten molar ratios (1:21, 1:46, 1:75, and 1:109) and were coded PA-1 to PA-4. In the case of the controls, the same micromole quantity of PS-DCC and PS-EDC was used as coupling reagents. The protein stock solution (10 mg/mL; 0.15 μmol/mL) was prepared in borate buffer (pH 9.0), and the final reaction volume of the protein-hapten conjugates was kept constant at 1 mL for each preparation. The hapten was conjugated to the protein by adding different amounts of protein (10 mg/mL) to a final volume of 1 mL to prepare protein-hapten conjugates of different molar ratios, as shown in Table 1. All four conjugates were incubated overnight at rt and centrifuged for 5 min at 10,000 rpm to remove the coupling reagent. They were purified by passing through a P10 gel filtration column (Pharmacia, Sweden). Fractions with the highest protein concentration were determined by absorbance measurements at 280 nm using a molar extinction coefficient 43,824/M/cm on a UV spectrometer. The final protein concentration of the conjugate was determined using a Micro BCA™ protein assay kit (Pierce).
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27. Mass analysis of the hapten-protein conjugates was carried out by MALDI mass-spectrometry (Applied Biosystems MALDI 4700 TOF). Protein solutions were prepared by interaction with C4 resin (3-5 mg, Jupiter 15u C4 300 Å, Phenomenex) in order to desalt and concentrate the protein content in the conjugates. The crystal matrix, 2,5-dihydroxybenzoic acid (Aldrich Chemical Co., Milwaukee, WI), was prepared at a concentration of 15 mg/mL in acetonitrile. Protein samples were typically 10-50 pmol/μL in a 2:1 water/acetonitrile solution. Sample and matrix solutions were mixed in equal volumes (typically 1.5 μL each) directly on the stainless steel probe tip (target) and allowed to dry (∼10 min) in a fume hood at room temperature. The crystallised analyte-matrix sample was then rinsed with 0.1% TFA solution by placing approximately 2 μL of the solution on the probe sample at room temperature, allowing it to stand for about 5 s, and then gently drying the crystals with a stream of nitrogen gas. Spectra were recorded at threshold laser irradiance for 50-150 shots in the linear mode at 30 kV. The resulting data were analyzed using the software supplied by Applied Biosystem.
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