Squalene Antibodies with Lipid Antigens
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Squalene Antibodies with Lipid Antigens |
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Reagents SQE and squalane (SQA) were purchased from Sigma Chemical Co., St. Louis, Missouri. DMPC, DMPG, and cholesterol were purchased from Avanti Polar Lipids, Alabaster, Alabama. Lipid A from Salmonella minnesota R595 was purchased from List Biological Laboratories, Campbell, California. Arachidic acid, behenic acid, palmitic acid, stearic acid, lignoceric acid, lauric acid, myristic acid, hexadecane, and pristane were purchased from Sigma Chemical Co., St. Louis, Missouri. Fetal bovine serum (FBS) was purchased from GIBCO BRL, Grand Island, New York and was heat inactivated at 57°C for 45 minutes. Peroxidase-linked anti-mouse IgM was purchased from The Binding Site, San Diego, California. ABTS substrate was purchased from Kirkegaard & Perry Laboratories, Gaithersburg, Maryland. Gelatin was from BioRad Laboratories, Richmond, California. Seal plate adhesive film was from PGC Scientific, Gaithersburg, Maryland. Non-sterile phosphate buffered saline, pH 7.4, (PBS) was prepared from standard laboratory salts. Hydrophobic polyvinyldiene fluoride (PVDF) Multi-Screen-IP plates were purchased from Millipore Corp., Bedford, Massachusetts. Immulon II U-bottom plates, Immulon II flat bottom plates, and Immulon 4HBX plates were purchased from Dynex Technologies Inc., Chantilly, Virginia. Tissue culture U-bottom plates and tissue culture flat bottom plates were purchased from Costar, Corning, New York. Nunc-Immunoplate F96 Maxisorp plates were purchased from Nalge Nunc International Corp., Naperville, Illinois. ELISA Development PVDF Multi-Screen-IP, Immulon II U-bottom, Immulon II flat bottom, Costar tissue culture U-bottom, Costar tissue culture flat bottom, Immulon 4HBX, and Nunc-Immunoplate F96 Maxisorp plates were tested in the ELISA. SQE was diluted in isopropanol at a concentration of 300 nmol/0.1 ml. One-tenth of ml of SQE solution was added to each well of a plate. The plates were allowed to incubate overnight in a biological safety cabinet to allow the isopropanol to evaporate. The plates were blocked with 0.25 ml/well (U-bottom plates) and 0.30 ml/well (flat bottom plates) of PBS-4% FBS or 0.25 ml/well and 0.30 ml/well of PBS-0.3% gelatin for 2 hours at room temperature. Culture supernatants were diluted at the concentrations indicated in PBS-4%FBS or PBS-0.3% gelatin. Normal mouse serum and mouse post-immune serum containing anti-SQE were diluted 1:200 in PBS-4%FBS or PBS-0.3% gelatin. Blocking buffer was removed and 100 ml/well of diluted culture supernatants or diluted mouse serum was added. The plates were incubated at room temperature for 1 hour. The plates were washed 3 times with PBS using a plate washer (Skatron Inc., Sterling, Virginia). The PVDF plates were hand washed 3 times with PBS. Peroxidase-linked anti-mouse IgM was diluted 1000-fold in PBS-4% FBS or PBS-0.3% gelatin and added 0.1 ml/well to each plate. The plates were incubated for 1 hour at room temperature. The plates were washed 3 times with PBS using a plate washer. The PVDF plates were hand washed 3 times with PBS. ABTS substrate was then added 100 ml/well to each plate and the plates were incubated in the dark for 1 hour at room temperature. Absorbance at 405 nm was measured using a UVmax Kinetic Microplate Reader (Molecular Devices, Palo Alto, California). Assay background was determined by the absorbance values of wells lacking antigen. This background was subtracted from the absorbance values of experimental wells. Titers were selected as dilutions at which absorbance values were twice background. ELISA for Testing Antibody Cross-Reactivity with Lipid Antigens SQE, SQA, DMPC, DMPG, cholesterol, lipid A, arachidic acid, behenic acid, palmitic acid, stearic acid, lignoceric acid, hexadecane, or pristane, were dissolved in isopropanol at a concentration of 300 nmol/0.1 ml. One-tenth of a ml of the appropriate antigen solution was placed in each well of a Costar tissue culture U-bottom plate. Plates were then allowed to incubate overnight in a biological safety cabinet to allow the isopropanol to evaporate. The plates were blocked with 0.25 ml/well of PBS-4% FBS for 2 hours at room temperature. Culture supernatants were diluted at the concentrations indicated in PBS-4%FBS. Normal mouse serum and mouse post-immune serum containing anti-SQE were diluted 1:200 in PBS-4%FBS. Blocking buffer was removed and 100 ml/well of diluted culture supernatants or diluted mouse serum was added. The plates were incubated at room temperature for 1 hour. All plates were then washed 3 times with PBS using a plate washer. Peroxidase-linked anti-mouse IgM was diluted 1000-fold in PBS-4% FBS and added 0.1 ml/well to each plate. The plates were then placed in incubation for 1 hour at room temperature. The plates were washed 3 times with PBS using a plate washer. ABTS substrate was then added 100 ml/well to each plate and the plates were incubated in the dark for 1 hour at room temperature. Absorbance at 405 nm was measured using a UVmax Kinetic Microplate Reader. Assay background was determined by the absorbance values of wells lacking antigen. This background was subtracted from the absorbance values of experimental wells. Titers were selected as dilutions at which absorbance values were twice background. |
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