2,6,10,14-tetramethylpentadecane

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: In 100 ml of an aqueous solution containing 20% by volume of ethanol were incorporated 100 ml of a mixed liquid comprising trioctyl phosphate and 2,6,10,14-tetramethylpentadecane at a weight ratio of 2:1 and 0.2 g of sodium laurylsulfate, and ultrasonic vibration having a frequency of 28 KHz was applied to the liquid mixture for 2 minutes to form an emulsion in which particles of the trioctyl phosphate/2,6,10,14-tetramethylpentadecane mixed liquid having an average particle diameter of 0.3 μm were dispersed in the aqueous solution of ethanol. The emulsion was filtered at a potential gradient of 4.5 V/cm under a pressure of 0.01 kg/cm2 through a polytetrafluoroethylene porous membrane having a nominal average pore size of 2.0 μm. Only the phase of trioctyl phosphate/2,6,10,14-tetramethylpentadecane dispersed in the form of particles permeated through the membrane at a flow rate of 48 kg/m2.hr, and the permeated liquid formed a continuous phase.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: In 100 ml of an aqueous solution containing 20% by volume of ethanol were incorporated 100 ml of a mixed liquid comprising trioctyl phosphate and 2,6,10,14-tetramethylpentadecane at a weight ratio of 2:1 and 0.2 g of sodium laurylsulfate, and ultrasonic vibration having a frequency of 28 KHz was applied to the liquid mixture for 2 minutes to form an emulsion in which particles of the trioctyl phosphate/2,6,10,14-tetramethylpentadecane mixed liquid having an average particle diameter of 0.3 μm were dispersed in the aqueous solution of ethanol. The emulsion was filtered at a potential gradient of 4.5 V/cm under a pressure of 0.01 kg/cm2 through a polytetrafluoroethylene porous membrane having a nominal average pore size of 2.0 μm. Only the phase of trioctyl phosphate/2,6,10,14-tetramethylpentadecane dispersed in the form of particles permeated through the membrane at a flow rate of 48 kg/m2.hr, and the permeated liquid formed a continuous phase.

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: Materials: All inorganic chemicals and organic solvents were of reagent grade or better. Bovine serum albumin (BSA) (fatty acid free and fraction V) ovalbumin (OA) (crude and fraction VII), polyethylene sorbitan monolaurate (Tween 20), 2,2'-azino-di-(3-ethylbentzthiazoline)sulfonic acid (ABTS), hydrogen peroxide dicyclohexylcarbodiimide, N-hydroxysuccidimide, dimethylformamide (DMF), polyethylene glycol (PEG) (MW 1450), insulin, oxaloacetate, hypoxanthine (H), amino-pterin (A), thymidine (T) and 2,6,10,14 tetramethylpentadecane (pristane), horseradish peroxidase (HRP) Type VI were purchased from Sigma Chemical Company (St. Louis, Mo.); tetrahydrofuran from Aldrich Chemical Company (Milwaukee, Wis.); Freund's complete and incomplete adjuvants from Difco (Detroit, Mich.); goat anti-mouse IgG conjugated to horseradish peroxidase from Cooper Biomedical (Malvern, Pa.); Dulbecco's Modified Eagle's Medium (DMEM), Penicillin/Streptomycin Solution (Pen/Strep) (100,000 U/ml), NCTC Medium 135, fetal bovine serum (FBS), and sodium pyruvate from Gibco Laboratories (Grand Island, N.Y.); microculture plates (96- and 24-well plates) from Costar (Cambridge, Mass.); microtiter plates from NUNC, Vangard International (Neptune, N.J.). Aflatoxins B1, B2, G1, G2 were purchased from Sigma Chemical Company (St. Louis, Mo.); aflatoxin M1 was generously provided by the Department of Botany and Plant Pathology, Michigan State University, East Lansing, Mich. Subclass identification kit was purchased from Boehringer Mannheim Biochemicals (Indianapolis, Ind.). The myeloma cell line P3/NS 1/1-Ag-4 (NS-1) (ATCC TIB 18) was purchased from The American Type Culture Collection (Rockville, Md.). Mice were purchased from Charles River Laboratories (Wilmington, Mass.).

Conditions: temp:0.00; pressure:0.00; stir:; See reaction.notes.procedure_details.

Procedure: ELISAs were performed in polyvinyl 96-well microtiter plates (Dynatech, Alexandria, Va.); 200-μl volumes were used for each step. Wells were coated with a cell envelope preparation (10 μg/ml) of nontypable H. influenzae strain 3524 prepared by the method of Johnston, "Immunobiology of Neisseria gonorrhoeae", American Society for Microbiology, 1978, 121-9. Plates were incubated at 37° C. for 1 hour followed by overnight incubation at 4° C. Wells were washed three times with PBS (phosphate buffered saline) plus 0.05% Tween 20® surfactant between each step. Unbound sites on the plastic were blocked with 3% bovine serum albumin in PBS for 2 hours at 37° C. Tissue culture supernatants (or dilutions of mouse ascites fluid in subsequent experiments) containing monoclonal antibody were incubated in the wells overnight at 4° C. Rabbit antibody to mouse IgG and IgM was then incubated for 2 hours at 37° C. followed by protein A-peroxidase for 2 hr at 37° C. Two hundred microliters of substrate was then added to each well. Substrate was prepared by dissolving 10 mg of o-phenyl-enediamine in 1 ml of methanol and adding this solution to 99 ml of citrate-phosphate buffer, pH 5.0, plus 0.1 ml of 3 % H2O2. After the substrate was incubated for 45 min in the dark at room temperature, the reaction was stopped with 50 233 μl of 4N H2SO4. The OD490 was measured. Each set of ELISAs was performed with a control in which NS-1 tissue culture supernatant or ascites fluid was used in place of the monoclonal antibody being tested. On the basis of the results of ELISA screening, selected clones were propagated by subsequent transfer to larger tissue culture wells. Large quantities of antibody were produced in tissue culture and by ip injection of 105 hybridoma cells into pristane-primed BALB/c mice. The resulting ascitic fluid was harvested in three to four weeks and tested for specificity. Wells were coated with a cell envelope preparation (10 μg/ml) of nontypable H influenzae strain 3524 prepared by the method of Johnston, "Immunobiology of Neisseria gonorrhoeae", American Society for Microbiology, 1978, 121-9 followed by overnight at 4° C Wells were washed three times with PBS (phosphate buffered saline) plus 0.05% Tween 20® surfactant between each step were blocked with 3% bovine serum albumin in PBS for 2 hours at 37° C Tissue culture supernatants (or dilutions of mouse ascites fluid in subsequent experiments) containing monoclonal antibody were incubated in the wells overnight at 4° C Rabbit antibody to mouse IgG and IgM was then incubated for 2 hours at 37° C. followed by protein A-peroxidase for 2 hr at 37° C Two hundred microliters of substrate was then added to each well Substrate was prepared After the substrate was incubated for 45 min dark at room temperature On the basis of the results of ELISA screening