This video demonstrates the steps involved in performing the lips. Assay antigens are expressed in CO one cells as recombinant ran vanilla luciferase or rook antigen fusions. Crude extracts are obtained and used without purification.
The lips assay is initiated by incubating crude rook antigen extracts with patient SRA. In Microtiter wells the antibody antigen mixture is then transferred to a 96 Well filter plate containing protein A G beads to capture IgG molecules after washing the filter plate containing the protein AG beads. Antibody bound rook antigen is measured by the addition of linine substrate and light units are measured with a luminometer.
Hi, I'm Dr.Catherine Chang from the Laboratory of Sensory Biology at the National Institute of Dental and C Craniofacial Research. Today we'll show you the procedure for lips luciferase immunoprecipitation systems. We use lips in our laboratory to measure antibody titers in order to diagnose disease and or infection.
Subst stratify disease states monitor vaccination and other applications involved in generating antibody profiles. So let's get started. Prior to beginning transfection plasmids for vanilla luciferase fusions are prepared in advance as described in the BMC biotechnology paper.
BYO etal 2005 plasma DNA is prepared using a MIDI prep kit with a yield of one to three milligrams and stored as a one milligram per milliliter stock solution at minus 20 degrees Celsius. Cost one cells used in transfection are cultured in DMEM 10%FCS using standard tissue culture protocols. One day before transfection split costs one cells into new 100 by 20 millimeter dishes to approximately two times 10 of the six cells per plate and incubate at 37 degrees Celsius on the following day.
The cost one cells should be 80 to 95%Confluent allow the FU gene six transfection reagent which is stored at four degrees Celsius to warm up to room temperature while waiting for the FU gene to warm label 1.5 milliliter polypropylene micro fuge tubes for each plasma DNA to be transfected being careful not to touch the sidewall of the tubes. Add six microliters of fusion gene six to the tubes, which should already contain 94 microliters of optimum media incubate for five minutes. At room temperature, add one to two micrograms of plasmid for ran Lucifer antigen fusion construct mixed by inverting the tube several times, centrifuge briefly and incubate at room temperature for 15 minutes.
Transfer the entire DNA fusion six optimum solution to the cells by dripping it evenly into the media of the cos. One cells incubate transfected cos one cells for two days then harvest. To begin harvesting fusion protein, remove media and wash the cells with six milliliters of PBS.
Pipe it away any residual PBS from the tissue culture dish add 1.4 milliliters of cold lysis, buffer and harvest cells with a cell scraper and quickly transfer half of the lysate to each of two 1.5 milliliter micro fuge tubes on ice. A Branson SOLIDIFIER one 50 is used to break the cells open. Place the micro centrifuge tube containing the cell lysate on ice and pulse for five seconds.
Five seconds and five seconds. With sonication settings of two, two and four respectively. Centrifuge the cell lysate at 12, 500 RPM for two four minutes.
Spins at four degrees Celsius. Gently inverting the tubes between spins to remove the loosely attached debris from the sidewall of the tube. Then carefully transfer the supernatant without disrupting the pellet from the two tubes to a new micro fuge tube on ice.
To measure the light units or lu of the lysate dilute one microliter of lysate with eight microliters of PBS in a new micro fuge tube directly add 100 microliters of one celent ine substrate to the diluted mixture and immediately measure luminescence in the tube using a tube luminometer with a five second read from this step forward. Remember to use safety precautions when working with human and animal S samples to make a serum master plate the source of patient antibodies for antigen profiling. First add 450 microliters of buffer A to each well of a 96 deep well polypropylene microtiter plate.
At this step, a dye phenol red can also be included in buffer A to act as a tracer from monitoring future SRA sample edition and other steps of the lips assay. Next, add 50 microliters of serum from each sample to each well containing 450 microliters of buffer. A note, this is a one to 10 dilution of the SRA in buffer.
A typically SRA is not added to the last two wells of the master plate because this is reserved for the buffer blanks before storing the master plate for future use. It is extensively shaken for one to two hours on a rotator platform. At 650 RPM, the serum is stable for at least one month or longer at four degrees Celsius and the plate can be sealed with parfum to prevent evaporation if it will be stored prior to running the lips assay, organize a spreadsheet containing the sample identification numbers and their associated wells on the master plate polypropylene 96 shallow well microtiter plates are used as working plates to test sera.
In the first step, add 40 microliters of buffer A to each well of the 96 well plate using an eight channel micro pipette. Next, take 10 microliters of diluted serum from the master plate and add it directly to each well of the working plate using an eight channel micro pipette. After use seal the master plate with paraform, replace the lid on the master plate, then cover with Saran wrap and store at four degrees Celsius.
A master mix containing the rook antigen extract is next Formulated such that one times 10 to the seventh lu is added in 50 microliters of buffer A to each. Well make this master mix mixture and pipette 50 microliters of rook antigen mixture to each. Well cover the plate with a polystyrene plate cover and incubate on a rotary shaker at 100 RPM for one hour.
At room temperature during the incubation retrieve a 1.5 milliliter aliquot of a 30%ultra link protein ag bead suspension in PBS from the fridge. Add five microliters to the bottom of each well of a new 96 well filter HTS plate After the one hour plate incubation transfer the 100 microliters of rook antigen antibody reaction mixture to 96. Well filter HTS plates containing the protein ag beads using an A channel micro pipette incubate the 96 well filter plate on a rotary shaker for one additional hour at 100 RPM at room temperature.
Next, wash the filter plate using a Biome FX automation workstation With a vacuum manifold, each well is washed eight times with 100 microliters of buffer A followed by two times with 100 microliters of PBS following the last wash, the vacuum is turned off. Remove the filter plate and blotted dry using a stack of paper towels or filter paper, making sure to remove moisture on the top and bottom of the plate. A Burt hold LB nine 60 Centro Microplate luminometer is used for determining luminescence in each well of the plate.
Turn the machine on and rinse the injector with distilled water. Using the injector wash cycle INE substrate is used for priming and running the machine and is prepared using the Promega ranil substrate kit following manufacturer's instructions, typically six milliliters of one excellent tine substrate mix is needed for one plate. Load the substrate mixture into the luminometer open a program file containing the setting for injecting the substrate and reading the plate.
For these measurements. 50 microliters of soine substrate is injected. The plate is shaken for two seconds, followed by a five second read of luminescence.
Start the program which initiates reading of the plate after the run, remove the microtiter filter plate promptly to prevent spillage in the luminometer. The data is automatically exported from the microwind program into an Excel format for further analysis. Averaging results for two different runs with the same sera.
In order to generate lu titer values for each sample is recommended. The duplicate samples should show high concordance in titer values between the two runs. For data plotting, the lu titer values can further be adjusted to subtract the buffer blanks as shown here.
The sensitivity and specificity of the assay can also be calculated by utilizing cutoffs derived from the mean plus three or five standard deviations of the control samples. We've just shown you how to measure antibody titers Using lips, LIPS requires minimal assay optimization and because of its simplicity can be used to generate high quality data in under two weeks for any given antigen. When working with human Sarah, it is important to remember to take appropriate safety precautions.
And keep in mind, lifts is highly scalable and because of this can be used to measure a small number of samples using manual washing or a full plate using a robotic working station with such versatility, we think lips will be highly useful in sero antigenic analysis and have broad implications in disease analysis and the public health in general. Well, that's it. Thanks for watching and good luck with your antibody profiling experiments.
