The overall goal of the following experiment is to detect the cardio tropic antibodies in the plasma of patients with dilated cardiomyopathy or other heart diseases with an autoimmune background. This is achieved by an initial mini immuno absorption of patient plasma on anti IgG spheros columns to purify immunoglobulin G as a second step. Ventricular cardiomyocytes are isolated from adult wistar rats providing the cellular base for this bioassay.
Next, the cardiomyocytes are allowed to attach to the chamber cover slips and stained with URA 2:00 AM in order to enable the observation of intracellular calcium transient during contractions, then the patient's IgG is tested to observe its effect on the cardiomyocyte contractility. The results demonstrate whether a patient's IgG contains tropic antibodies based on the changes in contractility and calcium transient in the isolated cardiomyocytes. The main advantage of this technique over existing methods like standard immunoassays for the detection of antibodies is that it is independent of a specific antigen and that it allows to discriminate between stimulating and blocking antibodies.
Demonstrating the procedure will bera Google and experience technical assistance from our laboratory. To begin this procedure, prepare the mini immuno absorption columns by filling three milliliters, anti IgG spheros per two milliliters patient EDTA plasma into an empty econopak column. Next, place a filter on top of the anti IgG spheros.
Cut open the lower tip of the column and press the filter to reach a flow rate of approximately one drop per second. Then wash the column three times with one plasma volume of 0.9%Sodium chloride pipette the plasma in the column when the plasma has completely immersed in the anti IgG Spheros wash with the same volume of 0.9%sodium chloride. Next, pipette one plasma volume of 0.2 molar glycine hydrochloride at pH 2.8 in the column and let it immerse into the phos.
Then add another volume of glycine hydrochloride to the column. Collect the flow in a 15 milliliter tube and adjust the pH to 7.3 with 0.5 molar tris hydrochloride at p pH 8.0 to regenerate the column. Wash it with three plasma volumes of glycine hydrochloride after a final wash with two volumes of 0.9%sodium chloride.
The column may be used for the next plasma sample for the use on cardiomyocytes. The collected IgG fraction has to be dialyzed against experimental buffer by first filling the IgG fraction to a cellulose ester dialysis membrane tube with a molecular weight cutoff at 100 kilodaltons. Then put the tube in one liter EB and stir slowly with a magnetic stir at four degrees Celsius After eight hours, transfer the dialysis tube into three liters of fresh EB and dialyzed for another 20 hours at four degrees Celsius following dialysis.
Boil the sample for 30 minutes at 57 degrees Celsius in a water bath. To inactivate the complement factors, determine the total IgG concentration and prepare aliquots with each containing 330 micrograms IgG. In this procedure, prepare calcium free isolation buffer according to the accompanying manuscript.
Then fill 80 milliliters of IB in the upper reservoir of a thermostat lanor perfusion system and maintain the buffer at 37 degrees Celsius and aerate it with 95%oxygen, 5%carbon dioxide for 30 minutes. Dissolve approximately 7, 000 to 10, 000 units of collagenase type two and 174 milligrams fatty acid-free BSA in 20 milliliters. IB containing 112.5 micromolar calcium chloride and adjust the pH of the isolation buffer in the upper reservoir if required.
Next, anesthetize a wistar rat of 175 to 200 grams using thiopental at 375 micrograms per kilogram with 2, 500 IU heparin added to the thiopental solution. After thoracotomy, excise the heart carefully with an intact aortic arch and transfer it into ice cold, zero point percent sodium chloride in ice cold sodium chloride. Remove the blood and the surrounding tissue from the heart.
Then transfer it to fresh 0.9%sodium chloride and expose the aorta. Turn on the flow reducer of the langor system to obtain a dropping speed of two to three per second. Then attach the heart to the cannula.
Perfuse it for up to three minutes with a flow rate of one drop per second to wash out the remaining blood. After that, circulate the IB in the LOR system. Now remove 20 milliliters buffer from the upper reservoir.
Fill the reservoir with the collagenase solution and refill as much buffer as required to reach 80 milliliters. Then circulate the digestion solution for another 27 minutes. Next, detach the heart from the cannula.
Remove the atria and aorta, then chop the ventricles into small pieces. Allow the digestion buffer to run into the lower reservoir. Reduce the volume to a maximum of 40 milliliters to further digest the chopped ventricles for 10 to 15 minutes under the continuous aeration with 95%oxygen, 5%carbon dioxide.
Afterwards, filter the cell suspension through a 200 micron mesh size gauze into a 50 milliliter tube. Lastly, restore the calcium content of the cells by centrifuge the cell suspension at 40 3G for two minutes. At room temperature, resuspend the cells in 10 milliliters.
IB containing 200 micromolar calcium chloride centrifuge again and resuspend cells in 10 milliliters. IB with 500 micromolar calcium chloride. After a final centrifugation, resus, suspend the cardiomyocytes in sterile filtered EB to a density of 50, 000 cells per milliliter.
Coat the cover glasses in a four well chamber with 10 micrograms laminin per well, and wait until it is fully dried. Then fill one milliliter of the cardiomyocyte suspension into each well. Using a micro pipette with a cut tip and allow the cells to adhere for one hour at room temperature, dilute 10 microliters of the one milligram per milliliter fira 2:00 AM stock solution in five milliliters, eb, and keep the staining solution in the dark.
Next, remove the buffer and the unattached cells from the cover glasses pipette 0.5 milliliters of the staining solution. In each, well incubate the cells for 10 minutes under moderate shaking. Subsequently, remove the staining solution and wash the cells once with one milliliter eb.
Then add 0.5 milliliters EB to each well in this procedure, place the cover glass under an inverse fluorescence microscope connected to a myocyte calcium and contractility recording system. Position a custom made electrode with an influx and influx tube in the first well and super. Fuse the cardiomyocytes with one milliliter per minute eb.
Begin the electric stimulation at 20 volts, one hertz and five milliseconds duration per pulse, and allow the cells to adapt to the stimulation for about two minutes. Choose an intact rod-shaped cardiomyocyte with clear striation and constant contraction and position it in the center of the visual field. Then open the camera channel and orientate the cell horizontally within the video area by rotating the cell framing adapter, and moving the microscope stage.
Adjust the boundary of the detecting control elements of the video area. Turn on the fluorescent channel and record 10 to 15 contractions to obtain the initial cell shortening and calcium transient. After that, turn off the microscope light channel to avoid bleaching of the fluorescent stain.
Switch the flow through from EB to the sample bypass with a three-way valve and super Fuse the cardiomyocytes with one milliliter of patient IgG. Stop the pump just before air reaches the, well turn on the light channel and record for another 10 to 15 contractions to obtain the acute cell response. Then pause the recording and turn off the light channel again, repeat the recordings after two and five minutes and withdraw the electrode from the well when they are finished at the end, analyze the cell shortening and calcium transient with the ion wizard software using the edge length slash length and the URA two numeric subtracted slash ratio window respectively.
Next, calculate the percentage changes for the acute, the two minute and the five minute responses from the initial cell shortening and calcium transient peak height. Here is a representative example of the control measurement. Cell shortening and calcium transient were monitored online before the superfusion with IgG and immediately two minutes and five minutes after the superfusion with IgG.
And here is an example of the measurement of an IgG sample containing cardio depressive antibodies. Cell shortening and calcium transient were measured online before the superfusion with IgG and immediately two minutes and five minutes after the superfusion with IgG. After watching this video, you should have a good understanding of how to detect cardio traffic antibodies in patient plasma by measuring the effect of isolated IgG on cell shortening in calcium transient of isolated adult red cardiomyocytes.
