Measuring the 50%hemolytic complement CH 50 activity of serum. You'll need a pet able to deliver 200 microliters and one that can deliver 1000 microliters test serum, which has been stored on ice, sensitized sheep, red blood cells, varone buffered saline and distilled water tubes for the test, sir, and tubes for the baseline and turtle lysis, A vortex mixer and a nine six well flat bottom plate labeled the tubes individually with control and the final dilution. In this case, we begin with a one in eight dilution.
Continue with twofold dilution giving rise to a one in 16, one in 32, 1 in 64, and one in 1 28 dilution labeled the remaining tubes blank and total lysis. Once all of the tubes have been labeled, you can now begin to dilute your test serum. Start by transferring 300 microliters of R buffer saline Into a fresh tube.
Now transfer 100 microliters of serum into the 300 microliters of R buffer saline. This will give you a one in four Starting dilution. Ensure that the serum sample is thoroughly mixed.
Now Transfer 200 microliters of roal buffer saline into the tubes that have been labeled previously. Also, Add 200 microliters of roal buffer saline to the two blank tubes to the total lysis tubes. Add 200 microliters of distilled water, not Verona buffet saline.
Now transfer 200 microliters of the one in four diluted serum into the tube labeled one in eight. We have previously transferred 200 microliters of al buffet saline into the one in eight through to one in hundred 28 series of tubes. This will now give rise to a one in eight dilution.
Mix the sample well and then transfer 200 microliters into the tube labeled one in 16. Continue until the entire series of tubes have been diluted. Discard 200 microliters from the one in 1 28 dilution so that the final volume is 200 microliters.
The sensitized sheep red blood cells may have settled over time. These will need to be resuspended by gently inverting the tube a number Of times. Once thoroughly resuspended transferred 200 microliters of the sensitized sheep, red blood cells into each of the prepared tubes.
Ensure the serum is thoroughly mixed with the red blood cells. Sensitized sheep, red Blood cells added to the blank tube will allow you to determine spontaneous lysis while blood added to the total lysis will allow you to determine the 100%value using a vortex mixer set on low gently mix each of the tubes. Now place the tubes into a 37 degrees Celsius water bath and incubate for 30 minutes.
After 15 minutes, Incubation gently vortex the tubes once again and incubate for further 15 minutes After incubation, transfer the tubes to a centrifuge set at four degrees Celsius and Centrifuge at 1500 G for five minutes. After centrifugation, Carefully remove the tubes and place them into an appropriate holder. Here you'll see the pellet of red blood cells while the SNA appears red.
Due to the release of hemoglobin transfer, 100 microliters of distilled water into the required number of wells of a nine to six well flat Bottom plate. Being careful to avoid The pellet transfer 100 microliters of S supernatant into an appropriate well of the nine six Well plate. Once all the samples have been Loaded onto the plate, read the absorption Using a plate spectrophotometer set at five 40 nanometers.
Here we have the raw absorption Data for the two replicates. A one and B one. Refer to the one in eight dilution.
Then we have the data for the one in 16, one in 32, 1 in 64, and one in 1 28. A six and B six are the blank. While A seven and B seven are the total lysis.
Once the data has been collected, you need to calculate the mean absorption for each duplicate sample. Then subtract the blank absorbance, which is your spontaneous lysis from all samples. Once all the data have been collected, you can now calculate the percentage lysis for each dilution using the following formula.
Once all the data have been calculated, plot the percentage lysis on the vertical axis against the serum dilution on the horizontal axis. The resulting graph can then be used to calculate the dilution of serum required to rise 50%of the sensitized she Blood cells. This graph shows the percentage lysis plotted versus the fold dilution of the serum.
A line can then be drawn from the 50%lysis until it intersects with the curve. At the point where the curve is intersected, a vertical line is drawn down to the horizontal axis. This value represents the fold dilution that the serum must be diluted to ensure 50%lysis when comparing two serum samples in order to determine if there is a difference in the CH 50, the dilution, which provides 50%lysis, can be used as a me.
