IgM ELISA Protocol
Acquired splenocytes from lab technicians. We placed an equal amount of splenocytes into two test tubes. We used two separate test tubes in case of contamination or an insufficient amount of B cells in one tube..We mixed the cells with lab media. ACK buffer was then mixed was then added to each test tube. The ACK buffer changes the pressure of liquid, causing the red blood cells to rupture. The two test tubes were then placed into a centrifuge at 15 000 RPM. The immune system cells are the heaviest cells in our mixture, after being the spun the immune system cells would sink to the bottom of the tubes. The liquid was then poured out by decantation and a small white pellet remained in the bottom of the tube. The cells were then resuspend using a Pipette. This process was repeated four times to wash the cells free of the ACK buffer. Once all the liquid was removed we counted the cells. We then chose the test tube that contained the highest concentration of live b-cells. The concentration was then adjusted to a dilution of 2,000,000,000 cells/ml. We then pipette 200,000 cells or 100ul per well on a flat-bottom 96 well sterile plate. We then prepared our treatments in triplicate. We used media as our negative control, 1ul/ml of LPS as the positive control, our unknowns are 500ug echinacea polysacarride(EP), 500ug B- Glucan, 100ug B- Glucan, 50ug B- Glucan, 500ug B- Glucan + EP, 100ug B- Glucan + Ep, 50ug B -Glucan + EP. The plate was then incubated for 72 hours at 36C. This allows time for possible b-cell stimulation. After three days we attempted to collect 150ul of supernatant from each well. Unfortunately this was not always possible, as not all treatments yielded 150ul. Instead the supernatants were dilated to the appropriate amount with PBS. We pipette the supernatant into an ELISA coated with capture antibodies. The IgM antibodies would then adhere to the capture antibody. We then store the plate at -20C overnight. Next day, pipette 200ul of standards (see Appendix A). Incubate plate for half an hour at 37C. We then washed the plate three times with PBS (Phosphate buffer solution, see Appendix A) With 300ul per well or 25cc per plate. This removes any supernatants that did not adhere to the capture antigen. The plate is then blotted to remove any liquid. We then pipetted 100ul of detector antibody into each well. The plate is incubated for 30 minutes at 37C. Another three PBS washes remove any unused capture antibody and the plate is blotted again. We then pipette 100ug of the substrate; the substrate reacts with the IgM causing the colour change(blue). The darker the colour the greater the amount of IgM present. We then incubate the plate for 30 minutes at room temperature. We then pipetted 100ug of the stop solution. The stop solution stops the colour changes and turns the blue to a yellow colour. We then wait 15 minutes and placed the plate in the optical density microplate reader.
Proliferation Assay
Acquired splenocytes from lab technicians. We placed an equal amount of splenocytes into two test tubes. We used two separate test tubes in case of contamination or an insufficient amount of B cells in one tube..We mixed the cells with lab media. ACK buffer was then mixed was then added to each test tube. The ACK buffer changes the pressure of liquid, causing the red blood cells to rupture. The two test tubes were then placed into a centrifuge at 15 000 RPM. The immune system cells are the heaviest cells in our mixture, after being the spun the immune system cells would sink to the bottom of the tubes. The liquid was then poured out by decantation and a small white pellet remained in the bottom of the tube. The cells were then resuspend using a Pipette. This process was repeated four times to remove all liquid and non-immune cells. The other immune system cells were ignored since only B cells should under go mitosis, which is our variable for this experiment. Once all the liquid was removed we counted the cells. We then chose the test tube that contained the highest concentration of live b-cells. The concentration was then adjusted to a dilution of 2,000,000,000 cells/ml. We then pipette 200,000 cells or 100ul per well on a flat-bottom 96 well sterile plate. We then prepared our treatments in triplicate. We used media as our negative control, 1ul/ml of LPS as the positive control, our unkowns are 500ug echinacea polysacarride(EP), 500ug Beta Glucan, 100ug B- Glucan, 50ug B-glucan, 500ug B-glucan + EP, 100ug B-glucan + Ep, 50ug B -glucan + EP. Incuabte plate at 37C for 48 hours. After 48 hours add 1ug/ml of tritiated thymidine. Tritated thymidine is radioactive therefore when the cells under go mitosis the radioactive particles are absorbed into the b-cell DNA. After 12 hours of incubation the cells are harvested by a cell harvester and the cells are pressed unto glass fibre paper. The radioactivity is checked by a scintillation counter. Since the particles are absorbed at a known rate, it is possible to tell how many new b-cells have formed by how radioactive the paper is.
We performed the IgM ELISA and proliferation experiment, as outlined in our lab journal, with an IgM ELISA Testing Kit. Our materials included EP(Echinacea polysaccride) LPS( lippolsacaride) B -Glucan and Lab Media. We prepared a sandwich ELISA comparing LPS, a known B cell stimulant and our positive control, EP, which we expected a negative reaction from, lab media, as our negative control, and B -Glucan, as our unknown. We tested for the IgM antibodies the B cells release and whether or not they multiplied by mitosis. These procedures are outlined in our lab journal. The Optical density reader gave us the amount of IgM produced by each stimulant in ng/ml.
The proliferation test requires the use of radiation and we therefore could not count the cells. An assistant counted the cells for us and we graphed and sorted the data using Microsoft Excel. The Igm ELISA data was organized in the same way.