The overall goal of this procedure is to identify antigens for antibodies with therapeutic potential with a focus on the IgM isotype. This method fills a knowledge gap in the biomedical field and may help to identify novel biomarkers and immunotherapeutic agents for cancer research and diseases of the central nervous system. The main advantage of this technique is that it opens the door to a neglected class of antibodies with great therapeutic potential.
The implications of this technique extend toward the diagnoses and treatment of all diseases that can be targeted by antibody-based therapeutics. What this matter can provide insight into antigens of IgM antibodies and growing tissue and subcultures. It can also be a blight to other antibody isotypes from all species.
I first had the idea for this method when I realized specific and prominent binding of the IgM antibody in different biochemical applications. Demonstrating the procedure will be Robert Kahoud and Jens Watzlawik. To begin this procedure, make a cut on the skull toward the front at the ear level and peel back the skull.
Then, gently place the brain into a 15 milliliter tube, and immediately store it on dry ice. After five minutes, transfer the brain to a clean weighing paper and quickly remove the cerebellum and brain stem using a clean razor blade. Next, weigh the frozen cerebrum before transferring it back into the 15 milliliter tube on ice.
Add ice-cold lysis buffer to a final concentration of 150 micrograms of brain tissue per microliter of lysis buffer. Then, homogenize the brain in lysis buffer by trituration through a one milliliter pipette tip followed by trituration through a five milliliter syringe equipped with a 27 gauge needle. After that, incubate the brain lysate for an additional 30 minutes on ice to allow complete tissue lysis.
After 30 minutes, remove detergent, insoluble material, and brain lipids to serial centrifugation. Discard the myelin and cell debris containing pellet, but retain the supernatant after every centrifugation step. Aliquot the brain lysate, and store at 80 degrees Celsius.
In this step, prepare 200 milliliters of IP Buffer with BSA and another 200 milliliters of IP Buffer without BSA. For the immunoprecipitation reaction, transfer 100 microliters of Protein L agarose slurry into a 1.5 milliliter microcentrifuge tube. Add one milliliter of IP buffer with BSA to the tube.
Then, mix it manually by inversion. Next, wash Protein L agarose beads four times by centrifugation. After that, remove the supernatant without disturbing the agarose pellet and repeat the washing procedure three more times.
Then, add fresh BSA containing IP buffer to the Protein L agarose and incubate it overnight on a roller at four degrees Celsius. In 800 microliters of IP buffer containing BSA and 30 milligrams of lysed brain tissue, add 20 micrograms of experimental antibody, control antibody, or PBS. Incubate 30 milligrams of lysed brain tissue in ice cold IP buffer with IgM antibody in a 1.5 millimeter microcentrifuge tube overnight at four degrees Celsius.
The next day, spin down Protein L agarose beads in a benchtop centrifuge. Discard the supernatant without disturbing the agarose pellet. Then, add the chilled antibody brain lysate solution to the Protein L agarose.
Incubate the antibody antigen, Protein L agarose suspension for two hours on a roller at four degrees Celsius. After two hours, wash the antibody antigen complex attached to agarose L through centrifugation. Carefully discard the supernatant without disturbing the pellet and add one milliliter of ice cold IP buffer containing 0.2%BSA to it.
Repeat the washing step one more time using ice cold IP buffer containing 0.2%BSA and two additional times using ice cold IP buffer without BSA. After that, spin down the antibody antigen complex attached to agarose L.Use a 200 microliter pipette to discard the supernatant until about 50 microliters of the solution is left on top on top of the Protein L agarose pellet. Then, use a 10 microliter pipette to completely remove it.
Keep all the samples on ice. Next, add 40 microliters of IP elution buffer to each 1.5 milliliter microcentrifuge tube. Finger flick the tube six times and heat it for five minutes at 95 degrees Celsius.
After that, place the sample on ice for two minutes and spin it down for 30 seconds. Then, transfer 35 microliters of the supernatant into a fresh 1.5 millimeter microcentrifuge tube without disturbing the pellet. Next, confirm the absence of Protein L agarose beads by rinsing down a small amount of diluted sample at the inner tube wall.
If Protein L agarose beads are present, spin down the sample for another 30 seconds at 13, 000 times g and transfer the supernatant to a clean tube. Repeat this step until no agarose beads are detectable. Then, load 10-20 microliters of diluted samples per well on an SDS polyacrylamide gel and a tris-glycine SDS buffer system and run the gel for about one hour at 100 volts on the benchtop without additional cooling.
Susequently, transfer the proteins to a PVDF membrane. Proteins are transferred for two and a half hours in the cold room at 100 volts using cold transfer buffer. Block the membrane with 10%weight per volume dry milk powder in PBS-T for one hour at 25 degrees Celsius on a benchtop orbital shaker.
Wash it twice for 10 minutes each time with PBS-T. Probe the membrane with primary antibody in 5%BSA in PBS-T. Incubate on an orbital shaker in the cold room overnight.
The next morning, wash the membrane once with excess PBS-T at 25 degrees Celsius followed by three washes with PBS-T for 10 minutes each on an orbital shaker. Next, add secondary antibody and 5%dry milk powder in PBS-T to the membrane and incubate it for one hour at 25 degrees Celsius on an orbital shaker. After that, wash the membrane once with excess PBS-T at 25 degrees Celsius followed by three washes with PBS-T for 10 minutes each on an orbital shaker.
Mix one milliliter of chilled enhanced chemiluminescence HRP substrate component A with one milliliter of component B at 25 degrees Celsius. Transfer the membrane on a paper towel to remove excess liquid. Then, dry the container with paper towels before transferring the membrane back to it.
Immediately, add two milliliters of the pre-mixed enhanced chemiluminescence HRP substrate to the membrane and incubate it for two minutes at 25 degrees Celsius. Tilt the container by hand every 20 seconds to moisten the membrane uniformly. After that, transfer the membrane into a transparent plastic sleeve and remove excess liquid and air bubbles with paper towels.
Tape the plastic sleeve into the cassette and close it. This figure shows that HigM12 immunoprecipitates its antigen from the cerebral brain lysates and acts as a detecting agent in Western blots. Neither isotype control antibody nor agarose L beads immunoprecipitate a similar antigen which demonstrates the specificity of the pull down.
Here, Western blots using CNS lysates from wild type and NCAM knockout mice demonstrate the specificity of HIgM binding to PSA containing NCAM`Enzymatic digestion of CNS tissue from wild type mice using ENDO-NF identifies PSA attached to NCAM as the specific binding apitope for HIgM12. Using the method outlined herein, IBA-1 positive rat microglial cultures of high purity are obtained. In contrast to microglia, HIgM12 and A2B5 target cell surface antigens were highly enriched in rat OPC cultures under live cell conditions.
Once mastered, this technique can be done within 13 working hours. Following this procedure, other methods like Biacore or surface plasmon resonance technology can be employed to identify the antibodies binding affinities to association and association constants. After its development, this technique paved the way for researchers in the field of cancer research, immunotherapies, and natural autoantibodies to identify carbohydrate apitopes on like proteins as antigens for therapeutic IgM antibodies in humans.
After watching this video, you should have a good understanding of how to identify glycoprotein antigens for research work containing human IgM antibodies. The data provides a new target for MS therapy. PSA ENCAP, the antibody treatment we have developed is one of the first to show functional improvement in an activity box using the rigorous model that's in the disease and devaluing disease similar to Multiple Sclerosis.
