The overall goal of these procedures is to produce a fusion protein of the anti-inflammatory cytokines IL4 and IL10, control its quality, and evaluate its functional activity and analgesic properties in mirroring models of chronic inflammatory pain. This detailed methods aid in developing novel biologics and test their quality and functionality. The advantage of these techniques is that you can produce a sufficient amount of recombinant fusion proteins, which enable its assessment for its analgesic properties in vivo.
To evaluate the therapeutic potential of the new developed fusion protein, it is important to have a well-characterized batch and to use a variety of behavioral tests. The implication of these methods is that they provide insight in highly needed new therapies for chronic pain but also extend the potential towards other inflammatory diseases. Generally, scientists new to these methods will struggle to be able to perform all procedures shown here, since expertise in different fields is required.
After subculturing 100 milliliters of HEK 293F cells in 500-milliliter Erlenmeyer flasks according to the text protocol, add 6.6 milliliters of DNA transfection reagent mixture to each flask of cells. Culture the cells at 37 degrees Celsius, 8%CO2, and 95%humidity on an orbital shaker rotating at 125 rpm for three days. Then, harvest the culture supernatant containing secreted IL4-10 fusion protein.
To carry out protein purification, pass 100 milliliters of 10-fold concentrated cell culture supernatant through a previously prepared, IL4-coupled Sepharose bead column at a flow rate of one milliliter per minute. Use 50 milliliters of PBS to wash the column. Then, apply 12.5 milliliters of 0.1 molar glycine buffer to elute the bound IL4-10 fusion protein into 2.5 milliliter fractions at a flow rate of one milliliter per minute.
Next, add 350 microliters of one molar Tris buffer, pH 9, to each fraction to bring the pH to 7. Use dialysis tubing with a 16-millimeter dry diameter and a 3.5K molecular weight cut-off to dialyze the neutralized fractions against two liters of PBS overnight. Then, use disposable filters with 0.45-micrometer pore diameter to filter sterilize the dialyzed fractions.
Aliquot the sterile IL4-10 fusion protein solution and store it at negative 80 degrees Celsius. Evaluate the purity of eluted IL4-10 fusion protein by first running a sample on a 12%SDS-PAGE gel and stain the gel with Coomassie blue. Then, carry out high-pressure size-exclusion chromatography by loading 40 microliters of one milligram per milliliter protein onto the column.
Determine the concentration of each batch of purified IL4-10 fusion protein using an IL10 ELISA and a BCA protein assay kit according to the manufacturer's protocols. Use RPMI medium to prepare three-fold serial predilutions of the purified IL4-10 fusion protein over a concentration range of five to 1, 215 nanograms per milliliter. Into the wells of a 48-well plate, add 50 microliters of IL4-10 fusion protein, 75 microliters of RPMI medium, 25 microliters of LPS, and 100 microliters of human blood.
Incubate the plate at 37 degrees Celsius, 8%CO2, and 95%humidity for 18 hours. Then, use a TNF-alpha ELISA according to the manufacturer's protocol to evaluate the concentration of TNF-alpha in culture supernatants. Maintain prepared carrageenan or CFA solutions in syringes with 30-gauge needles at room temperature for at least 15 minutes before injection.
Then, into the thumb of the hind paw of a non-anesthetized mouse, insert the needle approximately 0.5 centimeters, directing it towards the heel and inject 20 microliters of the compound. To carry out pain measurements using the Von Frey test, place an animal into an acrylic cage on a wire-mesh stand and allow the mouse to acclimatize for at least 15 minutes. Apply the Von Frey filaments ranging from 0.02 to four grams perpendicular to the paw surface with sufficient force to cause slight bending against the skin and hold them for approximately three seconds.
Measure the paw withdrawal threshold in response to each hair, considering any movement of the paw away from the applied hair as a positive withdrawal response. Place the animals into cages on a pre-warmed glass plate and allow the mice to acclimate for at least 15 minutes prior to measurements. Determine the heat withdrawal latency times by using a Hargreaves apparatus to heat the paw of the animals with a focused visible light beam.
Before the dynamic weight-bearing test, measure the body weight of each animal. Place an animal in a floor-instrumented dynamic weight-bearing system assembled onto a cover supporting a camera that will record mouse movements. Allow the animal to acclimate for 0.5 to one minute before recording mouse movements for five minutes.
Then, carry out analysis of each video, ensuring that each limb is recognized correctly by the software at the timeframe indicated by the software. Backfill the injectable compounds into a clean 25-microliter glass Hamilton syringe connected to a 27-gauge needle. Place the mouse on the table with its head placed in the nosecone of the apparatus and maintain the mouse sedation with 1.5 to 2%isoflurane with an oxygen flow rate of two liters per minute.
Check that the mouse is properly anesthetized by pinching the paw and ensuring that it is nonresponsive. Hold the mouse firmly by spine posterior to the ribs. Position the needle vertically between the L5 and L6 lumbar vertebrae and carefully insert it through the skin into the intervertebral space.
Confirm correct targeting by verifying that a tail flick occurred. If only a single paw flick is observed, the needle is likely not placed correctly. Then, slowly inject five microliters of the compound, wait a couple of seconds, and slowly retract the needle.
A representative picture of an SDS-PAGE gel containing affinity chromatography fractions is shown here. Two bands of 35 and 37-kilodaltons are observed in the elution fraction corresponding to two different glycoforms of IL4-10 fusion protein. This figure illustrates the results of a potency assay in a whole-blood assay of two different IL4-10 fusion protein batches.
A dose-dependent inhibition of LPS-induced TNF-alpha release is observed, showing comparable bioactivity for the two IL4-10 fusion protein batches. In these experiments, persistent inflammatory pain was induced by intraplantar injection of 2%carrageenan. On day six after induction, mice were injected intrathecally with IL4-10 fusion protein.
IL4-10 significantly resolved both mechanical and thermal hyperalgesia and reached its maximal effect 24 hours after injection. Here, inflammatory pain was induced by an intraplantar injection of 20 microliters of CFA. Weight-bearing on each paw was measured using the dynamic weight-bearing device.
These results show that intraplantar CFA injection reduced weight-bearing of the affected paw. Seven days after intraplantar CFA injection, intrathecal injection of IL4-10 fusion protein was carried out. Two days later, the reduction in weight-bearing of the affected paw was attenuated compared to vehicle-injected mice.
Once mastered, using these procedures, three milligram of fusion protein can be obtained from every liter of cultured cells. It is good to keep in mind to choose expression system compatible with future GMP-production of fusion proteins. To test the analgesic potential of this novel fusion protein, other models of chronic pain can be used, like nerve injury or chemotherapy-induced neuropathy.
A variety of behavioral tests is shown in this video, but other pain tests suggest conditioned place preference could also be performed. Don't forget that intrathecal injections require well-trained personnel and that the injection volume is rather limited. After watching this video, you should have a good understanding of how to produce a fusion protein and test its efficacy in vivo in various pain models.
