Instructions For UseNote:
All reagents should be brought to room temperature before use. Sample Preparation: Dilute all monoclonal antibody samples to a concentration of 1.0 ug/ml in PBS containing 1% w/v bovine serum albumin (BSA).
If the concentration of the sample is entirely unknown, make dilutions based on the following estimates:
Typically, serum contains between 10-15 mg/ml IgG and ascites can be as high as 10 mg/ml . Hollow fibre bioreactor culture supernatants contain approximately 0.5-1.0 mg/ml, whereas static flask tissue culture supernatants usually contain 10-50 ug/ml. Using these estimates, the appropriate dilutions can be made.
150ul of the diluted sample will be added to the development tubes. Assay Protocol:
1. Remove the required number of isotyping strips from the desiccant vial and replace thecap. Remove the caps from an equal number of development tubes. Note:
the tubes may be labeled with a marker for identification.
2. Pipette 150ul of the freshly diluted sample into each development tube and incubate at room temperature for 30 seconds. Vortex the tube briefly to ensure that the coloured micro particle solution is completely re-suspended.
3. Place one isotyping strip, with the solid red end at the bottom, into each development tube. Interpretation of Results:
Interpret the results at 5-10 minutes once the positive flow control bands have appeared. Within 5-10 minutes, a blue band will appear above the letters in one of the class or subclass windows as well as in the kappa window of the strip, indicating the heavy and light-chain composition of the monoclonal antibody (lambda light chains are very rare in rat monoclonal antibodies, but if present no positive band will be seen in the kappa window). The intensity of the blue bands will increase as the sample continues to flow up the strip. The positive flow control bands on one side of the isotyping test strip should also appear, indicating that the antibody-coated micro particles are functional and have flowed up the strip. In cases where the sample is very dilute, the development time may take up to 10 minutes.Note:
For a permanent experimental record or for an easier interpretation of results when testing multiple samples, the solid red area may be cut off the bottom of the strip to prevent further band development once the positive flow control bands have appeared. A gentle stream of air can be applied to the membrane portion of the strip to assist in drying the membrane and preventing any further development. Do not wash the strip to stop the reaction.