Directions For Use PTX-001

Product #: PTX-001 anti-GBM

Sheep Anti-Rat Glomeruli (GBM)

For the Introduction of Passive Anti-GBM Nephritis

Materials | Directions For Use | Download Full Data Sheet

Directions For Use:

This package contains sufficient antibody to induce disease in at least 25 rats when used according to the instructions below. Please read carefully before starting the procedure.

1. Preimmunization: To induce an accelerated passive anti-GBM nephritis it is necessary to first immunize rats with sheep immunoglobulin(1). This will allow the rats to produce antibodies to sheep IgG prior to administering the anti-GBM antibody, thus accelerating the course of the disease. If this step is omitted the rats will develop disease, however the response will be weak and more protracted.

A. Mixing the emulsion:

Attention! Always use gloves the FCA contains killed tuberculosis bacterium cell wall. Exposure to skin can permanently sensitize the user.

Each rat will be injected with 0.5 ml of emulsion containing 5 mg sheep immunoglobulin as immunogen. The emulsion is made with equal volumes of FCA and Sheep immunoglobulin solution. For 25 rats we recommend making a total of 15 ml of emulsion 7.5 ml FCA plus 7.5 ml sheep immunoglobulin solution, because the final mixture will be quite thick and recovery is not efficient. Mix the immunoglobulin solution by inversion then withdraw 7.5 ml in 10 ml syringe and put aside. Place a 20 ml syringe with the tip blocked to prevent drainage (included in kit) upright in a bucket of ice. Place 7.5 ml of FCA in the syringe. Firmly grip syringe and insert the homogenizer probe into the syringe bore down into the FCA. Turn on the homogenizer to approximately 15,000 rpm moving the syringe up and down to completely mix the emulsion from top to bottom of mixture. Slowly inject the immunoglobulin solution into the FCA while continuing to mix the emulsion (this should take at least 10 minutes, stop to cool the syringe and emulsion on ice intermittently while mixing). Continue to mix another 5 minutes. The emulsion will be ready when the syringe can be inverted at a 45 angle and the mixture is too thick to run down the side. At this point remove the blockage at the syringe tip and secure an 18-gauge needle. Put the needle in an inverted glass syringe (not included) with plunger open to make space for the emulsion to flow into. 5 ml glass syringes are recommended because 0.5 ml volumes will be injected in rats and larger syringes generally do not have appropriate graduations. Glass is recommended because the thick emulsion in a plastic syringe with a rubber plunger becomes excessively difficult to push and accurate injection volumes are not attainable. Place plastic plunger into the 20 ml syringe and eject the emulsion into the open space in the glass syringe to fill the glass syringe bottom up, without forming an air pocket. When the glass syringe is filled, secure a 20 gauge needle and push the plunger up until the air is fully expelled and the emulsion is at the tip of the needle. The emulsion is now ready for injection. If fewer rats are to be immunized, adjust the volumes proportionately. However the user is advised that recovery of the emulsion is less efficient with smaller volumes and the supplied reagents may be insufficient for a total of 25 rats. Excess sheep immunoglobulin and FCA have been provided to partially compensate for this usage.

B. Immunization: This product has been determined to produce glomerulonephritis in 175-200 gm male Sprague-Dawley rats. Anti-GBM disease can be elicited in other rat strains, however, production of nephritis is genetically predisposed and we cannot guarantee success with other strains or gender. To immunize the rats, one person should hold the rat gently, but firmly by placing a gloved hand over the shoulders of the animal with the head toward the palm. Lift the skin on the nape of the neck, insert the needle and inject no more than 0.1 ml of the emulsion. Repeat in additional subcutaneous sites until a total of 0.5 ml has been injected. Multiple injections of the emulsion are required to elicit a good immune response and to prevent open abscesses.

2. Injection of anti-GBM: Five days after preimmunization with sheep immunoglobulin anesthetize rat(s). Inject 1.0 ml of anti-GBM antibody into a tail vein as a bolus over a 30 second period (Larger volumes are required for heavier rats). Allow the rat to recover and return to cage. Production of disease is dose dependent. For a more fulminant disease inject more volume of antibody. Doses over 2 ml are not recommended. It is important that the complete dose of antibody is delivered iv. If the investigator is not experienced with tail vein injections, we recommended a number of practice injections in sham rats before going forward antibody experiments.

1. Salant DJ, Cybulsky AV: Experimental glomerulonephritis. Meth Enzymol 162:421-461, 1988.

Materials | Directions For Use | Download Full Data Sheet