IN VITRO PYROGEN TEST (IPT)

Charles River’s patented assay, Endosafe -IPT, is an alternative to the rabbit pyrogen test. IPT can effectively test for gram negative and positive organisms and other biological pyrogens, including yeast, parasitic and viral pyrogens in pharmaceuticals as well as complex biologicals such as HSA and other plasma products. The IPT works by accurately predicting human response to pyrogens on the more relevant basis of human fever rather than animal models.

Test Technology
The IPT is an ELISA assay that utilizes fresh or cryopreserved human whole blood. Immune cells within the blood will recognize exogenous pyrogen and in response release fever-inducing signal molecules that can then be measured.

Interleukin-1 Beta (IL1- ) is a molecule produced in response to gram negative, gram positive and other infectious/inflammatory challenges. It possesses a wide spectrum of immunologic and non-immunologic activities including the capacity to induce fever. If the sample being tested contains any pyrogenic activity, the formation of IL1- is induced and measured by ELISA. A spectrophotometer can then be used to measure the presence of pyrogens. The assay can be quantitative with a standard curve or as a pass/fail rabbit equivalency test.
 
Rabbit Pyrogen Test Replacement
Limitations of the rabbit pyrogen test include species variations by a factor of up to 10,000 and the absence of standardized controls. It is also not possible to test certain drugs such as cytokines, antibiotics, certain sedatives/analgesics, plasma proteins and radiopharmaceuticals. The rabbit pyrogen test does not always identify human pyrogens. A collaborative effort that examines the utility of the IPT as a replacement to the rabbit test is being performed between academic, governmental and industrial partners in Europe.

IPT Product Uses
  • Complex biologics
  • Blood products
  • Cellular therapies
  • Medical devices
  • Environmental air pollutants
  • Veterinary products

Advantages of IPT

  • Testing in relevant species (human – human)

  • Testing in physiological environment

  • Broad pyrogen spectrum, not only LPS

  • High sensitivity

  • Low variability between donors

  • Simple incubation steps

  • Substitution of animal testing

 

 
 

1. Prepare Blood
Draw fresh blood in heparinized tubes. Blood must be used within four (4) hours of collection.

2. Prepare Reagents
Prepare standards, positive and negative controls and samples following package inserts and Certificates of Analysis.

3. Load Samples, Standards and Controls
Using the tissue culture microplate, add 0.2 mL NaCl to wells for standards, negative control and samples and 0.18 mL NaCl to wells for positive sample controls. Next add 0.02 mL of samples and controls to designated wells.

4. Load Blood and Incubate Sample
Add 0.02 mL of blood to each well. Mix the wells
thoroughly using a plate mixer and incubate overnight at 37°C. The next day, remove from incubator and mix the plate until cells are resuspended. Allow cells to settle for approximately 45 minutes.

5. Transfer to Coated Plate
Using the antibody-coated microplate, add 0.1 mL of conjugate to each well. Add 0.1 mL of supernatant to antibody plate. Incubate the plate on a plate mixer at room temperature for 90 minutes.

6. Add Substrate
Wash the plate 4-5 times with wash solution until clear, tapping plate between washes to remove excess wash solution. Add 0.2 mL of TMB substrate to each well. Incubate at room temperature in the dark for 30 minutes. Add 0.1 mL of STOP solution to all wells. Read plate at 405 nm within 15 minutes.

Materials Required

  • Heparinized blood collection tubes/devices

  • Microplate reader

  • Vortex and microtiter plate mixers

  • Plastic containers, troughs and pipette tips

  • Adjustable pipettors and 8-channel micropipettor

  • Microtiter plate washer

  • Heating block or 37 C incubator

  • Non-pyrogenic borosilicate tubes
    and pipette tips

Test Results And Validitys

  • Refer to package insert for specific acceptance criteria.

  • Pyrogen controls must react and/or be linear.

  • Negative control optical density (OD) must be < the OD of the 0.5 EU/mL standard.

  • Sample positive product control OD must be the OD of the 0.5 EU/mL standard.

  • Samples are considered pyrogenic if the OD readings are the OD reading of the 0.5 EU/mL standard.

  • IPT is not a licensed product and should not be used for the release vof drugs.