-1-INTENDED USE CANINE T&S IgE TEST KIT is designed to determine the levels of Total IgE (T IgE) and Specific IgE (S IgE) against different allergens in canine serum or plasma. -2-DESIGN AND PRINCIPLE For one sample testing, one Solid Array Unit, one Solution Unit and one Substrate should be used together. The Solid Array Unit, which contains an array composed of location markers, anti-canine IgE antibody, and allergens on a membrane and a protective cap, is packaged in one aluminum foil bag with a desiccant. The Solution Unit contains all the necessary reagents for forming enzyme linked complex of antibody-antigen reaction that are deposited separately in the different compartments of a plastic cartridge and sealed with a protective aluminum foil. The Substrate is deposited in a small substrate bottle. Briefly, pull open the Solution Unit and deposit the serum sample in the compartment 1 of the Solution Unit and mix well. After tearing aluminum foil bag, take the Solid Array Unit out and pull off the protective cap. Immobilized location markers, anticanine IgE antibody, and allergenic substances can be observed as pink spot array on the membrane in the window of the Solid Array Unit. Then insert the Solid Array Unit into the compartment 1 and have it absorb the solution in the compartment 1 for a few minutes. After the absorption, the pink dye will disappear from the membrane in the window, which indicates successful specific antibody-antigen reaction finished. -3- Then the Solid Array Unit will be transferred to the remaining compartments at timed intervals step by step. The bound canine IgE antibodies on the spot array will be labeled with enzyme in the compartment 3, which contains anti-canine IgE-enzyme conjugate. For a satisfactory result, wash steps are introduced. In the compartment 2, the unbound canine IgE antibodies and other substances in the serum sample will be removed. In compartment 4,5and 6,the unbound or excess enzyme conjugate will be adequately removed. At the end, pipette substrate in the substrate bottle, and slowly drop the substrate on the membrane at the window center to develop purple-blue spots if there were enzyme bound there. To confirm the validation of the performance, purple-blue color of the location markers on the membrane should be visible above a certain level after finishing a successful testing process. The location markers will be always visible on the membrane in the window of the Solid Array Unit after successful testing. By placing the transparent Locator on the window of the Solid Array Unit in correct position, the Total IgE and Specific IgE spots can be located. By comparing the visible spots with the Color Scale provided, the signal strength can -4-be obtained and the levels representing the clinical interpretation can be recorded by hand in the Result Card provided according to the INTERPRETING TEST RESULTSTEST PROCEDUREPreparation before performing the test: 1. Bring one Solid Array Unit, one Solution Unit and one Substrate to room temperature (20*C-30*C) for 30 minutes before using. 2. Prepare a dispenser and two pipette tips proper for 200pL and lOOOpL. 3. Stand upright the Solution Unit on a work bench and confirm that compartment numbers, from 1 to 8, can be seen in correct direction. Stamp the Solution Unit slightly to make sure the solutions in the compartments, -5-from 1 to 6, turn back to the bottom.Performing the test: 1. Hold tightly the solution cartridge with one hand and pull the protective foil along the horizontal direction carefully with another hand from the compartment 1 to 8 to remove whole the protective foil off. 2. Obtain 200pL of the tested serum or plasma sample with a proper dispenser set with a pipette tip. EDTA or heparin anticoagulant tubes are recommended for plasma sample collection. -6- 3. Deposit the sample into the compartment 1. Then raise and lower dispenser plunger several times to achieve mixing 4. Tear open the aluminum foil bag, take the solid-state array unit out, and then remove the protective cap. 5. Insert the Solid Array Unit into the compartment 1 for 20 minutes. 6. Pick up the Solid Array Unit and insert it into the compartment 2 for 10 minutes. 7. Pick up the Solid Array Unit and insert it into the compartment 3 for 20 minutes.

8. Pick up the Solid Array Unit and insert it into the compartment 4 for 10 minutes. 9. Pick up the Solid Array Unit and insert it into the compartment 5 for 10 minutes. INTERPRETING TEST RESULTS Specific IgE Comparing with provided Color Scale, there are four conditions illustrated as the following table. Notes: Total IgE Comparing with provided Color Scale, there are three conditions illustrated as the following table. 10. Pick up the Solid Array Unit and insert it into the compartment 6 for 10 minutes. 11. Pick up the Solid Array Unit and lay it flat on a work bench. 12. Pipette the Substrate...