Step by Step with the ImmunoComb®

The ImmunoComb®: A Diagnostic Tool The ImmunoComb® is based on solid-phase ELISA principle. It provides serological results; namely, measuring antibody levels in blood or serum to specific disease agents (e.g., viruses, mycoplasma, rickettsia).

ImmunoComb® Antibody Test Kit - Contents Developing Plate* Comb* Specimen Paper* Specimen Paper* Specimen Paper* Specimen Paper* Tweezers* Comb Scale* Capillary Tube & piston Note: * Included in all kits. Amount varies according to kit.

Main Components Antigens are attached to each tooth of the Comb. The placement of the antigens is shown by the labeling on the Comb. The developing plate contains the reagents needed for Comb development.

Preparing ImmunoComb® Kit for Use The ImmunoComb® kit is based on an enzymatic reaction, therefore it requires warm temperature. There are two options to reach proper temperature: Another option: Warm it in a 37° C/98.6° F incubator for 20 minutes. One option: Allow to stand in room temperature of 20° – 25° C (68° – 77° F) for 1 - 2 hours.

Obtaining Blood/Serum from Animal A. When using specimen paper Take a specimen paper with prepunched disks. Saturate one pair thoroughly (both sides) with blood. Allow to air dry. 4. Use the tweezers to pierce protective aluminum cover of row A. One well for each sample.

A. When using specimen paper - Continued 5. Punch out one disk of the pair, saturated with blood and immerse it into well of row A. 6. Store the second saturated disk in a refrigerator for possible later use. 7. Wait 60 minutes before proceeding. ATTENTION: Do not open any well that you do not intend to use.

Obtaining Blood/Serum from Animal B. When using serum or whole blood Use the tweezers to pierce protective aluminum cover of well in row A. One well for each sample. For testing whole blood use 10l. For testing serum use 5l. To transfer blood/serum sample from test tube into a well in row A, use either capillary tube or pipette. Capillary tube with piston ATTENTION: Do not open any well that you do not intend to use.

B. When using serum or whole blood - Continued 4. Deposit a sample into well in row A. Raise and lower the piston/pipette plunger several times to achieve mixing. 5. No need for an-hour wait.

Developing the Kit after Inserting Sample(s) Remove the Comb from its protective envelope. For testing less than 12 samples, cut Comb in allocated notches for the number of tests required. Insert the Comb into the open well(s) with the sample(s) in row A (printed side facing you) and incubate. To improve mixing, gently jiggle the Comb up and down at the start of each incubation. Repeat this motion every 2-3 minutes for achieving best results. Timing of incubation depends on type of kit.

Developing the Kit after Inserting Sample(s) 4. Transfer the Comb to the remaining wells (B-F) at timed intervals, according to instruction manual enclosed in kit. 5. Before moving the Comb from one well to the next, pierce with tweezers the foil of next well. Gently shake off excess liquids from Comb’s teeth on a clean paper towel. 6. When developing process is over, allow Comb to air dry completely.

Analyzing Results Example of a Developed Comb for testing Canine Parvovirus & Distemper (3 Teeth) Positive Reference CPV CDV The color intensity of spots corresponds to antibody level.

Example of a Developed Comb for testing Analyzing Results Example of a Developed Comb for testing Poultry IBD – ND –IB C+ Positive control 1 2 3 4 IBD ND IB The color intensity of spots corresponds to antibody level. C- Negative control

Example of a Developed Comb for testing Analyzing Results Example of a Developed Comb for testing Bovine Leptospira C+ Positive control Internal Control L. Hardjo The color intensity of spots corresponds to antibody level. 1 2 3 C- Negative control

Scoring Results There are three ways to read results: (1) by eye, (2) by using the Comb Scale and (3) by using the CombScan 2000. Provides qualitative results Reading results by eye Provides semi-quantitative results Reading results using the Comb Scale Provides quantitative results Reading results using the Comb Scan 2000

Reading Results by Eye Compare the color tone of the test spot with either the positive reference spot (in pet kits) or with the positive control (livestock and poultry kits). There are 4 levels of interpretation:  Negative  Low Positive (suspicious)  Positive  High Positive Charts in manual instruction help user with interpretation of results.

Reading Results using the CombScale Look for the color tone of the positive reference spot on the color scale of the Comb Scale. Slide the yellow ruler until the C+ mark appears in the window corresponding to that color of the positive reference spot.

Continued – using CombScale 3. Hold the ruler in this position while finding the matching color tone of each test spot. 4. The number that appears in the window above the color tone just found is the Comb Scale score (S0 – S6).

Reading Results using the CombScan 2000 The CombScan 2000 is a software program, which utilizes a computer and an office scanner. When a Comb is placed on the scanner the program translates the color results into numerical values. It is free of charge product and it is most useful for large number of tests.

Summary  Contents of kit  Preparing the kit for use  Obtaining blood/serum a. Using paper disks b. Using capillary tubes or pipette  Developing the kit  Reading results and interpreting a. By eye b. Using the CombScale c. Using the CombScan 2000

This is an introductory presentation This is an introductory presentation. In order to achieve best results using the ImmunoComb® test Kit you must read and follow the instructions in the manual instruction provided in each kit.

Thank You! For Technical Support Please Contact: Biogal – Galed Labs Tel: 972-4-9898605, E-mail: info@biogal.co.il www.biogal.co.il