Mycobacterium bovis – Mycobacterium tuberculosis Antibody Test Kit PrimaTB STATPAK^® Assay

INTENDED USE

The PrimaTB STATPAK Assay is a qualitative, single use, twostep, immunochromatographic screening test for the detection of antibodies to Mycobacterium tuberculosis and Mycobacterium bovis in Macaque serum, plasma or whole blood. The test is used for the diagnosis of active tuberculosis (TB) in conjunction with other diagnostic methods.

If specific antibodies are present in the sample, the expected test result is reactive. A reactive result is suggestive of active TB. In the absence of antibodies, the expected test result is nonreactive.

SUMMARY AND EXPLANATION

TB is considered to be one of the most important bacterial diseases of macaques because of its frequency of occurrence, its ability to spread rapidly, its high fatality rate, and its zoonotic potential. TB in macaques is most often caused by the same organism that causes TB in humans – M. tuberculosis – and the disease spreads readily from simian to man and vice versa. The second most common type of TB in macaques, accounting for about 15% of cases, is due to M. bovis that is also transmissible to and from humans and/or other animals [15].

The present method of monitoring macaques for TB the USDAapproved test for delayed hypersensitivity in response to an intradermal injection of mammalian tuberculin into the skin of the eyelid – has very serious shortcomings. While the incidence of false positives and false negatives varies from batch to batch of tuberculin, false results have tended to be a problem with all skin tests aimed at diagnosing simian TB [6, 7].

Serological methods constitute an attractive alternative, as they are simple, inexpensive, relatively noninvasive, and they do not depend on detection of mycobacteria. None of the existing TB tests alone is sufficient to diagnose disease. Therefore, new TB diagnostic algorithms are being developed, in which serological assays may play an important role (see PERFORMANCE CHARACTERISTICS below) [810].

The Chembio PrimaTB STATPAK Assay is a rapid immunochromatographic test for antibody detection that is safe, simple, and easy to perform.

PRINCIPLE OF TEST

The Chembio PrimaTB STATPAK Assay is based on immunochromatographic (lateralflow) technology. The test employs a unique cocktail of recombinant M. tuberculosis proteins that are bound to the membrane solid phase. Blue latex particles conjugated with protein are used as the detection system. The PrimaTB STATPAK Assay can be used with serum, plasma or whole blood. Once a test sample is applied to the SAMPLE (S) well followed by the addition of a diluent, it flows laterally through the membrane strip. When it reaches the conjugate pad, antibodies, if present, bind to proteinlatex conjugate and then the migrating immune complex binds to the antigens on the solid phase in the TEST (T) area producing a blue line. In the absence of antibodies there is no line in the TEST (T) area. The sample continues to migrate along the membrane and produces a blue line in the CONTROL (C) area demonstrating that the reagents are functioning properly.

MATERIALS PROVIDED

Each kit contains the following items:

• 5 PrimaTB STATPAK test devices
• 5 Disposable pipettes
• 1 Sample Diluent vial (5ml)
• 1 Product insert
• Additional Material Required But Not Provided
• Clock, watch or other timing device
• Pipettor capable of delivering 30 �L
• Disposable gloves
• Sterile gauze (for fingerstick whole blood specimens)
• Sterile lancets (for fingerstick whole blood specimens)
• Antiseptic wipes
• Biohazard disposal container
• Collection devices for specimens (other than fingerstick whole blood specimens)

STORAGE AND STABILITY

The PrimaTB STATPAK Assay should be stored at 8 to 30^oC in the original sealed pouch. The diluent should be stored in the original vial at 8 to 30^oC. The kit is stable until the date imprinted on the box label and/or pouch. NOTE: Do not use expired test kits. CAUTION: Do not freeze test kits.

PRECAUTIONS

1. The test is designed FOR IN VITRO DIAGNOSTIC USE only. Use the test only in accordance with instructions supplied with the kit.
2. Handle all specimens as recommended for any potentially infectious human serum or blood specimen in the CDCNIH manual, Biosafety in Microbiological and Biomedical Laboratories, 4^th ed., 1999.
3. Use suitable protective clothing (gloves, lab coat, safety glasses) when handling samples or test devices after samples have been applied. Avoid any contact between hands, eyes, nose or mouth during specimen collection and testing.
4. Do not pipette any material by mouth. Do not smoke, eat or drink in areas where specimens or kit material are kept.
5. All testing should be performed at a temperature of 18 to 30^oC.
6. After the completion of the assay, carefully dispose of materials treating them as biohazardous waste.
7. Do not use expired test kits. Do not freeze test kits.
8. Do not mix reagents from different kit lots.

SPECIMEN COLLECTION

The PrimaTB STATPAK Assay can be performed on whole blood, serum or plasma.

Whole Blood: Collect whole blood into tubes containing heparin or EDTA. Follow test procedure instructions. Optionally, fingerstick blood may be used via the disposable pipettes included with the test kit. If so, follow the sampling and sample application procedure for fingerstick specimens in the TEST PROCEDURE section.

Serum: Serum is used from whole blood collected aseptically by venipuncture into a clean tube without anticoagulant. Allow the blood to clot at room temperature, centrifuge at 2000 rpm for 10 minutes at room temperature, 18 to 30^oC, and separate the serum from the clot.

Plasma: Collect whole blood with anticoagulant (heparin, EDTA or sodium citrate), centrifuge at 2000 rpm for 10 minutes at room temperature, 18 to 30^oC, and isolate the plasma supernatant.

Samples perform best when tested immediately after collection. Specimens should be immediately refrigerated at 2 to 8°C following collection and can be used up to 3 days. If testing within 3 days is not possible, the specimens should be frozen at 20ºC or colder until use. Avoid repeated freezing and thawing. DO NOT FREEZE WHOLE BLOOD.

NOTE: If specimens are to be shipped, they should be packed in compliance with regulations covering the transportation of etiologic agents. Venous whole blood, serum and plasma specimens should be shipped refrigerated with cold packs or wet ice.

Figure 1

Fill line indicates total sample collected

Step 1: Prepare to perform the fingerstick blood collection procedure. Clean the finger with an antiseptic wipe. Allow the finger to dry thoroughly or wipe dry with a sterile gauze pad. Using a sterile lancet, puncture the skin just off the center of the finger and wipe away the first drop with sterile gauze. Avoid squeezing the fingertip to accelerate bleeding as this may dilute the blood with excess fluid. Collect the sample from the second drop as explained in Step 2.

Step 2: Once the drop of blood has formed on the finger, hold the tube horizontally and touch the tip of the pipette to the sample. Capillary action will draw the sample to the black fill line and stop. Test immediately.

CAUTION!

Filling is automatic. Never squeeze the pipette while sampling.

5a. For Venous Whole Blood, Serum or Plasma: Add the 30 �l of specimen onto the center of the SAMPLE (S) well. (See Figure 2 below)

Figure 2

5b. For Fingerstick Blood: Step 3: To expel the 30 �l sample, align the tip of the pipette with the sample well and squeeze the bulb.

ONLY IF THE SAMPLE DOES NOT COME OUT OF THE TUBE, hold the pipette vertically and slide the finger over the vent hole. Then align the tip with the sample well and squeeze the bulb.

6. Once the specimen has been applied to the SAMPLE

(S) well, invert the diluent bottle and hold it vertically (not at an angle) over the SAMPLE well. Add the diluent slowly dropwise; add 3 drops (~100 �l) into SAMPLE (S) well. (See Figure 3)

Figure 3

1. Read results at 20 minutes after the addition of diluent. Do not read any results after 30 minutes. Refer to INTERPRETATION OF RESULTS section below.
2. Discard the used disposable pipette, test device and any other test materials into a biohazard waste container.

Control Line No Test Line

Control Line

Test Line

Invalid Results

A blue line should always appear in the CONTROL (C) area, whether or not a line appears in the TEST (T) area. If there is no distinct blue line in the CONTROL (C) area, the test is invalid and should be repeated using a new device.

No Control Line

Test Line

No Control Line

No Test Line

LIMITATIONS OF THE PROCEDURE

1. The assay is designed for detecting antibodies against

M. tuberculosis and M. bovis only from macaque plasma, serum or whole blood. Any result from the testing of other body fluids or of pooled serum or plasma samples should not be used.

2. A reactive result suggests the presence of antibodies to

M. tuberculosis and/or M. bovis.

1. For a reactive result, the intensity of the test line does not necessarily correlate with the titer of antibody in the specimen.
2. Reading nonreactive results earlier than 20 minutes or any results later than 30 minutes may yield erroneous results.
3. Do not use hemolyzed blood samples.
1. Be careful to add only 30 �L of specimen and 3 drops of diluent after applying the specimen to the SAMPLE
2. (S) well.
4. Do not open the sealed test pouch until just prior to use.
5. Do not use kit contents beyond labeled expiration date.
6. Read results in a welllit area.

Table 1. Diagnostic sensitivity of PrimaTB STATPAK

Table 2. Diagnostic specificity of PrimaTB STATPAK

REFERENCES

1. Bernacky B.J., Gibson S.V., Keeling M.E. and Abee C.R. (2002) Nonhuman primates, p.676777 In J.G. Fox, L.C Anderson, F.M. Loew, and F.W. Quimby (ed.), Laboratory Animal Medicine, Second ed. Academic Press, San Diego, Calif.
2. CDC (1993) Tuberculosis in imported nonhuman primates – United States, June 1990May 1993. MMWR 42:572576.
1. Gibson, S.V. (1998) Bacterial and mycotic diseases, p.59
2. 110. In C. R. A. B. Taylor Bennett, and R. Henrickson (ed.), Nonhuman primates in biomedical research. Academic Press, San Diego, Calif.
3. Kalter S.S., Millstein C.H., Boncyk L.H., and Cummins L.B. (1978) Tuberculosis in nonhuman primates as a threat to humans. Dev. Biol. Stand. 41:8591.
4. Garcia M.A., Bouley D.M., Larson M.J., Lifland B., Moorhead R., Simkins M.D., Borie D.C, Tolwani R., and Otto G. (2004) Outbreak of Mycobacterium bovis in conditioned colony of rhesus (Macaca mulatta) and cynomolgus (Macaca fascicularis) macaques. Comp. Med. 54:578584.
5. Kaufmann A.F. and Anderson D.C. (1978) Tuberculosis control in nonhuman primate colonies. In R.J. Montali, (ed), Mycobacterial infections of zoo animals. Smithsonian Institution Press, Washington, D.C.
6. Walsh G.P., Tan E.C., DelaCruz E.C., Abalos R.M., Villahermosa L.G., Young L.J., Cellona R.V., Nazareno J.B., and Horwitz M.A. (1996) The Philliphine cynomolgus monkey (Macaca fasicularis) provides a new nonhuman primate model of tuberculosis that resembles human disease. Nature Med. 2:430436.8.
7. Lyashchenko K., Colangeli R., Houde M., Jahdali H.A., Menzies D., and Gennaro M.L. (1998) Heterogenous antibody responses in tuberculosis. Infect. Immun. 66: 39363940.
1. Lyashchenko K.P., Singh M., Colangeli R., and Gennaro
2. M.L. (2000) A multiantigen print immunoassay for the serological diagnosis of infectious diseases. J. Immunol. Methods 242: 91100.
8. Brusasca P.N., Peters R.L., Motzel S.L., Klein H.J., and Gennaro M.L. (2003) Antigen recognition by serum antibodies in nonhuman primates experimentally infected with Mycobacterium tuberculosis. Comp. Med. 53:165172.

FOR MORE INFORMATION, CONTACT:

CHEMBIO DIAGNOSTIC SYSTEMS, INC. 3661 HORSEBLOCK ROAD MEDFORD, NY 11763 USA U.S. Veterinary License No. 645 Tel : 18003273635 Tel: (631) 9241135 Fax: (631) 9246033 Email: info@chembio.com Web Site: www.chembio.com