Span Biotech Ltd.

INTENDED USE

The Leishmania Ab Rapid Test is a lateral flow immunoassay for the qualitative detection of antibodies including IgG, IgM, and IgA to the subspecies of the Leishmania donovani (L. donovani), the Visceral leishmaniasis causative protozoans in human serum or plasma. This test is intended to be used as a screening test and as an aid in the diagnosis of the disease of Visceral leishmaniasis. Any reactive specimen with the Leishmania Ab Rapid Test must be confirmed with alternative testing method(s).

PRINCIPLE

Visceral leishmaniasis, or Kala-azar, is a disseminated infection caused by several subspecies of the L. donovani. The disease is estimated by the World Health Organization (WHO) to affect approximately 12 million people in 88 countries¹. It is transmitted to humans by bites of the Phlebotomus sandflies, which acquire infection from feeding on infected animals. Though it is a disease for poor countries, in Southern Europe, it has become the leading opportunistic infection in AIDS patients^2-3.

Identification of L. donovani organism from the blood, bone marrow, liver, lymph nodes or the spleen provides a definite means of diagnosis. However, these test methods are limited by the sampling method and the special instrument requirement. Serological detection of anti-L. donovani Ab is found to be an excellent marker for the infection of Visceral leishmaniasis. Tests used in clinic include: ELISA, fluorescent antibody and direct agglutination tests^4-5. Recently, utilization of L. donovani specific protein in the test has improved the sensitivity and specificity dramatically^6-7.

The Leishmania Ab Rapid Test is a recombinant protein based serological test, which detects antibodies including IgG, IgM and IgA to the L. Donovani. This test provides a reliable result within 10 minutes without any instrumentation requirements.

TEST PRINCIPLE

The Leishmania Ab Rapid Test is a lateral flow chromatographic immunoassay. The test cassette consists of: 1) a burgundy colored conjugate pad containing recombinant L. donovani specific antigen conjugated with colloid gold (Leishmania conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose membrane strip containing a test band (T band) and a control band (C band). The T band is pre-coated with un-conjugated L. donovani antigen, and the C band is pre-coated with goat anti-rabbit IgG antibody.

When an adequate volume of test specimen is dispensed into the sample well of the test cassette, the specimen migrates by capillary action across the cassette.  Anti- L. donovani Ab if present in the specimen will bind to the Leishmania conjugates. The immunocomplex is then captured on the membrane by the pre-coated antigen, forming a burgundy colored T band, indicating a L. donovani Ab positive test result.

Absence of the T band suggests a negative result. The test contains an internal control (C band) which should exhibit a burgundy colored band of the immunocomplex of goat anti-rabbit IgG/rabbit IgG-gold conjugate regardless of the color development on the T band. Otherwise, the test result is invalid and the specimen must be retested with another device.

REAGENTS AND MATERIALS PROVIDED

1.Each foil pouch contains with three items inside:

a. One cassette device.     

b. One plastic dropper.

c. One desiccant.

2.Sample diluent

3.One package insert (instruction for use).

MATERIALS REQUIRED AND AVAILABLE FOR PURCHASE

1.        Positive Control

2.        Negative Control

MATERIALS REQUIRED BUT NOT PROVIDED

1.        Clock or Timer

WARNINGS AND PRECAUTIONS

·    For professional in vitro diagnostic use only.

·    Do not use after expiration date indicated on the package. Do not use the test if its foil pouch is damaged. Do not reuse tests.

·    This kit contains products of animal origin. Certified knowledge of the origin and/or sanitary state of the animals does not totally guarantee the absence of transmissible pathogenic agents. It is therefore, recommended that these products be treated as potentially infectious, and handled observing the usual safety precautions (do not ingest or inhale).

·    Avoid cross-contamination of specimens by using a new specimen collection container for each specimen obtained.

·    Read the entire procedure carefully prior to performing any tests.

·    Do not eat, drink or smoke in the area where the specimens and kits are handled. Handle all specimens as if they contain infectious agents. Observe established precautions against microbiological hazards throughout the procedure and follow the standard procedures for proper disposal of specimens. Wear protective clothing such as laboratory coats, disposable gloves and eye protection when specimens are assayed.

·    Buffered Saline contains sodium azide which may react with lead or copper plumbing to form potentially explosive metal azides. When disposing of buffered saline or extracted samples, always flush with copious quantities of water to prevent azide build up.

·    Do not interchange or mix reagents from different lots.

·    Humidity and temperature can adversely affect results.

·    The used testing materials should be discarded in accordance with local, state and/or federal regulations.

REAGENT PREPARATION AND STORAGE INSTRUCTIONS

All reagents are ready to use as supplied. Store unused test devices unopened at 2°C-30°C.  The positive and negative controls should be kept at 2°C-8°C. If stored at 2°C-8°C, ensure that the test device is brought to room temperature before opening. The test device is stable through the expiration date printed on the sealed pouch. Do not freeze the kit or expose the kit over 30°C.

SPECIMEN COLLECTION AND HANDLING

Consider any materials of human origin as infectious and handle them using standard biosafety procedures.

Plasma

1.Collect blood specimen into a lavender, blue or green top collection tube (containing EDTA, citrate or heparin, respectively in Vacutainer®) by veinpuncture.

2. Separate the plasma by centrifugation.

3.Carefully withdraw the plasma into new pre-labeled tube.

Serum

1.Collect blood specimen into a red top collection tube (containing no anticoagulants in Vacutainer®) by veinpuncture.

2.Allow the blood to clot.

3.Separate the serum by centrifugation.

4.Carefully withdraw the serum into a new pre-labeled tube.

Blood

1.Drops of whole blood can be obtained by either finger tip puncture or veinpuncture. Do not use any hemolized blood for testing.     

2.Whole blood specimens should be stored in refrigeration (2°C-8°C) if not tested immediately. The specimens must be tested within 24 hours of collection.

Test specimens as soon as possible after collecting. Store specimens at 2°C-8°C if not tested immediately.

Store specimens at 2°C-8°C up to 5 days. The specimens should be frozen at -20°C for longer storage.

Avoid multiple freeze-thaw cycles. Prior to testing, bring frozen specimens to room temperature slowly and mix gently. Specimens containing visible particulate matter should be clarified by centrifugation before testing. Do not use samples demonstrating gross lipemia, gross hemolysis or turbidity in order to avoid interference on result interpretation.

ASSAY PROCEDURE

Bring the specimen and test components to room temperature if refrigerated or frozen. Mix the specimen well prior to assay once thawed.

Step 2:      When ready to test, open the pouch at the notch and remove device. Place the test device on a clean, flat surface.

Step 3:  Be sure to label the device with specimen’s ID number.

Step 4:    For Plasma/ Serum:

               - Dispense 1 drop  (about 40 µL) of the specimen into the sample well

               - Then add 1 drop (about 40 µL) of Sample Diluent immediately

For Whole Blood:

               - Dispense 1 drop (about 40 µL)  of the specimen into the sample well

               - Then add 2 drops (about 80 µL) of Sample Diluent immediately          

Step 5:      Set up timer.

Step 6:   Results can be read in 15 minutes. Positive results can be visible in as short as 1 minute.  

Don’t read result after 30 minutes.  To avoid confusion, discard the test device after interpreting the result.

INTERPRETATION OF ASSAY RESULT

POSITIVE RESULT:

Two colored bands appear on the membrane. One band appears in the control region (C) and another band appears in the test region (T).

NEGATIVE RESULT:

Only one colored band appears in the control region (C). No apparent colored band appears in the test region (T).

INVALID RESULT:

Control band fails to appear. Results from any test which has not produced a control band at the specified reading time must be discarded. Please review the procedure and repeat with a new test. If the problem persists, discontinue using the kit immediately and contact your local distributor.

1. The appearance of any burgundy color in the T band, regardless of intensity, must be considered as presence of the band.

2. Samples with positive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made.

QUALITY CONTROL

Using individual Leishmania Ab Rapid Test cassettes as described in the Assay Procedure above, run 1 Positive Control and 1 Negative Control (provided upon request) under the following circumstances to monitor test performance:

1.        A new operator uses the kit, prior to performing testing of specimens.

2.        A new test kit is used.

3.        A new shipment of kits is used.

4.        The temperature used during storage of the kit falls outside of 2°C-30°C.

5.        The temperature of the test area falls outside of 15°C-30°C.

PERFORMANCE CHARACTERISTICS

Clinical Performance

A total of 234 patient samples from susceptible subjects were tested by the Leishmania Ab Combo Rapid Test and by a commercial L. donovani Ab ELISA kit. Comparison for all subjects is showed in the following table.

       Relative Sensitivity: 91.2 %, Relative Specificity: 99.5%, Overall Agreement: 98.3%

LIMITATIONS OF TEST

1.        The Assay Procedure and the Test Result Interpretation must be followed closely when testing the presence of antibodies to the L. donovani in serum or plasma from individual subjects. Failure to follow the procedure may give inaccurate results.

2.        The Leishmania Ab Combo Rapid Test is limited to the qualitative detection of antibodies to L. donovani in human serum or plasma. The intensity of the test band does not linear correlation with the antibody titer in the specimen.

3.        A negative result for an individual subject indicates absence of detectable anti-L. donovani antibodies. However, a negative test result does not preclude the possibility of exposure to Visceral leishmaniasis causative species of the L. donovani

4.        A negative result can occur if the quantity of the L. donovani antibodies present in the specimen is below the detection limits of the assay, or the antibodies that are detected are not present during the stage of disease in which a sample is collected.

5.        Some specimens containing unusually high titer of heterophile antibodies or rheumatoid factor may affect expected results.

6.        The results obtained with this test should only be interpreted in conjunction with other diagnostic procedures and clinical findings.