* For research use only, not for use in diagnostic procedures.
The lymphatic filariasis known as Elephantiasis, mainly caused by W. bancrofti and B. malayi, affects about 120 million people over 80 countries1,2. The disease is transmitted to humans by the bites of infected mosquitoes within which the microflariae sucked from an infected human subject develops into third-stage larvae. Generally, repeated and prolonged exposure to infected larvae is required for establishment of human infection.
The definitive parasitologic diagnosis is the demonstration of microflariae in blood samples3. However, this gold standard test is restricted by the requirement for nocturnal blood collection and lack of adequate sensitivity. Detection of circulating antigens is commercially available. Its usefulness is limited for W. bancrofti4. In addition, microfilaremia and antigenemia develop from months to years after exposure.
Antibody detection provides an early means to detect filarial parasite infection. Presence of IgM to the parasite antigens suggest current infection, whereas, IgG corresponds to late stage of infection or past infection5. Furthermore, identification of conserved antigens allows ‘panfilaria’ test to be applicable. Utilization of recombinant proteins eliminates cross-reaction with individuals having other parasitic diseases6. The Filariasis IgG/IgM Rapid Test uses conserved recombinant antigens to simultaneously detect IgG and IgM to the W. bancrofti and B. malayiparasites without the restriction on specimen collection.
The Filariasis IgG/IgM Rapid Test is a lateral flow chromatographic immunoassay. The test cassette consists of: 1) a burgundy colored conjugate pad containing recombinant W. bancrofti and B. malayicommon antigens conjugated with colloid gold (Filariasis conjugates) and rabbit IgG-gold conjugates, 2) a nitrocellulose membrane strip containing two test bands (T1 and T2 bands) and a control band (C band). The T1 band is pre-coated with monoclonal anti-human IgM for the detection of IgM anti-W. bancrofti and B. malayi, T2 band is pre-coated with reagents for the detection of IgG anti-W. bancrofti and B. malayi, and the C band is pre-coated with goat anti rabbit IgG.
When an adequate volume of test specimen is dispensed into the sample well of the cassette, the specimen migrates by capillary action across the cassette. W. bancrofti or B. malayi IgM antibodies if present in the specimen willbind to the Filariasis conjugates. The immunocomplex is then captured on the membrane by the pre-coated anti-human IgM antibody, forming a burgundy colored T2 band, indicating a W. bancrofti or B. malayi IgM positive test result.
W. bancrofti or B. malayi IgG antibodies if present in the specimen will bind to the Filariasis conjugates. The immunocomplex is then captured by the pre-coated reagents on the membrane, forming a burgundy colored T1 band, indicating a W. bancrofti or B. malayi IgG positive test result.
Absence of any test bands (T1 and T2) 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 any of the test bands. 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 But Not Supplied
MATERIALS REQUIRED AND AVAILABLE FOR PURCHASE
1. Positive Control
2. Negative Control
MATERIALS REQUIRED BUT NOT PROVIDED
1. Clock or Timer
2. Lancing device for whole blood test
Consider any materials of human origin as infectious and handle them using standard biosafety procedures.
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.
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.
Test specimens as soon as possible after collecting. Store specimens at 2°C to 8°C if not tested immediately.
Store specimens at 2°C to 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.
Drops of whole blood can be obtained by either finger tip puncture or veinpuncture. Do not use any hemolized blood for testing.
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.
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.
Step 1: 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 whole blood test##
Apply 1 drop of whole blood (about 40-50 µL) into the sample well. Then add 1 drop (about 35-50 µL) of Sample Diluent immediately.
##For serum or plasma test##
Fill the pipette dropper with the specimen.
Holding the dropper vertically, dispense 1 drop (about 30-45 µL) of specimen into the sample well making sure that there are no air bubbles.
Then add 1 drop (about 35-50 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 15 minutes. To avoid confusion, discard the test device after interpreting the result.##
Interpretation of Results
1. In addition to the presence of C band, if only T1 band is developed, the test indicates for the presence of anti-W. bancrofti or B. malayi IgG antibody. The result is positive.
2. In addition to the presence of C band, if only T2 band is developed, the test indicates for the presence of anti-W. bancrofti or B. malayi IgM antibody. The result is positive.
3. In addition to the presence of C band, both T1 and T2 bands are developed, the test indicates for the presence of both IgG and IgM anti-W. bancrofti or B. malayi. The result is also positive.
##*NOTE:## Samples with positive results should be confirmed with alternative testing method(s) and clinical findings before a positive determination is made.
##NEGATIVE RESULT:## If only the C band is present, the absence of any burgundy color in the both test bands (T1 and T2) indicates that no antiW. bancrofti or -B. malayiantibody is detected in the specimen. The result is negative.
##INVALID RESULT:## If no C band is developed, the assay is invalid regardless of any burgundy color in the test bands. Repeat the assay with a new device.
Using individual Filariasis IgG/IgM Rapid Testcassettes 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.
##1. Clinical Performance For IgM Test##
24 samples from patients with acute lymphatic filariasis and 200 samples collected from a non-filariasis region were tested by the Filariasis IgG/IgM Rapid Test.Comparison for all subjects is showed in the following table: Relative Sensitivity: 95.8%; Relative Specificity: 100%; Overall agreement: 99.6%
##2. Clinical Performance For IgG Test##
26 samples from patients with chronic lymphatic filariasis and 200 samples collected from a non-filariasis region were tested by the Filariasis IgG/IgM Rapid Test. Comparison for all subjects is showed in the following table: Relative Sensitivity: 92.3%; Relative Specificity: 100%; Overall agreement: 99.1%