The CapiliaTM - TiBiliaTM rapid test kit for MPB64 is a immunochromatographic (ICA) assay using anti-MPB64 monoclonal antibodies for direct qualitative detection of the antigen MPB64, which present in high concentrations in culture fluids of M. tuberculosis complex strains in human sputum, bronchial aspirate, bronchoalveolar lavage, gastric fluid, and racheal aspirate specimens. The kit is also for discriminating between the Mycobacterium tuberculosis complex and mycobacteria other than tubercle bacilli (MOTT).
PRINCIPLE:
The ICA test kit for MPB64 consists of a plastic plate with a sample pad, a reagent pad, a nitrocellulose membrane, and an absorbent pad. On the test plate, there is a sample well where the sample is applied and two test reaction zones where the results of the test and control are observed as pink to reddish purple bands. An aliquot of 100 μl sample is applied to the test well, and it flows through the nitrocellulose membrane laterally. The monoclonal antibody against MPB64, which is conjugated with colloidal gold and is immobilized at the membrane, is rehydrated by the liquid from the sample and reacts with the MPB64 antigen if present in the sample. The MPB64-antibody complex then flows further laterally and is captured by the test band, where anti-MPB64 antibody is present, producing a color band. A secondary anti-mouse immunoglobulin G antibody immobilized at the control band reacts with the free gold conjugate antibody when the sample continues to migrate through the control band area and gives another reddish purple color band. The test is read after 15 min. If the test band is positive it indicates the presence of the M. tuberculosis complex. The control band color indicates that all of the test reactions performed satisfactorily and should be positive in every testing.
REAGENTS AND MATERIALS PROVIDED:
1. 20 Individual pouches, each containing:
(1) Test device: Membrane assembly pre-dispensed with MPB64McAb - colloidal gold conjugate, MPB64McAb and anti-mouse antibody at the respective regions.
(2) Desiccant Pouch.
2. Package Insert
No instrumentation was required.
SPECIMEN COLLECTION AND PREPARATION
*Use only for the detection of members of the M. tuberculosis complex using sediments prepared following the NALC-NaOH or NaOH procedures recommended by the Centers for Disease Control and Prevention (CDC). This test may only be used with concentrated sediments prepared from sputum (induced or expectorated), tracheal aspirates, or bronchial specimens (e.g., bronchoalveolar lavages or bronchial aspirates).
1. SPECIMEN COLLECTION:
Sputum
(1) 4% NaOH method
·To x ml of sputum add 2x ml of 4% NaOH.
·Tighten cap of container and shake to digest.
·Take 0.1ml deposit on to two slopes of L-J or take 0.5ml to liquid medium.
(2) N-acetylcysteine (NaOH-NALC) method
- Prepare NALC-NaOH-NaC/sputum mixture : 2ml/2ml
- Votex completely
- Let stand 15-20 minutes at room temperature
- Add PBS(pH6.8) to bring to 15 ml and invert tube 4 times
- Centrifuge x 15 minutes @ 3000 x g
- Decant supernatant and re-suspend pellet in 1ml PBS(pH6.8)
- Take 0.1ml deposit on to two slopes of L-J or take 0.5ml to liquid medium.tracheal aspirates, or bronchial specimens (e.g. bronchoalveolar lavages or bronchial aspirates)
- To x ml of specimen add x ml of 4% NaOH.
- Tigid if no bands appear on the device. Repeat the test with a new device ensuring that the test procedure has been followed accurately.
LIMITATIONS:
1. A negative result does not rule out infection by Tuberculosis. All M. tuberculosis and M. africanum cultures were found to be positive, whereas the results of M. bovis and M. bovis BCG cultures were variable since some of the BCG strains are known to lack MPB64 antigen production.
2. As with all diagnostic tests, the test results must always be correlated with clinical findings. The results of the test are to be interpreted within the epidemiological, clinical and therapeutic context.
3. Any modifications to the above procedure and / or use of other reagents will invalidate the test procedure.
4. Do not compare the intensity of the test line and control line to determine the concentration of the MPB64 protein in the test specimen.
PRECAUTIONS:
1. For In Vitro Diagnostic Use.
2. Avoid contact of samples with skin, eyes, and mucous membranes. Wash with water if contact with these samples.
3. Use universal precautions when performing this test. Preparation of digested and decontaminated sediments, and MTD procedures, should be done using Biosafety Level 2 practices. It is strongly recommended that the biosafety cabinehten cap of container and shake to digest.
4.Let stand 30 minutes at 37℃.
5.Take 0.1ml deposit on to two slopes of L-J or take 0.5ml to liquid medium.
2 Sample preparation
The digested, decontaminated, and concentrated specimens were inoculated into liquid broth or on L-J slopes at 37°C. Löwenstein-Jensen slants were examined weekly for eight weeks for visible appearance of colonies. All cultures, positive in liquid media or L-J, will be applied to the ICA test slide directly without any manipulation.
PROCEDURE:
1. Open a pouch containing a cassette and lay the cassette on a flat surface.
2. Carefully apply 100μl culture sample onto test well.
3. Wait and let sample solution absorb on the membrane.
4. Read the result in 15 minute.
INTERPRETATION OF RESULTS:
Negative: Only one pink band appears on control region of the Cassette. This indicates that there is no detectable MPB64 protein in the sample.
Positive: Two pink bands appear on control and test region of the Cassette. This indicates that the specimen contains detectable amount of MPB64 protein.
Invalid: The test should be considered invalt used for specimen processing not be used for performing the MTD test.*For more inquiries about the product, please contact manufacturer's official website below.
Product Manufacturer: Hangzhou Genesis Biodetection & Biocontrol CO., Ltd.
Website: www.hgb.com.cn
0 comments :
Post a Comment