Leishmania Antigen ELISA (VL ELISA)
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1 Leishmania Antigen ELISA (VL ELISA) A double antibody sandwich enzyme immunoassay for the detection of the Visceral Leishmaniasis antigen in human urine. For Research Use Only Catalogue Number E M Kalon Biological Ltd Unit G Perram Works Merrow Lane Guildford GU4 7BN United Kingdom Telephone: +44 (0) Fax: +44 (0) info@kalonbio.co.uk
2 INTENDED USE An enzyme-linked immunosorbent assay for the detection of a leishmania antigen found in urine specimens collected from patients with visceral leishmaniasis (kala-azar). This product is intended For Research Use Only by trained laboratory personnel. INTRODUCTION Visceral leishmaniasis (VL) or kala-azar is a disease caused by infection with various species of the intracellular protozoan parasite Leishmania. The disease is transmitted by sandflies from reservoirs of infection which include domestic dogs, cats and wild animals. The disease is endemic on the Indian subcontinent, as well as parts of East Africa, South America and around the Mediterranean basin. It is characterised by a widespread infection of the monocytes, particularly in the liver and spleen. The symptoms of VL are not specific and include a prolonged, unexplained fever, hepatosplenomegaly, pancytopaenia and weight loss. In co-infections with HIV many of these clinical signs are missing and over 40% are VL antibody negative, yet co-infected patients have been found to have high urinary concentrations of the leishmania antigen. Identification of the parasite in bone marrow or spleen aspirates by microscopy is the gold standard of diagnosis This test has been developed in collaboration with FIND and DNDi to determine the levels of antigen present in the urine of patients diagnosed with VL before, during and after treatment. As the antigen detected in urine remains largely uncharacterised, a novel unit, the Urinary Antigen Unit (UAU) has been created. The calibrators included in the kit cover the range 0 to 50 UAU/mL. In the two studies conducted to date, the observed range of the Leishmania spp. antigen was found to range from 1 to UAU/mL. These two studies also showed a rapid decrease in Leishmania antigen levels in most individuals following treatment with anti-leishmanial drugs. From these two studies an initial dilution of 1 in 20 is considered appropriate for urine specimens. Urine specimens giving results above the top of the range will need to be re-assayed at the higher dilution of 1 in 400. The current field study has been designed to measure the Leishmania antigen levels at 14 time points (daily from days 1 to 12 then at days 14, 28, 56 and 210). PRINCIPLE OF THE ASSAY The assay is based on the double antibody sandwich format. Polystyrene microtitre plate wells are supplied pre-coated with sheep anti-leishmania antibodies. During the first incubation in which calibrators and urine specimens are incubated in the microwells, leishmania antigen (if present) is immobilised onto the plate. Unbound urine components are removed in the following wash step. The surface is then probed for bound leishmania antigen with enzyme conjugated sheep anti-leishmania antibody (Tracer). Following a second wash step to remove unbound Tracer, any bound Tracer is detected by incubation with enzyme substrate and chromogen (TMB Substrate). The enzyme reaction generates a coloured product which is halted by the addition of dilute acid. The final colour is measured photometrically at 450 nm. The optical density is proportional to the level of leishmania antigen present in the test sample. This is quantified by the construction of a standard curve.
3 KIT PRESENTATION q E q E Antibody coated 96 well plate 8-well strips (microwell strips) sensitised with sheep antileishmania antibody. VL ELISA CAL (Calibrators) A set of six, pre-diluted calibrators. They contain approximately 0, 2.0, 5.0, 12, 20 and 50 UAU/mL leishmania antigen: the exact value is printed on each vial. Ready to use do not dilute. q E Tracer (41 ) Sheep anti-leishmania antibody labelled with peroxidase. Requires dilution before use. q R1-01 Assay Diluent Buffered saline with protein stabiliser, surfactant and 0.1 g/l Thiomersal preservative. q R2-01 Wash Concentrate (40 ) Buffered saline and surfactant. Requires dilution before use. 96 wells 500 µl 500 µl 50 ml 50 ml q R7-01 q R5-01 TMB Substrate Contains a peroxide source, TMB chromogen and stabilisers. Ready to use. Stop Solution 0.5M HCl Solution. Ready to use. q Resealable plastic bags 2 q Sterilin 96-well microplate An untreated solid microplate for diluting urine samples only. Do not confuse with E Store the kit refrigerated. Do not use beyond the expiry date printed on the label. Do not mix with components from different batches of kits. 12 ml 15 ml 1 ADDITIONAL REQUIREMENTS q Precision micropipettes with disposable tips to cover the range 10 to 1000 µl (preferably multichannel pipettes for volumes of 10 and 100 µl). q Vortex mixer. q 37 C incubator. q Microtitre plate washer. q Microtitre plate reader with a 450 nm filter. q Timer q Laboratory-grade purified water q Tubes suitable for diluting urine samples or blank microtitre plates q Reagent dispensing troughs. q Clean volumetric laboratory plastic or glassware. q Absorbent paper towels.
4 q General laboratory consumables and equipment including protective clothing and biohazardous waste containers. SAFETY PRECAUTIONS Treat all urine samples - and the materials they come into contact with - as potentially infectious. Wear appropriate protective clothing and dispose of used materials and reagents accordingly. The kit reagents are all classified as non-hazardous however and in general, avoid contact with eyes, skin and mucous membranes. If any reagents do come in contact with these sites rinse with copious amounts of water. If symptoms persist seek medical attention. KIT STORAGE AND STABILITY Once opened, microwell strips may be stored at 2 to 8 C until the expiry date on the label provided that desiccated conditions are maintained. Unused strips should be returned to the original foil pouch along with the desiccant sachet and re-sealed. If correct humidity conditions are not maintained the desiccant will change colour from orange to green. If this occurs the microwells should not be used. All components should be stored at 2 to 8 C and should not be used beyond the expiry date as indicated on the label. The Wash Concentrate contains no preservatives and therefore the working strength wash solution should not be stored for longer than 4 weeks at 2 to 8 C or 1 week at room temperature. If the TMB Substrate turns blue on storage or shortly after dispensing, it is likely caused by chemical contamination and should not be used. SPECIMEN COLLECTION AND STORAGE All urine samples must be frozen overnight to -20 C or below prior to testing. Fresh (unfrozen) urine samples may give false positive results. Thaw all urine samples and mix thoroughly before testing. Extra freeze-thaw cycles do not affect the results obtained. ASSAY PROCEDURE Preparation 1) Thaw all urine samples and bring to room temperature before use. Note: all urine must be stored frozen prior to use. Prepare an appropriate dilution of each specimen to be tested (see the Procedural Notes). To dilute 1 in 20, add 10 µl urine sample to 190 µl Assay Diluent. Mix each well immediately by gently pumping the diluted urine in and out of the micropipette five times. To dilute 1 in 400, add 10 µl of the 1/20 sample dilution to 190 µl Assay Diluent and mix as above. 2) Prepare the required volume of working strength Tracer by diluting Tracer (41 ) in Assay Diluent , e.g. add 300 µl Tracer (41 ) concentrate to 12 ml Assay Diluent. 3) Prepare the required volume of working strength wash solution by diluting Wash Conc. (40 ) 1 in 40 with purified water, e.g. make 25 ml Wash Conc. (40 ) to 1 L with purified water.
5 Primary Incubation 4) Dispense 100 µl of each VL Antigen Calibrator and test sample in duplicate into designated microwells. 5) Place the plate in a Resealable Plastic Bag and incubate at 37 C for 30 minutes. 6) Wash the microwells - Alternately fill and aspirate the microtitre wells with 350 µl working-strength wash solution a total of four times. Tap out residual wash solution on clean absorbent towelling ready for the next step. Do not allow the plate to dry before the next step. Secondary Incubation 7) Dispense 100 µl working strength Tracer into each microwell. 8) Return the plate to the Resealable Plastic Bag and incubate at 37 C for 30 minutes. 9) Wash the microwells as before. Enzyme Incubation 10) Dispense 100 µl TMB Substrate solution into each microwell. 11) Incubate the plate uncovered at 18 to 25 C for 30 minutes. 12) Dispense 100 µl Stop Solution into each microwell. Assay Completion 13) Read the microwell optical densities (OD) at 450 nm within 30 minutes of adding Stop Solution. The microtitre plate reader should be blanked on air or else blanked using the nm reading. 14) Construct a standard curve using all the VL Antigen Calibrator points. It is recommended to fit the curve using four parameter logistic curve fitting software. Alternatively a standard curve can be created on graph paper with a flexicurve and pen. 15) Verify the assay. The assay is considered to have been executed correctly when the procedure has been followed correctly and the OD for the 50 UAU/mL standard is more than 1.5 and the OD for the 0 UAU/mL calibrator is less than ) All wells with an OD value < 2.0 UAU/mL calibrator should be classified as Antigen Not Detected (ND). 17) Read the remaining test urine values off the standard curve and correct for the dilution factor. Urine sample S1 tested at 1/20 dilution. Value read off standard curve = 10 UAU/mL; S1=10 20=200 UAU/mL. Urine sample S2 tested at 1/400 dilution. Value read off standard curve = 18 UAU/mL; S2=18 400=7200 UAU/mL.
6 EXPECTED VALUES Figure 1. A typical standard curve The leishmania antigen was detected in 100 out of 105 (95.2%) urine samples in a study conducted on parasite-confirmed visceral leishmaniasis patients from Bangladesh. A smaller number of samples collected from patients from Kenya, where the leishmania urinary antigen was detected in 18 out of 18 (100%) parasite-confirmed patients, indicates a similarly, high sensitivity. Leishmania Antigen was not detectable in urine samples from healthy individuals from Bangladesh (48/48) or Kenya (17/17). We do not have data on this test for patients with alternative diagnoses (such as malaria, tuberculosis, HIV) and so cannot comment on the specificity of this test. PROCEDURAL NOTES These notes are provided as supplementary information to the instructions detailed in the Protocol. Each kit contains sufficient VL ELISA Calibrators to perform two assay runs. Ensure all reagents are at room temperature before use. When opening the foil pouch containing the Antibody Coated 96-Well Plate, check that the desiccant sachet is orange. A green colour indicates that moisture has entered the bag and in this instance the plate should be discarded. Select the correct number of 8-well strips required: For example, to test 18 urine specimens and six VL ELISA Calibrators in duplicate will require a total of 6 strips (48 wells). Remove surplus 8-well strips from the frame and reseal inside the foil pouch with the desiccant sachet. Do not mix strips from different kit boxes. Each assay run should contain the six VL ELISA Calibrators in duplicate, so smaller runs should not be considered.
7 When a large number of urine samples are to be diluted it is recommended to use a blank microtitre plate, the diluted urine samples can then be efficiently transferred to the assay wells. Gently but completely mix all dilutions: this is essential for accurate test results. In a study conducted in Bangladesh, leishmania antigen levels declined by more than 90% 14 days after commencing treatment and decreased by more than 96% after 45 days. Estimate of the proportion of urine samples which will fall with the assay range (from 2 to 50 UAU/mL) at different times post treatment. Day 0 Day 3 Day 7 Day 14 Day 18 1/20 61% 75% 68% 31% 19% 1/400 44% 31% 6% 0% 0% The decrease in antigen levels seen in this study should be used as a guide to estimate the proportion of urine samples which are expected to fall inside the range of the Leishmania Antigen ELISA. q Samples collected on day 0 and the following first 3 days of treatment: initially test at dilutions of 1/20 and 1/400. q Use the results available from previous time points as a guide to the best dilution to use. q Retest samples at higher dilution if the test OD is too high (greater than 50 UAU). Do not contaminate kit reagents. Always use fresh pipette tips when drawing from stock reagent bottles. Liquids should be dispensed with the tip of the micropipette touching the side of the microwell at a point about midway down. Incubate the assay plate inside a sealed polythene plate bag provided with the kit to avoid evaporation during incubation. Dilute the Tracer (41 ) in Assay Diluent one part in 40 before use. For example, add 150 µl Tracer to 6 ml Assay Diluent for 6 strips. Add 300 µl to 12 ml Assay Diluent for 12 strips. Dilute Wash Concentrate (40 ) in purified water one part in 39 before use. For example, make 25 ml Wash Concentrate up to 1 Litre in a measuring cylinder. Efficient rinsing to remove the unbound components after each incubation step is a fundamental requirement of the ELISA procedure. The plate washer must be well maintained to prevent contamination from previous use. The manufacturer s cleaning procedures must be followed. After washing ensure that all excess wash buffer has been removed from the microwells by tapping the inverted plate onto a pad of paper towelling. Immediately proceed on to the next step of the protocol and do not allow the plate to dry between steps. Dispense 1 ml TMB Substrate per strip used into a reagent trough. If the TMB Substrate appears blue or starts to turn blue before use, discard and dispense fresh reagent into a new clean reagent trough. Never return unused TMB Substrate to the bottle. Measure the microwell optical density (OD) at 450 nm and subtract either the air blank or nm reading to correct for the background OD. The stopped substrate solution (yellow) will fade slowly in bright light so take the OD measurements within 30 minutes of adding the Stop Solution.
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