H1N1 HA ELISA Kit

Influenza virus Hemagglutinin

RUO, The H1N1 ( A/PuertoRico/8/1934 ) HA ELISA kit is for the quantitative determination of H1N1 (A/Puerto Rico/8/1934 ) HA. This ELISA kit contains the basic componentsrequired for the development of sandwich ELISAs. Each kit contains sufficientmaterials to run ELISAs on five 96-well plates.

Bring allreagents to room temperature before use. 1.Capture Antibody – 0.3 mg/mL of mouse anti-H1N1 ( A/Puerto Rico/8/1934 ) HAmonoclonal antibody. Dilute to a working concentration of 1 ug/mL inCBS before coating. 2.Detection Antibody – 0.5 mg/mL mouse anti-H1N1 ( A/Puerto Rico/8/1934 ) HAmonoclonal antibody conjugated to horseradish-peroxidase ( HRP ). Dilute toworking concentration of 2 ug/mL in detection antibody diluteionbuffer before use. 3.Standard – Each vial contains 50 ng of recombinant anti-H1N1 ( A/PuertoRico/8/1934 ) HA. Reconstitute with 1 mL detection antibody dilution buffer.After reconstitution, store at -20C to -70Cin a manual defrost freezer . A seven-point standard curve using 2-foldserial dilutions in sample dilution buffer, and a high standard of 4 ng/mL isrecommended. 4. CBS - 0.05M Na2CO3, 0.05M NaHCO3, pH 9.6, 0.2 um filtered 5. TBS - 20 mM Tris, 150 mM NaCl, pH 7.4 6. Wash Buffer -0.05% Tween20 in TBS, pH 7.2 - 7.4 7. Blocking Buffer -2% BSA in Wash Buffer 8. Sample dilutionbuffer - 0.1% BSA in wash buffer, pH 7.2 - 7.4, 0.2 umfiltered 9. Detectionantibody dilution buffer - 0.5% BSA in wash buffer, pH 7.2 - 7.4, 0.2 umfiltered 10. SubstrateSolution: To achieve best assay results, fresh substrate solution isrecommended 11. Substrate stocksolution - 10 mg/ml TMB ( Tetramethylbenzidine ) in DMSO 12. Substratedilution buffer - 0.05M Na2HPO4 and 0.025M citric acid, adjust pH to 5.5 13. Substrateworking solution - For each plate dilute 250 ulsubstrate stock solution in 25ml substrate dilution buffer and then add 80 ul 0.75% H2O2, mix it well 14. Stop Solution -2 N H2SO4

1. Plate Preparation 1) Dilute thecapture antibody to the working concentration in CBS. Immediately coat a96-well microplate with 100uL per well of the diluted captureantibody. Seal the plate and incubate overnight at 4C. 2) Aspirate eachwell and wash with at least 300ul wash buffer, repeating the processtwo times for a total of three washes. Complete removal of liquid at eachstep is essential for good performance. After the last wash, remove anyremaining wash buffer by inverting the plate and blotting it against clean papertowels. 3) Block plates byadding 300 uL ofblocking buffer to each well. Incubate at room temperature for a minimum of 1hour. 4) Repeat theaspiration/wash as in step 2. The plates are now ready for sample addition. 2. Assay Steps 1) Add 100 uL of sampleor standards in sample dilution buffer per well. Seal the plate and incubate2 hours at room temperature. 2) Repeat theaspiration/wash as in step 2 of plate preparation. 3) Add 100 uL of thedetection antibody, diluted in antibody dilution buffer, to each well. Sealthe plate and incubate 1 hour at room temperature. 4) Repeat theaspiration/wash as in step 2 of plate preparation. 5) Add 200 uL ofsubstrate solution to each well. Incubate for 20 minutes at room temperature( if substrate solution is not as requested, the incubation time should beoptimized ). Avoid placing the plate in direct light. 6) Add 50 uL of stopsolution to each well. Gently tap the plate to ensure thorough mixing. 7) Determine theoptical density of each well immediately, using a microplate reader set to450 nm.

SENSITIVITY The minimumdetectable dose of H1N1 ( A/Puerto Rico/8/1934 ) HA was determined to beapproximately 62.5 pg/ml. This is defined as at least three times standarddeviations above the mean optical density of 10 replicates of the zerostandard.