Alpha-amylase 1 ELISA 试剂盒

• ELISA Microplate(Dismountable)
• Lyophilized Standard
• Biotin-labeled Antigen(Concentrated, 100X)
• HRP-Streptavidin Conjugate(SABC, 100X)
• TMB Substrate
• Sample Dilution Buffer
• Antibody Dilution Buffer
• SABC Dilution Buffer
• Stop Solution
• Wash Buffer(Concentrated, 25X)
• Plate Sealer
• Product Description

1. Microplate reader (wavelength:450nm)
2. 37 °C incubator
3. Automated plate washer
4. Precision single and multi-channel pipette and disposable tips
5. Clean tubes and Eppendorf tubes
6. Deionized or distilled water

1. Wash the plate twice before adding the standard and sample.
2. Add 100ul standard/sample/biotin-labeled antigen working solution into each well. Seal the plate and statically incubate for 45 minutes at 37°C.
3. Wash the plate three times and immerse for 1min each time.
4. Add 100ul HRP-Streptavidin Conjugate (SABC) working solution into each well, seal the plate and statically incubate for 30 minutes at 37°C.
5. Add 90ul TMB substrate solution, seal the plate and statically incubate for 10-20 minutes at 37°C. (Accurate TMB visualization control is required.)
6. Add 50ul stop solution. Read at 450nm immediately and calculate.

Take the Elisa kit from the fridge around 20 minutes earlier and equilibrate to room temperature(18-25℃). For repeated assays, please just take the strips and standards required for the current assay, store the rest materials according to the relevant condition.

Wash Buffer

1. Dilute 30ml concentrated wash buffer to 750ml wash buffer with deionized or distilled water and mix well. (The recommended resistivity of ultrapure water is 18MΩ.)
2. Alternatively, take appropriate amount of concentrated wash buffer according to the assay requirement, then create a 25-fold dilution and mix well. Store the rest solution at 2-8℃.
3. Crystals formed in the concentrated wash buffer can be heated by water bath at 40℃till complete dissolution. (Heating temperature should be below 50℃.) Mix well for the next step. It's better to use up the prepared wash buffer in one day. Store the rest buffer at 2-8℃ within 48h.

1. Centrifuge standards tube for 1min at 10000xg. Label it as Zero tube.
2. Add 1ml sample dilution buffer into the standard tube. Tighten the tube cap and Let it stand for 2minatroom temperature. Invert the tube several times to mix gently. (Or you can mix it using a low speed vortex mixer for 3-5 seconds.)
3. Centrifuge the tubes for 1min at 1000xg, making the liquid towards the bottom of tube and removing possible bubbles.
4. Standard dilution: Label 7 EP tubes with 1/2, 1/4, 1/8, 1/16, 1/32, 1/64 and blank respectively. Add0.3ml of the sample dilution buffer into each tube. Add 0.3ml solution from zero tube into 1/2 tube and mix them thoroughly. Transfer 0.3ml from 1/2 tube into 1/4 tube and mix them thoroughly. Transfer 0.3ml from 1/4 tube into 1/8 tube and mix them thoroughly, so on till 1/64 tube. Now blank tube only contains 0.3ml sample dilution buffer. Notes: Store the zero tube with dissolved standards at 2-8℃ and use it within 12h. Other diluted working solutions containing standards should be used in 2h.

1. Dissolve: Centrifuge for 1min at 2000xg and bring down the concentrated biotin-labeled antigen to the bottom of tube. Add 70ul purified water into tube and mix them thoroughly, after the biotin-labeled antigen is dissolved, please store it at 2-8°C.
2. Calculate required total volume of the working solution: 55ul/well x quantity of wells. (It's better to prepare additional 100ul-200ul.)
3. Dilute the biotinylated detection antigen with antigen dilution buffer at 1/100 and mix themthoroughly. (e.g. Add 10ul concentrated biotin-labeled antigen into 990ul antigen dilution buffer.)

1. When samples and standards are not tested repeatedly: 1. Take 55ul prepared standard of each gradient and 55ul biotin-labeled antigen working Solution and mix them well in EP tube for later use. 2. Take 55ul diluted sample and 55ul biotin-labeled antigen working Solution and mix well in EP tube for later use.
2. When the sample and standard are tested in duplicate: 1. Take 110ul prepared standard of each gradient and 110 ul biotin-labeled antigen working Solution and mix them well in EP tube for later use. 2. Take110ul diluted sample and 110ul biotin-labeled antigen working Solution and mix well in EP tube for later use.

1. For unopened kit: All the reagents should be kept according to the labels on vials. The Reference Standard and the 96-well stripe plate should be stored at -20 °C upon receipt while the other reagents should be stored at 4 °C.
2. For used kit: When the kit is used, the remaining reagents need to be stored according to the above storage condition. Besides, please return the unused wells to the foil pouch containing the desiccant pack, and zip-seal the foil pouch.