Q: How should samples be stored?

A: For long-term storage of samples, store at -80°C or lower is recommended. For saliva samples, assay results did not change significantly after storage at room temperature for 6 hours, in a refrigerator for 24 hours at -20°C for 1 month or at -80°C for 6 months. (Fluctuations in room temperature were within ±10% of the temperature at hour 0.)

Please avoid repeated freeze-thaw cycles as much as possible. Based on our experience, for saliva samples collected via the passive drool method and stored at -80°C, if the number of freeze-thaw cycles is limited to no more than 5, the measured values will remain within 10% of the initial value.

Q: What is the recommended method of collection of saliva samples?

A: The passive drool method is recommended for the collection of saliva samples. (the passive drool method requires subjects to salivate into a vial or tube.)

It has been confirmed that samples collected using cotton swabs or inert polymer swabs can be used similarly to samples collected by the passive drool method. However, caution should be taken because some swab materials may absorb target analytes.

Q: What is the pretreatment method for the samples?

A: Please dilute the samples adequately. Heat treatment or similar processes are not necessary.

• Human Serum/Plasma: 2-fold dilution
• Human Saliva: 4-fold dilution
• Mouse Serum/Plasma: 2-fold dilution
• Rat Serum/Plasma: 2-fold dilution

Q: How should I pretreat highly viscous saliva samples?\

A: For highly viscous saliva samples, first freeze-thaw the sample, then centrifuge (e.g., 1,500 x g, 15 minutes, 4°C). Use the supernatant as the sample and proceed with the preprocessing (dilution) as described in the instructions provided with the kit.

Freeze-thawing will precipitate mucin, a substance that interferes with the measurement.

Q: What reagents, instruments, and equipment are required for the assay using this kit?

A: The following are required.

• Purified water (distilled water)
• Tubes for dilution of standard solutions and samples
• Glassware for dilution of Wash Solution (graduated cylinders, beakers)
• Micropipettes (for 10μL disposable tips and 200 to 500 μL disposable tips)
• Continuous dispensing pipette, capable of dispensing 100 μL continuously (preferred if available)
• Paper towels or other absorbent material (to remove liquid left on the plate after washing)
• Vortex-type mixer
• Microplate shaker (range approx. 600 to 800 rpm)
• Automatic washer for 96-well plate (if available) or washing bottle
• Microplate reader for 96-well plates (for absorbance measurement)
• Software for data processing

Q: What type of plate shaker should be used?

A: A capacity to shake the ELISA plates at about 500 rpm is required.

Q: Is it possible to purchase the antibody provided in this kit?

A: This antibody is not sold separately.

Q: Can I divide plates?

A: Since the plate in this kit are separable and each row (8 wells) is detachable, divided use of the kit is possible.