Best Practices for ELISA Sample Handling

Storage of Samples:

• Avoid Contamination: Follow the manufacturer’s guidelines for storing samples.
• Label and Seal: Ensure samples are properly labeled and sealed to prevent drying out or evaporation.
• Timely Analysis: Analyze samples as soon as possible for accurate results.

Sample Collection:

• Follow Rules: Adhere to legal guidelines for sample collection.
• Inform Customers: Let customers know how much sample is needed.
• Reflect the Condition: Ensure results accurately reflect the condition of the received sample.

Sample Preparation:

• Follow Instructions: Adhere to test kit instructions for mixing and extracting the analyte.
• Use Proper Equipment: Ensure all equipment is suitable and well-maintained.
• Timing: Prepare samples just before analysis or store them as directed.

Using Frozen Samples:

• Thawing: Thaw samples completely at 4°C or room temperature, depending on the stability of the analyte.
• Mixing: Mix liquid samples thoroughly without creating foam.
• Avoid Repeated Freezing: Store samples in aliquots at -20°C to avoid multiple freeze-thaw cycles and maintain sample quality.

Cell Culture Supernatant:

• Centrifuge: Spin media at 1,500 rpm for 10 minutes at 4°C.
• Store: Aliquot and store the supernatant at -80°C. Avoid repeated freeze/thaw cycles.

Cell Extract:

• Prepare: Place tissue culture plates on ice and wash cells with ice-cold PBS.
• Extract: Add 0.5 mL extraction buffer per 100 mm plate, scrape cells, collect in a chilled tube, vortex briefly, and incubate on ice for 15-30 minutes.
• Centrifuge: Spin at 13,000 rpm for 10 minutes at 4°C.
• Store: Aliquot the supernatant and store at -80°C. Avoid repeated freeze/thaw cycles.

Conditioned Medium:

• Grow Cells: Use serum-containing medium until cells reach desired confluence.
• Prepare Medium: Wash with warm PBS, add serum-free medium, and incubate for 1-2 days.
• Centrifuge: Spin medium at 1,500 rpm for 10 minutes at 4°C.
• Store: Aliquot and store the supernatant at -80°C. Avoid repeated freeze/thaw cycles.

Milk, Urine, Saliva:

• Centrifuge: Spin at 10,000 x g for 2 minutes at 4°C.
• Store: Aliquot the supernatant and store at -80°C. Avoid repeated freeze/thaw cycles.

Plasma:

• Collect Blood: Use an anti-coagulant tube and centrifuge at 3,000 rpm for 10 minutes at 4°C.
• Store: Aliquot the supernatant (plasma) and store at -80°C. Avoid repeated freeze/thaw cycles.

Serum:

• Collect Blood: Use an untreated tube and incubate at room temperature for 20 minutes.
• Centrifuge: Spin at 3,000 rpm for 10 minutes at 4°C.
• Store: Aliquot the supernatant (serum) and store at -80°C. Avoid repeated freeze/thaw cycles.

Tissue Extract:

• Dissect Tissue: Work on ice, snap freeze in liquid nitrogen.
• Extract: Add ~300 µL extraction buffer per ~5 mg tissue, homogenize, and rinse the blade.
• Agitate: Mix for 2 hours at 4°C, then centrifuge for 20 minutes at 13,000 rpm at 4°C.
• Store: Aliquot the supernatant and store at -80°C. Avoid repeated freeze/thaw cycles.

General Recommendations:

• Protein Concentration: Aim for a concentration of ≥1-2 mg/mL.
• Dilution: Dilute serum, plasma, cell, and tissue extracts 50% with binding buffer.
• Centrifuge Thawed Samples: Spin at 10,000 rpm for 5 minutes at 4°C before use.

Storage Duration Limits:

• 2-8°C: Suitable for up to 5 days.
• -20°C: Suitable for up to 6 months.
• -80°C: Suitable for up to 2 years
• Liquid nitrogen: Suitable for indefinite storage with proper methods.

Avoiding Repeated Freeze-Thaw Cycles:

• Divide samples into smaller aliquots to reduce the need for multiple freeze-thaw cycles.
• Thaw samples quickly in a 15-25°C water bath and use them immediately.
• If refreezing, do so promptly after thawing to minimize sample degradation.

Storage of Kits:

• Store at 2-8°C in a dry place; avoid freezing components.
• Follow the expiry date on the label.
• Prevent kits from freezing to avoid damage and invalid results.

First In, First Out:

• Use kits with the shortest expiry date first.
• Note the date of first use on the kit box to prevent mix-ups.

Pre-Warming:

• Bring all reagents to room temperature (20-25°C) before use.
• Return components to storage conditions promptly after use.

Storage of Unused Components:

• Store unused microtiter plates in a resealable bag with desiccant.
• Store all components upright and securely closed.