Ranibizumab ELISA Kit

MATERIALS PROVIDED & STORAGE CONDITIONS

* Provided this is within the expiration date of the kit.

OTHER SUPPLIES REQUIRED

• Microplate reader capable of measuring absorbance at 450 nm, with the correction wavelength set at 630 nm or 620 nm.

• Pipettes and pipette tips.

• Deionized or distilled water.

• 500 mL graduated cylinder.

• Squirt bottle, manifold dispenser, or automated microplate washer.

• Test tubes for dilution of standards.

SAMPLE COLLECTION & STORAGE

The sample collection and storage conditions listed below are intended as general guidelines. Sample stability has not been evaluated.

Handle all blood and serum as if capable of transmitting infectious agents. The NCCLS provides recommendations for handling and storing serum and plasma specimens (Approved Standard-Procedures for the Handling and Processing of Blood Specimens, H18-A. 1990).

Serum - Use a serum separator tube (SST) and allow samples to clot for 30 minutes at room temperature before centrifugation for 15 minutes at 1000 x g. Remove serum and assay immediately or aliquot and store samples at ≤ -20℃. Avoid repeated freeze-thaw cycles.

Plasma - Collect plasma using EDTA, heparin, or citrate as an anticoagulant. Centrifuge for 15 minutes at 1000 x g within 30 minutes of collection. Assay immediately or aliquot and store samples at ≤ -20℃. Avoid repeated freeze-thaw cycles.

REAGENT PREPARATION

Bring all reagents to room temperature before use.

20-fold Wash Buffer Concentrate - If crystals have formed in the concentrate, warm to room temperature and mix gently until the crystals have completely dissolved. Add 25 mL of Wash Buffer Concentrate to 475 mL of deionized or distilled water to prepare 500 mL of Wash Buffer.

Serum and Plasma - Serum and plasma samples require a 10-fold dilution. A suggested 10-fold dilution is 10 μL of sample + 90 μL of Standard Diluent (diluted 1:9). If the sample value is outside the range of the standard curve, the dilution can be adjusted appropriately and the assay can be redetermined. If the antibody concentration in the sample can be estimated and the assay can be performed simultaneously by diluting several gradients prior to the experiment.

Standard - Reconstitute with 1mL Standard Diluent, this reconstitution produces a stock solution of  15,000 ng/mL. Allow the standard to sit for a minimum of 15 minutes with gentle agitation prior to making dilutions. 15,000 ng/mL is the first standard point, and the concentration of the 7 standard sample were  15,000 ng/mL, 7,500 ng/mL, 3,750 ng/mL, 1,875 ng/mL, 937.5 ng/mL, 468.8 ng/mL, 234.4 ng/mL respectively. The appropriate Standard Diluent serves as the zero standard (0 ng/mL).

Detection A (working solution) - Shake and mix before used. Centrifuge instantaneously with palm centrifuge to make the liquid at the bottom of the tube. Dilute the Detection A 1: 100 times to the working concentration with Assay Diluent.

Detection B (working solution) - Shake and mix before used. Centrifuge instantaneously with palm centrifuge to make the liquid at the bottom of the tube. Dilute the Detection B 1: 100 times to the working concentration with Assay Diluent.

ASSAY PROCEDURE

Bring all reagents and samples to room temperature before use. It is recommended that all standards and samples be assayed in duplicate.

1. Prepare all reagents and working standards as directed in the previous sections.

2. Remove excess microplate strips from the plate frame, return them to the foil pouch containing the desiccant pack, and reseal.

3. Add 100 μL premixed solution (50 μL diluted standard/sample + 50 μL Detection A working solution) to each well . Cover with the adhesive strip provided. Incubate for 2 hours at 37℃.

4. Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (300 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

5. Add 100 μL of Detection B (working solution) to each well. Cover with a new adhesive strip. Incubate for 30 minutes at 37℃.

6. Aspirate each well and wash, repeating the process five times. Wash by filling each well with Wash Buffer (300 μL) using a squirt bottle, manifold dispenser, or autowasher. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.

7. Add 100 μL of Color Reagent to each well. Incubate for 15 minutes at 37℃. Protect from light.

8. Add 50 μL of Stop Solution to each well. The color in the wells should change from blue to yellow. If the color in the wells is green or the color change does not appear uniform, gently tap the plate to ensure thorough mixing.