ELISA/assay

Eculizumab ELISA Kit

DEIAZ0063

96T

Eculizumab ELISA Kit

Eculizumab

Eculizumab ELISA Kit

sELISA

Eculizumab

NA

Quantitative

Serum, plasma, cell culture samples

96T

The kit is shipped at ambient temperature and should be stored at 2-8°C. Keep away from heat or direct sun light. The storage and stability of specimen and prepared reagents is stated in the corresponding chapters. The microtiter strips are stable up to the expiry date of the kit in the broken, but tightly closed bag when stored at 2-8°C.

Eculizumab

NA

Enzyme immunoassay for the specific and quantitative determination of free Eculizumab (Soliris) in serum, plasma and cell culture samples.

This ELISA assay is based on Eculizumab-specific mouse monoclonal antibody (capture Ab). Diluted standards and samples are incubated in the microtiter plate coated with Eculizumab mAb. After incubation, the wells are washed. A Biotinylated mouse anti-Eculizumab monoclonal antibody is added and binds to the CDR part of Eculizumab. Following incubation and wash steps, a streptavidin-enzyme conjugate is added and binds to the biotinylated anti-Eculizumab antibody. Unbound streptavidin-enzyme conjugate is removed during a wash step, and substrate solution is added to the wells. The color developed is proportional to the amount of Eculizumab in the sample or standard. Results of samples can be determined by using the standard curve.

1. 1×12×8 Microtiter ELISA Plate, Break apart strips coated with Anti-Eculizumab. 2. 1 × 120 μL Eculizumab Standard, Concentrate (6.4 μg/ml). Used for construction of the standard curve. Contains Eculizumab, proteins, preservative and stabilizer. 3. 1 × 120 μL Anti-Eculizumab Biotin (100×). Contains biotinylated mouse anti-Eculizumab monoclonal antibody, proteins, preservative and stabilizer. 4. 1 × 120 μL Streptavidin-enzyme Conjugate, Concentrate (100×). Contains horseradish peroxidase (HRP) - streptavidin, proteins, preservative and stabilizer. 5. 1 × 50 mL Dilution Buffer, Ready to use. Contains proteins and preservative. 6. 2 × 6 mL Substrate Solution, Ready to use. Contains 3,3',5,5'-Tetramethylbenzidine (TMB). 7. 1 × 7 mL Stop Solution, Ready to use. Contains 0.5M sulfuric acid (H 2 SO 4 ). 8. 1 × 50 mL Wash Buffer, Concentrate (20×).

1. Micropipettes ( 2. Bidistilled or deionised water and calibrated glasswares (e.g. flasks or cylinders). 3. Wash bottle, automated or semi-automated microtiter plate washing system. 4. Microtiter plate reader capable of reading absorbance at 450 nm (reference wavelength at 600-650 nm is optional). 5. Absorbent paper towels, standard laboratory glass or plastic vials, and a timer.

This kit is compatible with EDTA, heparin or citrate plasma, and serum samples. Samples can be stored at or below -20°C for up to 1 year. Serum: To collect serum, use a serum separator tube and allow the whole blood to clot for 25-35 minutes. Then centrifuge blood for 15 minutes at 1500 ×g and remove serum immediately. Plasma: To collect plasma, use EDTA, heparin, or citrate as an anticoagulant. Within 30 minutes of whole blood collection, centrifuge for 15 minutes at 1500 ×g and remove plasma immediately. Sample Storage: Store assay samples immediately after collection, or aliquot and store at or below -20°C for up to 1 year. Avoid repeated freeze-thaw. We have demonstrated sample stability up to five freeze-thaw cycles. We strongly recommend each lab generate their own stability data. Avoid using samples with gross hemolysis or lipemia.

1. 1× Wash Buffer: Dilute the 20× Wash Buffer to 1× with deionized water. Stir to homogeneity. 2. 1× Anti-Eculizumab Biotin and Streptavidin-enzyme Conjugate: Immediately before use dilute the Anti-Eculizumab Biotin 1:100 and the Streptavidin-enzyme Conjugate 1:100 with Dilution Buffer. Do not store diluted solutions. 3. Preparation of Standard Curve: Prepare a dilution series of Eculizumab Standard in the concentration range of 2.6 ng/mL-640 ng/mL by diluting the Eculizumab Standard (6.4 μg/ml) in Dilution Buffer.

Note: 1. Any improper handling of samples or modification of the test procedure may influence the results. The indicated pipetting volumes, incubation times, temperatures and pre-treatment steps have to be performed strictly according to the instructions. Use calibrated pipettes and devices only. 2. Once the test has been started, all steps should be completed without interruption. Make sure that required reagents, materials and devices are prepared readily at the appropriate time. Allow all reagents and specimens to reach room temperature (20-25 °C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming. 3. Avoid contamination of reagents, pipettes and wells/tubes. Use new disposable plastic pipette tips for each reagent, standard or specimen. Do not interchange the caps of vials. Always cap not used vials. Do not reuse wells or reagents. 4. Use a pipetting scheme to verify an appropriate plate layout. 5. Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipette for pipetting of solutions in all wells. 6. Microplate washing is important. Improperly washed wells will give erroneous results. It is recommended to use a multichannel pipette or an automatic microplate washing system. Do not allow the wells to dry between incubations. Do not scratch coated wells during rinsing and aspiration. Rinse and fill all reagents with care. While rinsing, check that all wells are filled precisely with Wash Buffer, and that there are no residues in the wells. 7. Humidity affects the coated wells. Do not open the pouch until it reaches room temperature. Unused wells should be returned immediately to the resealed pouch including the desiccant. Steps: 1. Before performing the assay, samples and assay kit should be brought to room temperature (about 30 minutes beforehand) and ensure the homogeneity of the solution. 2. Add 100 μL of 1:50 diluted samples and diluted Eculizumab standards to the respective wells of the microtiter plate. Each sample, standard and blank should be assayed in duplicate. 3. Cover with a plate cover and incubate at room temperature for 1 hour. 4. Remove plate cover and empty wells. Wash microwell strips 5 times with 300 μL 1× Wash Buffer per well with thorough aspiration between each wash. After the last wash, empty wells and tap microwell strips on absorbent pad or paper towel to remove excess 1× Wash Buffer. 5. Add 100 μL of the diluted Anti-Eculizumab Biotin to each well. 6. Cover with a plate cover and incubate at room temperature for 1 hour. 7. Remove plate cover and empty wells. Wash the strip wells 5 times according to step 4 above. 8. Add 100 μL of the diluted Streptavidin-enzyme Conjugate to each well. 9. Cover with a plate cover and incubate at room temperature for 1 hour. 10. Remove plate cover and empty wells. Wash microwell strips 5 times according to step 4 above. Proceed immediately to the next step. 11. Warm Substrate Solution to room temperature. Add 100 μL of Substrate Solution to each well, including the blank wells. incubate at room temperature for 15 minutes. 12. Stop the enzyme reaction by adding 50 μL of Stop Solution into each well, including the blank wells. Results should be read immediately (color will fade over time). 13. Read absorbance of each microwell on a spectrophotometer using 450 nm as the primary wave length.

The test results are only valid if the test has been performed following the instructions. Moreover the user must strictly adhere to the rules of GLP (Good Laboratory Practice) or other applicable standards/laws. All standards/controls must be found within the acceptable ranges as stated above and/or label. If the criteria are not met, the run is not valid and should be repeated. In case of any deviation, the following technical issues should be reviewed: Expiration dates of (prepared) reagents, storage conditions, pipettes, devices, incubation conditions and washing methods.

1. For the calculation of the Eculizumab concentration, polynomial regression is recommended. Standard curve is constructed by plotting the units of the 7 standard points (ng/mL) along the abscissa (X axis) and the corresponding OD450nm values along the ordinate (Y axis). 2. The Eculizumab concentration of samples can be reported based on the principles indicated below. 3. The Eculizumab concentration of pre-diluted (at the ratio of 1:50) samples can be directly read on the standard curve. In order to report the Eculizumab concentration for the corresponding undiluted sample, the value must be multiplied by the factor of 50. Example: If a 1:50 pre-diluted sample results in an Eculizumab concentration of 100 ng/mL on the standard curve, then its undiluted sample value corresponds to 5000 ng/mL (100 ng/mL x 50 = 5000 ng/mL). 4. If any diluted sample is reading OD450nm higher than that of highest standard supplied, it should be further diluted (in such a case 1:500 dilution of the sample is proposed) appropriately with Dilution Buffer and then retested. Also this second dilution has to be used for calculation of the final result. 5. It is recommended that each laboratory determines the best dilution ratio for their samples in order to minimize retesting.

The following figures demonstrate typical Eculizumab ELISA results. One should use the data below for reference only. This data should not be used to interpret actual results.

Intra-assay and inter-assay CVs <20%

2.6 - 640 ng/mL

0.218 ng/mL

Recovery rate was found to be 80-120%.

1. Before starting the assay, read the instructions completely and carefully. Use the valid version of the package insert provided with the kit. Be sure that everything is understood. 2. Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents. 3. All reagents should be handled as if potentially infectious. Operators should wear gloves and protective clothing when handling any patient sera or serum-based products. 4. Some reagents within the kit may contain antimicrobial agents, and acid. Avoid contact with the skin and eyes. Rinse immediately with plenty of water if any contact occurs. 5. Any liquid brought into contact with potentially infectious material needs to be discarded in a container with a disinfectant. Disposal must be performed in accordance with local regulations. 6. Only trained laboratory personnel should execute this test.

Enzyme immunoassay for the specific and quantitative determination of free Eculizumab (Soliris) in serum, plasma and cell culture samples.

No. Components Size Storage Conditions 1. Microtiter ELISA Plate 1×12×8 2-8°C 2. Eculizumab Standard, Concentrate (6.4 μg/ml) 1 × 120 μL 2-8°C 3. Anti-Eculizumab Biotin (100×) 1 × 120 μL 2-8°C 4. Streptavidin-enzyme Conjugate, Concentrate (100×) 1 × 120 μL 2-8°C 5. Dilution Buffer, Ready to use 1 × 50 mL 2-8°C 6. Substrate Solution, Ready to use 2 × 6 mL 2-8°C 7. Stop Solution, Ready to use 1 × 7 mL 2-8°C 8. Wash Buffer, Concentrate (20×) 1 × 50 mL 2-8°C

The following figures demonstrate typical Eculizumab ELISA results. One should use the data below for reference only. This data should not be used to interpret actual results.

See individual product datasheet

0.5M

See individual product datasheet

See individual product datasheet

Enzyme immunoassay for the specific and quantitative determination of free Eculizumab (Soliris) in serum, plasma and cell culture samples.