Intended Use

The Romiplostim (Nplate™) ELISA is a rapid and easy method for the quantitative determination of romiplostim in human serum, plasma and cell culture supernatant

General Description

Romiplostim (NPlate™) is a fusion protein analog of thrombopoietin, a hormone that regulates platelet production. Romiplostim is an Fc-peptide fusion protein (peptibody) that signals and activates intracellular transcriptional pathways via the TPO receptor (also known as cMpl) to increase platelet production. The peptibody molecule is comprised of a human immunoglobulin IgG1 Fc domain, with each single-chain subunit covalently linked at the C-terminus to a peptide  chain containing 2 TPO receptor-binding domains. Romiplostim has no amino acid sequence homology to endogenous TPO. In pre-clinical and clinical trials no antiromiplostim antibodies cross reacted with endogenous TPO. Romiplostim is indicated for adult chronic immune(idiopathic)thrombocytopenic purpura (ITP) patients who are refractory to other treatments (e.g. corticosteroids, immunoglobulins).

Principles of Testing

Romiplostim ELISA is a two-step enzyme immunoassay on the basis of humanized monoclonal antibodies specific to c-Mpl. In the first step, Assay Diluent and Standard or Samples are dispensed simultaneously into the wells of a microtitration plate coated with humanized monoclonal antibodies specific to c-Mpl. In the second step, horseradish peroxidase (HRP) labelled humanized monoclonal antibodies specific to -Mpl are dispensed in respective wells. After incubation unbound components are removed by a washing step. HRP converts the subsequently added substrate solution of 3,3’,5,5’- Tetramethylbenzidine (TMB) within a 30 min reaction time into a blue product. The enzyme reaction is terminated by stop solution dispensed into the wells turning the solution from blue to yellow. The optical density (OD) of the solution read at 450 is directly proportional to the specifically bound amount of Romiplostim.

Reagents And Materials Provided

• Anti-c-Mpl Coated Microtiter Plate (12X8 wells) – 1 no
• Romiplostim Standard, (0.5 ml/vial) – 0, 10, 20, 40, 80, 160, 320 and 640 ng/ml
• Anti-c-Mpl:HRP Conjugate – 12 ml
• Assay Diluent – 6 ml
• Sample Diluent – 50 ml
• Wash Buffer (20X) – 25 ml
• TMB Substrate – 12 ml
• Stop Solution – 12 ml
• Instruction Manual

Materials Required But Not Supplied

• Microtiter Plate Reader able to measure absorbance at 450 nm.
• Adjustable pipettes and multichannel pipettor to measure volumes ranging from 25 ul to 1000 ul
• Deionized (DI) water
• Wash bottle or automated microplate washer
• Graph paper or software for data analysis
• Timer
• Absorbent Paper

 Storage

• All reagents should be stored at 2ºC to 8ºC for stability.
• All the reagents and wash solutions should be used within 12 months from manufacturing date.
• Before using, bring all components to room temperature (18-25ºC). Upon assay completion ensure all components of the kit are returned to appropriate storage conditions.
• The Substrate is light-sensitive and should be protected from direct sunlight or UV sources

Specimen Collection And Preparation

Sample Preparation and Storage:

Blood is taken by venipuncture. Serum is separated after clotting by centrifugation. Plasma can be used, too. Lipaemic, hemolytic or contaminated samples should not be run. Repeated freezing and thawing should be avoided. If samples are to be used for several assays, initially aliquot samples and keep at - 20°C. For Cell Culture Supernatant – If necessary, centrifuge to remove debris prior to analysis. Samples can be stored at 20°C or -80°C. Avoid repeated freeze-thaw cycles.

Preparation Before Use:

Test sample preparation- Samples have to be diluted 1 in 200 (v/v), e.g. 5μl sample in 1ml sample diluent, prior to assay. The samples may be kept at 2 - 8 °C for up to three days. Long-term storage requires - 20°C.

Reagent Preparation

• Label any aliquots made with the kit Lot No and Expiration date and store it at appropriate conditions mentioned.
• Bring all reagents to Room temperature before use.
• To make Wash Buffer (1X); dilute 50 ml of 20X Wash Buffer in 950 ml of DI water

Assay Procedure

Procedural Notes:

• 1. In order to achieve good assay reproducibility and sensitivity, proper washing of the plates to remove excess unreacted reagents is essential.
• 2. Avoid assay of Samples containing sodium azide (NaN3), as it could destroy the HRP activity resulting in underestimation of the amount of Romiplostim.
• 3. It is recommended that all Standards and Samples be assayed in duplicates.
• 4. Maintain a repetitive timing sequence from well to well for all the steps to ensure that the incubation timings are same for each well.
• 5. If the Substrate has a distinct blue color prior to use it may have been contaminated and use of such substrate can lead
• to compromisation of the sensitivity of the assay.
• 6. The plates should be read within 30 minutes after adding the Stop Solution.
• 7. Make a work list in order to identify the location of Standards and Samples.

Assay Procedure:

Bring all reagents to room temperature prior to use. It is strongly recommended that all Standards and Samples be run in duplicates or triplicates. A standard curve is required for each assay.

• 1. Dispense 50 ul of Assay Diluent per well.
• 2. Dispense 100 ul Romiplostim Standard or Sample and mix gently.
• 3. Cover plate and incubate for 1.0hr at 37°C and aspirate.
• 4. Dispense 100 ul of Anti-c-Mpl:HRP Conjugate per well.
• 5. Decant, then wash each well 5x with 300 ul Wash Buffer (1X) (with 10s soak time) and tap dry onto absorbent paper.
• 6. Dispense 100 ul TMB substrate per well.
• 7. Incubate for 30 min at Room Temperature protected from light.
• 8. Dispense 100 ul Stop Solution per well, mix gently.
• 9. Read OD at 450 nm with a microplate reader within 30 min after reaction stop

Quality Control

It is recommended that for each laboratory assay appropriate quality control samples in each run to be used to ensure that all reagents and procedures are correct.

Calculation

Determine the Mean Absorbance for each set of duplicate or triplicate Standards and Samples. Using Semi-Log graph paper, plot the average value (absorbance 450nm) of each standard on the Y-axis versus the corresponding concentration of the standards on the X-axis. Draw the best fit curve through the standard points. To determine the unknown Romiplostim concentrations, find the unknown’s Mean Absorbance value on the Y-axis and draw a horizontal line to the standard curve. At the point of intersection, draw a vertical line to the Xaxis and read the Romiplostim Concentration. If samples were diluted, multiply by the appropriate dilution factor. Software which is able to generate a cubic spline curve-fit or using a polynomial regression to the 2nd order is best recommended for automated results.

Note:

It is recommended to repeat the assay at a different dilution factor in the following cases: - If the sample absorbance value is below the first standard. - If the absorbance value is equivalent or higher than the 640 ng/ml standard

Detection Range

0-640 ng/ml

Precautions

• − For professional use only.
• − In case of severe damage of the kit package please contact Creative Diagnostics or your supplier in written form, latest one week after receiving the kit. Do not use damaged components in test runs but keep safe for complaint related issues.
• − Obey lot number and expiry date. Do not mix reagents of different lots. Do not use expired reagents.
• − 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. For further information (clinical background, test performance, automation protocols, alternative applications, literature, etc.) please refer to the local distributor.
• − Follow good laboratory practice and safety guidelines. Wear lab coats, disposable latex gloves and protective glasses where necessary.
• − All reagents of this kit containing human serum or plasma (standards etc.) have been tested and were found negative for HIV I/II, HBsAg and Anti-HCV. However, a presence of these or other infectious agents cannot be excluded absolutely and therefore reagents should be treated as potential biohazards in use and for disposal.
• − Reagents of this kit containing hazardous material may cause eye and skin irritations. See “Reagents And Materials Provided”, MSDS and labels for details.
• − Chemicals and prepared or used reagents must be treated as hazardous waste according the national biohazard safety guidelines or regulations.