Filgrastim (GCSF) Pharmacokinetic ELISA | EL-1611-73196

Catalogue Number: EL-1611-73196

Size: 1X96 wells

Antigen: granulocyte colony stimulating factor (G-CSF)

Storage: -20°C, 1 year

Reactivity: Human

Detection Range: 9.375ng/mL - 500 ng/mL

Detection Minimum: 9.375 ng/mL

Protocol: This assay employs the indirect sandwich enzyme immunoassay technique. Anti-GCSF is coated onto a 96 well microplate. Calibrator and test samples prepared by dilution into assay buffer and are pipetted into the appropriate wells. GCSF present in biological matrices is bound by the immobilized anti- GCSF antibody. After washing away any unbound substances, tagged anti-GCSF secondary antibody is added to the wells. After washing, diluted detection reagent is added to the wells. The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of GCSF present in test samples. The color development is stopped and the intensity of the color is measured.

Components: Coated microtiter plate, 96 wells (1x8 strips) 1 Calibrator diluent 1.8ml Calibrator (1mg/ml) 12μl 20X wash buffer 25ml Assay buffer 50ml 1000X secondary antibody 1000X detection reagent 17μl TMB 12ml TMB stop solution 12ml

Method Type: Direct sandwich ELISA

Detection Method: Peroxidase / OD450

Principle: Granulocyte colony stimulating factor in biological matrices

Plate: Strip

Sample Type: Serum Plasma

Sample Volume: 15ul

Assay Time: 2.5 hours

Specificity: GCSF

Assay Precision: < 25% for ULOQ and LLOQ and <20% for the remaining concentrations

Preservative: None

Description: Granulocyte colony-stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream

Gene ID: 1440

Assay Procedure: This assay employs the indirect sandwich enzyme immunoassay technique. Anti-GCSF is coated onto a 96 well microplate. Calibrator and test samples prepared by dilution into assay buffer and are pipetted into the appropriate wells. GCSF present in biological matrices is bound by the immobilized anti- GCSF antibody. After washing away any unbound substances, tagged anti-GCSF secondary antibody is added to the wells. After washing, diluted detection reagent is added to the wells. The plate is washed to remove any unbound antibody-enzyme reagent and a substrate solution is added to the wells for color development. The color development is proportional to the amount of GCSF present in test samples. The color development is stopped and the intensity of the color is measured.

Reagent Preparation: Prepare only the appropriate amount of required reagent on the day of use. Store all reagents as per instructions stated on the label. 1. Wash Buffer (1X) Preparation: Dilute wash buffer
concentrate with ultra-pure water 1/20 before use
(for example add 25mL concentrate to 475mL ultrapure water). Mix well.
2. Secondary Antibody (1X) Preparation: Dilute secondary
antibody with assay buffer 1/1000 before use (for
example add 12μl to 12mL of assay buffer). Mix well.
3. Detection Reagent (1X) Preparation: Dilute detection
reagent with assay buffer 1/1000 before use (for
example add 12μl concentrate to 12ml of assay buffer).
Mix well.
4. Calibrator Preparation: Dilute the calibrator from 1mg/ml
down to 5µg/ml by pipetting 5µL of calibrator stock into
995µL assay buffer. Label "Cal. Int." Mix well. Prepare
calibrators with concentrations ranging from 500 ng/ml
to 9.375 ng/ml. The following is an example calibrator
curve.

Results Calculation: 1. Construct a standard curve by plotting the absorbance obtained from each standard against concentration. Use a 4 or 5 parameter curve fit. 2. The concentration of the unknowns can be back calculated directly from this standard curve using the absorbance value for each sample. 3. Any sample diluted more or less than the standard series will need additional data correction. For example, if the sample is diluted 1/50, then the concentration will be calculated by dividing by 2 due to the calibrators being 2 times more diluted. Similarly, if the sample is diluted 1/500, then the concentration will be calculated by multiplying by a correction factor of 5 due to the calibrators being 5 times more concentrated.

Sample Collection: This kit is compatible with EDTA-plasma, heparinplasma and serum samples. Samples can be stored at or below -20°C for up to 1 year.

Sample Preparation: Dilute calibrators and test samples 1/50 with assay buffer (for example add 5µL of prepared calibrator or sample to 245µL of assay buffer). Mix well. Do not store diluted samples.