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Quantitative Therapeutic Drug Monitoring (TDM) ELISA Sample Protocol

Quantitative Therapeutic Drug Monitoring ELISA Test Procedure Step-by-Step

The Assay Genie Quantitative Therapeutic Drug Monitoring (TDM) Enzyme-Linked-Immuno-Sorbent Assay (ELISA) Kit is intended for the quantitative determination of antibodies to the therapeutic of interest in serum and plasma. It is for professional use only.

Key Information & Technical Specifications

  • Quantitative determination of anti-drug antibodies (ADA)
  • Requires only 20 uL of serum/plasma sample
  • All common reagents
  • Easily adaptable to automation
  • Lowest detection limit: 62 ng/mL
  • Total incubation time is as short as 95 min

Storage and Stability

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

Specimen Collection and Storage

> Serum, Plasma (EDTA, Heparin)

The usual precautions for venipuncture should be observed. It is important to preserve the chemical integrity of a blood specimen from the moment it is collected until it is assayed. Do not use grossly hemolytic, icteric or grossly lipemic specimens. Samples appearing turbid should be centrifuged before testing to remove any particulate material.

Storage Stability

2-8°C

7 days

-20°C

6 months

Keep away from heat/direct sun light

Avoid repeated freeze-thaw cycles


Procedure Notes

  • 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 must be performed strictly according to the instructions. Use calibrated pipettes and devices only.
  • Allow all reagents and specimens to reach room temperature (18-25 °C) and gently swirl each vial of liquid reagent and sample before use. Mix reagents without foaming.
  • Incubation time affects results. All wells should be handled in the same order and time sequences. It is recommended to use an 8-channel Micropipettor for pipetting of solutions in all wells.

Quantitative Therapeutic Drug Monitoring ELISA Kit Contents

Size Kit Contents

1 x 12 x 8

Microtiter Plate

Break apart strips. Microtiter plate with 12 rows each of 8 wells coated with reactant

7 x 1 mL

Standards A-E, High Level Control, Low Level Control

3; 1; 0.3; 0.1; 0 microgram/mL Ready to use. Used for construction of the standard curve.Contains ADA, human serum and <0,1% NaN3

1 x 50 mL

Assay Buffer Blue coloured. Ready to use. Contains proteins, RF blockers and <0.1% NaN3.

1 x 12 mL

Confirmation Reagent Ready to use. Contains optimized concentration of the therapeutic antibody, proteins and stabilizer <0,1% NaN3

1 x 12 mL

Peroxidase Conjugate Red coloured. Ready to use. Contains peroxidase (POD) conjugate, stabilizer and preservatives.

1 x 12 mL

TMB Substrate Solution Ready to use. Contains TMB

1 x 12 mL

TMB Stop Solution Ready to use. 1N HCl

1 x 50 mL

Wash Buffer concentrate (20x) Contains Buffer with Tween 20.

2 x 1

Adhesive Foil For covering of Microtiter Plate during incubation.


Preparation of Components

Dilute Stability With Dilutent Relation Remarks Storage Stability

10mL

Wash Buffer

Up to 200 mL

ddH2O

1:20

Warm up at 37°C to dissolve crystals.

2-8 °C

2 w


Dilution of Samples (serum/plasma)

Sample To be Diluted With Relation Remarks

Serum/Plasma

1/10

Assay Buffer

1:10 – 1:100

For dilution 1:10 20µl Sample +180µl Assay Buffer For dilution 1:100 5µl Sample + 495µl Assay Buffer


Quantitative Therapeutic Drug Monitoring (TDM) ELISA Test Procedure

Steps Protocol

1

QUANTITATIVE ELISA TEST FORMAT

Pipette 100 µL of each ready-to use Standards, High Level Control, Low Level Control and Pre-diluted Samples into the respective wells of microtiter plate. Wells A1: Standard A B1: Standard B C1: Standard C D1: Standard D E1: Standard E F1: High Level Control G1: Low Level Control H1 and on: Sample (Serum / Plasma)

2

Cover the plate with adhesive film. Briefly mix contents by shaking the plate. Incubate 60 min at room temperature (18- 25°C).

3

Remove adhesive film. Discard incubation solution. Wash plate 3 times each with 300µL of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

4

Pipette 100 µL of ready-to use Peroxidase Conjugate into each well.

5

Cover the plate with adhesive foil. Incubate 60 min at room temperature (18- 25°C).

6

Remove adhesive foil. Discard incubation solution. Wash plate 3 times each with 300 µL of diluted Wash Buffer. Remove excess solution by tapping the inverted plate on a paper towel.

7

Pipette 100 µL of TMB Substrate Solution into each well.

8

Incubate 20 min (without adhesive foil) at room temperature (18-25°C) in the dark

9

Stop the substrate reaction by adding 100 µL of Stop Solution into each well. Briefly mix contents by gently shaking the plate. Colour changes from blue to yellow.

10

Measure optical density with a photometer at 450/650 nm within 30 min after pipetting of the Stop Solution.