DM1 ADC ELISA Kit

Catalog No. ABIN2014350

Product Details

Target: DM1 ADC

Reactivity: Chemical

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 0.024-1.0 μg/mL

Minimum Detection Limit: 0.024 μg/mL

Application: ELISA

Purpose: This test kit is intended for use in the quantitative determination of antibody-DM1-conjugate level in test sample. It is useful for pre-clinical and clinical pharmacology study of DM1 Antibody Drug Conjugate (ADC).
Samples from tissue/cell culture and serum samples from human, rat, mouse, primate, etc. can be used directly with this kit.- Both humanized monoclonal antibody based DM1-ADC and mouse monoclonal antibody based DM1-ADC can be measured with this kit.

Brand: EDI™

Sample Type: Tissue Samples, Serum

Analytical Method: Quantitative

Specificity: This DM1-ADC EIA doesn't show any cross reactivity to MMAE-ADC, MMAF-ADC, DUO-3 ADC, and DUO-6 ADC.

Cross-Reactivity (Details): This DM1-ADC EIA doesn't show any cross reactivity to MMAE-ADC, MMAF-ADC, DUO-3 ADC, and DUO-6 ADC.

Characteristics: This EIA kit is designed, developed and produced for the quantitative measurement of antibody DM1 conjugate in serum. The assay utilizes the competitive immunoassay technique with an antibody that exclusively binds to DM1. Assay calibrators (antibody DM1 conjugate) and test serum samples are added directly to wells of a microtiter plate that is coated with specific anti-DM1 antibody. Subsequently, a horseradish peroxidase (HRP) conjugated DM1 is added to each well. During the incubation period, the antibody DM1 conjugate competes with the HRP conjugated DM1 for the limited binding sites of anti-DM1 antibody. An immune complex of well coated ?anti-DM1 antibody HRP conjugated DM1 is formed. The unbound antibodies and buffer matrix are removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction, which is terminated with an acidic reagent (i.e. ELISA stop solution). The absorbance is then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is inversely proportional to the amount of antibody-DM1 conjugate in the test sample. A calibration curve is generated by plotting the absorbance versus the respective antibody-DM1 conjugate concentration for each calibrator on a 4-parameter or log-logit curve fitting. The concentration of antibody-DM1 conjugate in test samples is determined directly from this calibration curve

Components: 1. Anti-DM1 Antibody Coated Microplate serum based matrix with a non-azide preservative. Refer to the vial for exact concentration of the standard. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box.

Material not included: 1. Antibody-DM1 Conjugated Stock.
2. Precision single channel pipettes capable of delivering 25 µL, 50 µL, 100 µL, etc.
3. Disposable pipette tips suitable for above volume dispensing.
4. Aluminum foil.
5. Deionized or distilled water.
6. Plastic microtiter well cover or polyethylene film.
7. ELISA multichannel wash bottle or automatic (semi-automatic) washing system.
8. Spectrophotometric microplate reader capable of reading absorbance at 450/650 or 450/620 nm.

Application Details

Assay Time: 4 h

Plate: Pre-coated

Protocol: This EIA kit is designed, developed and produced for the quantitative measurement of antibody DM1 conjugate in serum. The assay utilizes the competitive immunoassay technique with an antibody that exclusively binds to DM1.Assay calibrators (antibody DM1 conjugate) and test serum samples are added directly to wells of a microtiter plate that is coated with specific anti-DM1 antibody. Subsequently, a horseradish peroxidase (HRP) conjugated DM1 is added to each well. During the incubation period, the antibody DM1 conjugate competes with the HRP conjugated DM1 for the limited binding sites of anti-DM1 antibody. An immune complex of well coated anti-DM1 antibody HRP conjugated DM1 is formed. The unbound antibodies and buffer matrix are removed in the subsequent washing step. For the detection of this immunocomplex, the well is then incubated with a substrate solution in a timed reaction, which is terminated with an acidic reagent (i.e. ELISA stop solution). The absorbance is then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to the wall of each microtiter well is inversely proportional to the amount of antibody-DM1 conjugate in the test sample. A calibration curve is generated by plotting the absorbance versus the respective antibody-DM1 conjugate concentration for each calibrator on a 4-parameter or log-logit curve fitting. The concentration of antibody-DM1 conjugate in test samples is determined directly from this calibration curve.

Reagent Preparation:

(1) Prior to use allow all reagents to come to room temperature. Reagents from different kit lot numbers should not be combined or interchanged.
(2) ELISA Wash Concentrate must be diluted to working solution prior to use. Please see REAGENTS section for details.
(3) Using EDI Calibrators: Reconstitute calibration stock 30702 with 0.5 mL DI-water. Dilute the reconstituted calibration stock (30702) 1:X* using the zero calibrator (30701) to obtain a level-8 standard at 1 g/mL. Further create calibrator level seven to two by 1:2 serial dilutions to obtain these calibrators with concentrations of 0.5 g/mL, 0.25 g/mL, 0.125 g/mL, 0.063 g/mL, 0.031 g/mL, 0.016 g/mL. Assay calibrators should be used within 2 hours and should be stored below -20 C. Do not exceed 3 freeze-thaw cycles.
(5) Place a sufficient number of Anti-DM1 antibody coated microwell strips in a holder to determine calibrators and unknown samples in duplicates.

Assay Procedure:

(1) Add 100 µL of calibrators and test samples into the designated microwells.
(2) Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 30 minutes at 400 to 450 rpm.
(3) Immediately add 25 µL of HRP Conjugated DM1 (cat# 30683) to each well. (Note: no wash step before add the HRP Conjugated DM1)
(4) Seal the plate wells securely, cover with foil or other material to protect from light, and rotate on an ELISA plate shaker (small orbit radius) for 2 hr. 10 minutes at 400 to 450 rpm.
(5) Wash each well 5 times by dispensing 350 µL of working wash solution into each well and then completely aspirating the contents. Alternatively, an automated microplate washer can be used.
(6) Add 100 µL of ELISA HRP Substrate into each of the wells.
(7) Cover the plate with aluminum foil or other material to avoid exposure to light. Incubate plate static, at room temperature for 20 minutes.
(8) Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
(9) Read the absorbance at 450 nm.

Calculation of Results:

It is recommended to use a 4-parameter or log-logit calibration curve fitting.
1. Calculate the average absorbance for each pair of duplicate test results.
2. The calibration curve is generated by the corrected absorbance of all calibration levels on the ordinate against the calibrator concentration. Appropriate computer assisted data reduction programs should be used for the calculation of results. The antibody-DM1 conjugate concentrations for the test samples are read directly from the calibration curve using their respective corrected absorbance.

Assay Precision: The intra-assay precision was validated by measuring three calibrators (L3, L5 and L7) in six replicate determinations. The CV% is 6.8%, 6.2% and 8.9%.

Restrictions: For Research Use only

Handling

Precaution of Use: The reagents must be used in professional laboratory. Source material for reagents containing bovine serum was derived in the contiguous 48 United States. It was obtained only from healthy donor animals maintained under veterinary supervision and found free of contagious diseases. Wear gloves while performing this assay and handle these reagents as if they are potentially infectious. Avoid contact with reagents containing TMB, hydrogen peroxide, or sulfuric acid. TMB may cause irritation to skin and mucous membranes and cause an allergic skin reaction. TMB is a suspected carcinogen. Sulfuric acid may cause severe irritation on contact with skin. Do not get in eyes, on skin, or on clothing. Do not ingest or inhale fumes. On contact, flush with copious amounts of water for at least 15 minutes. Use Good Laboratory Practices.

Storage: 4 °C

References

van der Lee, Groothuis, Ubink, van der Vleuten, van Achterberg, Loosveld, Damming, Jacobs, Rouwette, Egging, van den Dobbelsteen, Beusker, Goedings, Verheijden, Lemmens, Timmers, Dokter: "The Preclinical Profile of the Duocarmycin-Based HER2-Targeting ADC SYD985 Predicts for Clinical Benefit in Low HER2-Expressing Breast Cancers." in: Molecular cancer therapeutics, Vol. 14, Issue 3, pp. 692-703, (2015) (PubMed).

Target Details

Target: DM1 ADC

Target Type: Disease