MMAE Antibody Drug Conjugate (ADC) ELISA Kit

Catalog No. ABIN2014351

Product Details

Target: MMAE Antibody Drug Conjugate (ADC)

Reactivity: Chemical

Detection Method: Colorimetric

Method Type: Competition ELISA

Detection Range: 0.0435-4000 μg/mL

Minimum Detection Limit: 0.0435 μg/mL

Application: ELISA

Purpose: This test kit is intended for use in the quantitative determination of antibody-MMAE-conjugate level in test sample. It is useful for pre-clinical and clinical pharmacology study of MMAE 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 MMAE-ADC and mouse monoclonal antibody based MMAE-ADC can be measured with this kit.

Brand: EDI™

Sample Type: Tissue Samples, Serum

Analytical Method: Quantitative

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

Components: 1. Anti-MMAE Antibody Coated Microplate.One microplate with twelve by eight strips (96 wells total) coated with specific anti-MMAE antibody. The plate is framed and sealed in a foil zipper bag with a desiccant. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box.
2. HRP Conjugated MMAE.One vial containing 3 mL of ready to use HRP labeled MMAE in a stabilized protein matrix. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box.
3. ELISA Wash Concentrate.One bottle containing 3 mL of 30-fold concentrate. Before use the contents must be diluted with 87 mL of demineralized water and mixed well. Upon dilution, this yields a working wash solution containing a surfactant in phosphate buffered saline with a non-azide, non-mercury preservative. The diluted wash solution may be stored at room temperature and is stable until the expiration date on the kit box.
4. ELISA HRP Substrate.One bottle containing 15 mL of tetramethylbenzidine (TMB) with hydrogen peroxide. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box.
5. ELISA Stop Solution.One bottle containing 15 mL of stop solution. This reagent may be stored at 2-8 °C or room temperature and is stable until the expiration date on the kit box.
6. Antibody-MMAE Conjugated Zero Calibrator. One vial containing 30mL zero calibrator (30711) . This reagent is used for diluting the calibration stock t make assay calibrators, as well as for diluting test samples. This reagent should be stored at 2-8 °C and is stable until the expiration date on the kit box.
7. Antibody-MMAE Conjugated Calibration Stock. Not provided in the kit (optional) One vial (3071
2. containing the calibration stock of antibody-MMAE-conjugate in a lyophilized (0.5 mL) 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-MMAE Conjugated Stock
2. Precision single channel pipettes capable of delivering 2 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 450/2. 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 MMAE conjugate in serum. The assay utilizes the competitive immunoassay technique with an antibody that exclusively binds to MMAE.Assay calibrators (antibody MMAE conjugate) and test serum samples are added directly to wells of a microtiter plate that is coated with specific anti-MMAE antibody. Subsequently, a horseradish peroxidase (HRP) conjugated MMAE is added to each well. During the incubation period, the antibody MMAE conjugate competes with the HRP conjugated MMAE for the limited binding sites of anti-MMAE antibody. An immune complex of well coated anti-MMAE antibody HRP conjugated MMAE 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-MMAE conjugate in the test sample. A calibration curve is generated by plotting the absorbance versus the respective antibody-MMAE conjugate concentration for each calibrator on a 4-parameter or log-logit curve fitting. The concentration of antibody-MMAE 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 with 0.5 mL DI-water. Dilute the reconstituted calibration stock Place a sufficient number of Anti-MMAE 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 MMAE to each well. (Note: no wash step before add the HRP conjugated MMAE)
(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 calibrator levels on the ordinate against the calibrator concentration. Appropriate computer assisted data reduction programs should be used for the calculation of results.The antibody-MMAE 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, L6 and L8) in eight replicate determinations. The CV% is 7.1%, 6.7% and 11.0%.

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

Target Details

Target: MMAE Antibody Drug Conjugate (ADC)

Target Type: Antibody