MMAE Antibody Drug Conjugate (ADC) ELISA Kit

Catalog No. ABIN2014353

Product Details

Target: MMAE Antibody Drug Conjugate (ADC)

Reactivity: Chemical

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 1.12-390 ng/mL

Minimum Detection Limit: 1.12 ng/mL

Application: ELISA

Purpose: This highly sensitive sandwich test kit is intended for use in the quantitative determination of antibody MMAE conjugate level in serum or plasma of human, mouse, rat, primate, etc. It is useful for pre-clinical and clinical pharmacology study of MMAE Antibody Drug Conjugate (ADC).

Brand: EDI™

Sample Type: Serum, Plasma

Analytical Method: Quantitative

Specificity: This MMAE-ADC ELISA doesn't show any cross reactivity to DM1-ADC. This assay does cross react to MMAF-ADC.

Cross-Reactivity (Details): This MMAE-ADC ELISA doesn't show any cross reactivity to DM1-ADC. This assay does cross react to MMAF-ADC.

Components: 1. Anti-MMAE Antibody Coated Microplate
One vial containing lyophilized antibody MMAE conjugated calibrator 6 in a serum based matrix with non-azide preservative. Refer to the vial for exact concentration of the calibrator. This calibrator should be stored at 2-8 °C and is stable until the expiration date on the kit box.

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

Application Details

Sample Volume: 10 μL

Assay Time: 4 h

Plate: Pre-coated

Protocol: This ELISA kit is designed, developed and produced for the quantitative measurement of antibody MMAE conjugate in serum or palsma. The assay utilizes the sandwich immunoassay technique with an antibody that binds to MMAE. Briefly, a mouse monoclonal antibody specific to MMAE is coated onto a microtiter plate. In the assay system, the assay calibrators, controls and test specimen are added to this microtiter plate. During the first incubation period, the anti-MMAE monoclonal antibody captures the MMAE-Antibody Conjugate of calibrators, controls and test samples. Unbound proteins are washed away with a wash step. A HRP (horseradish peroxidase) conjugate anti-human IgG tracer antibody is added to each well of the microtiter plate. After the second incubation, a sandwich immunocomplex of Anti-MMAE antibody MMAE Antibody Conjugate HRP-conjugated anti-human IgG antibody is formed and attached to the wall of the plate. The unbound HRP-conjugated antibody is removed in a subsequent washing step. For the detection of this immunocomplex, each well is then incubated with a substrate solution in a timed reaction and then measured in a spectrophotometric microplate reader. The enzymatic activity of the immunocomplex bound to MMAE Antibody Conjugate on the wall of the microtiter well is directly proportional to the amount of MMAE Antibody Conjugate level in the sample.

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 Calibrator Stock (Cat 30794): Reconstitute calibration stock 30794 with 0.5 mL DI-water. Dilute the reconstituted calibration stock (30794) 1:X* using the zero calibrator (30759) to obtain a level six calibrator at 390 ng/mL. Further create calibrator level five to two by 1:3 serial dilutions to obtain these calibrators with concentrations of 130 ng/mL, 43.3 ng/mL,14.4 ng/mL, 4.8 ng/mL. Assay calibrators should be used within 2 hours and should be stored below -20 C. Do not exceed 3 freeze-thaw cycles.
(4) Each unknown sample needs to be diluted 1:100 using Antibody Conjugated Calibrator Zero.
(5) Prepare MMAE Tracer Antibody working solution by 1:21 fold dilution of the MMAE Tracer Antibody by adding the Tracer Antibody into the Tracer Antibody Diluent . Following is a table that outlines the relationship of strips used and antibody mixture prepared. NOTE: the Tracer Antibody should be prepared just prior to use.

Sample Collection: Serum or EDTA-plasma samples are suitable specimens for MMAE-ADC measurement. Only 10 L of samples is required for a duplicate determination of MMAE-ADC with this test kit. No special preparation of individual is necessary prior to specimen collection. Samples should be collected by standard technologies of clinical laboratory practice and recommended by manufacturer of sample collection tube. It is extremely important to carefully separate the plasma from blood cells to avoid hemolyzation, etc. Samples should be transferred to a clean test tube right after centrifugation and should be stored at 2 ? 8 C if the assay is to be performed within 72 hours. Otherwise, patient samples should be stored at -20 °C or below until measurement. Avoid more than three times freeze-thaw cycles of specimen. Do not use hemolyzed, hyperlipermic, heat-treated or any contaminated specimens.

Assay Procedure:

(1) Add 25 µL of calibrators and diluted 1:100 test samples into the designated microwells. Tap the plate gently.
(2) Immediately add 100 µL of Assay Buffer (cat# 30799)
(3) 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 1 hour at 400 to 450 rpm.
(4) 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.
(5) Add 100 µL of diluted MMAE Tracer Antibody (cat# 30753) to each well. Tap the plate gently.
(6) 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.
(7) 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.
(8) Add 100 µL of ELISA HRP Substrate into each of the wells.
(9) Cover the plate with aluminum foil or other material to avoid exposure to light. Incubate plate static, at room temperature for 20 minutes.
(10) Immediately add 100 µL of ELISA Stop Solution into each of the wells. Mix gently.
(11) Read the absorbance at 450 nm with reference filter at 620 nm.

Calculation of Results:

It is recommended to use a point to point standard curve fitting.
1. Calculate the average absorbance for each pair of duplicate test results.
2. The standard curve is generated by the corrected absorbance of all standard levels on the ordinate against the standard 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 standard curve using their respective corrected absorbance.

Assay Precision: The intra-assay precision was validated by measuring three spiked samples with 16 replicate determinations.The inter-assay precision was validated by measuring two control levels in duplicate in 8 individual assays.

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