Mouse Immunoglobulin Isotyping ELISA Kit

Catalog No. ABIN1305250

Product Details

Target: Ig

Reactivity: Mouse

Detection Method: Colorimetric

Conjugate: HRP

Application: Isotyping (IsoT)

Purification: Purified from tissue culture supernatant or ascites by affinity chromatography.

Material not included: 96-well ELISA-grade polystyrene or PVC microtiter plates, modular strips are also acceptable Precision pipettes capable of delivering between 50 and 200 µl 0.05% Tween-20 in PBS as washing solution

Application Details

Reagent Preparation:

1. Bring all reagents to room temperature (18 - 25°C) before use.
   2. Coating Buffer (1X PBS): Dilute required quantity of 10X PBS with deionized or distilled water, mix (50 ml for each plate).
   3. Blocking Buffer: Dilute required quantity of 10% BSA 1:10 with 1X PBS (35 ml for each plate).
   4. Dilute positive reference antigen mixture 1:50 with Blocking Buffer (1 ml for each plate).
   5. Dilute HRP-labeled rat anti-mouse Ig Ab 1:100 with Blocking Buffer (10 ml for each plate).
   6. Substrate Solution: Within 15 minutes prior to use, mix equal volumes of Substrate Reagent A and Substrate Reagent B (5 ml of each solution for each plate) in a clean glass tube or flask. Make only the amount required for each test. Discard any remaining working solution after use.

Assay Procedure:

For optimal results, the required amounts of the purified coating antibodies should be diluted immediately before use, and the diluted antibodies should not be stored for a long period of time. Diluted aliquots should not be frozen.
1. Dilute an appropriate amount of each isotype-specific rat anti-mouse purified monoclonal antibody in Coating Buffer and deliver 50 µl of each reagent to applicable rows (see Figure 1 for suggested layout).
2. Tap plate gently to ensure even distribution of antibody solution on the bottom of wells.
3. Incubate, covered, at 37°C for 1 hour or at 4°C overnight.
4. Use washing solution (0.05% Tween-20 in PBS) to wash out plate contents using a plate washer or similar device and taking care not to cross-contaminate wells with different capture antibodies. Then shake out remaining contents, and blot excess on a clean paper towel. Repeat the wash 3X.
5. Add 200 µl of Blocking Buffer (see Reagent Preparation, Step 3) to each well, and incubate at room temperature for 30 minutes.
6. To prepare for Step 13, wash 3X, shake out Blocking Buffer, and blot dry.

Sample Incubation:
7. Pipette 100 µl of each sample (e.g hybridoma culture supernatant) to be tested to the appropriate plate columns and incubate for 1 hour at room temperature. Positive controls (see Reagent Preparation, Step 4) should be included as desired, negative controls generally consist of parent myeloma culture supernatant (see Figure 1 for suggested layout).
8. To prepare for Step 15, wash 3X, shake out remaining contents, and blot dry.

Enzyme Conjugate Incubation:
9. Pipette 100 µl of HRP-labeled rat anti-mouse Ig mAb solution (see Reagent Preparation, Step 5) to each well, and incubate at room temperature for 1 hour.
10. To prepare for Step 17, wash 6X, soaking the wells for 30 seconds to 1 minute on each wash. Thorough washing at this step is very important.

Color Development:
11. Add 100 µl of prepared Substrate Solution (see Reagent Preparation, Step 6) to each well and incubate plate for 3 - 10 minutes at room temperature. Positive reaction wells will develop a greenish-blue color. Negative wells will be colorless.
12. Pipette 50µl of Stop Solution to each well. Positive wells will become yellow.
Plate Result Reading:
13. Read visually or spectrophotometrically at 450 nm. If wavelength correction is available, subtract A (570 nm) from A (450 nm). Figure 2 is an example of the visual readout for immunoglobulins of various isotypes.

Restrictions: For Research Use only