Rat Immunoglobulin Isotyping ELISA Kit

Catalog No. ABIN1305251

Product Details

Target: Ig

Reactivity: Rat

Detection Method: Colorimetric

Conjugate: HRP

Application: Isotyping (IsoT)

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

Material not included: 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 and Sample Diluent (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 mouse anti-rat Ig Ab 1:100 with Blocking Buffer (12 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 never be stored for a long period. Diluted aliquots should not be frozen.
1. Dilute an appropriate amount of each isotype-specific mouse anti-rat monoclonal antibody in Coating Buffer and deliver 100 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 crosscontaminate 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 hybridoma culture supernatant to be tested to 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 mouse anti-rat 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 50 µl 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

Handling

Storage: 4 °C

Target Details

Target: Ig

Alternative Name: Immunoglobulin (Ig Products)

Synonyms: ATPIG Kit, ATPIQ Kit, A330005H02Rik Kit, AI315324 Kit, Ig Kit, ATPase phospholipid transporting 11C Kit, ATPase, class VI, type 11C Kit, ATP11C Kit, Atp11c Kit

Background: BD Pharmingen monoclonal antibody-based Rat Immunoglobulin Isotyping ELISA Kit enables rapid, efficient identification of rat immunoglobulin isotypes. The Kit employs a direct horseradish peroxidase-labeled system, and the assay format eliminates the need for coating the plate with antigen. These features lead to a significant reduction in assay time without sacrificing sensitivity. See the enclosed Assay Protocol.
Each antibody was generated from rat immunoglobulin (Ig) antigen and has been extensively tested for reactivity toward rat Ig isotypes. Each monoclonal antibody is specific for its stated isotype. However, the anti-rat IgG2a antibody pair has been shown to cross-react with rat IgG1 isotype antibodies in some instances. The positive control is a mixture of purified monoclonal rat immunoglobulins of nine Ig heavy- and light-chain isotype combinations (IgG1kappa, IgG1lambda, IgG2akappa, IgG2alambda, IgG2bkappa, IgG2ckappa, IgMkappa, IgMlambda, and IgAkappa).
This kit was formerly known as the Monoclonal Antibody-based Rat Immunoglobulin Isotyping Kit.