|For100 tests||For1000 tests|
|Rabbit Anti-Mouse IgG1||20ul||200ul|
|Rabbit Anti-Mouse IgG2a||4ul||40ul|
|Rabbit Anti-Mouse IgG2b||20ul||200ul|
|Rabbit Anti-Mouse IgG3||20ul||200ul|
|Rabbit Anti-Mouse IgM||4ul||40ul|
|Rabbit anti-mouse IgG-HRP (Antigen Affinity Purified, sell alone, 50323-RP02)||10ul||100ul|
Dilution ratio 1:1000 in PBS for IgG1, IgG2b, and IgG3. Dilution ratio 1:5000 in PBS for IgG2a and IgM. Diflution ratio 1:5000~1:20000 for Rabbit anti-mouse IgG-HRP.
To determine the subclass of mouse monoclonal antibodies(IgG1, IgG2a, IgG2b, IgG3, IgM)derived from hybridoma supernatant or purified forms.
The Isotyping Reagents for Mouse Monoclonal Antibody (IRMMA) are the research tool intended for qualitative isotype determination of mouse immunoglobulins. This new generation product enables accurate identification of mouse immunoglobulin isotypes, including IgG1, IgG2a, IgG2b, IgG3, and IgM, from hybridoma cell culture supernatant or purified antibodies by capture Enzyme Linked Immunosorbent Assay (ELISA). The tool consists of rabbit monoclonal antibodies against mouse antibody isotypes, and has a higher specificity and sensitivity than most similar products available. In case mixed mouse hybridoma cell cultures, the IRMMA can quantitatively determine each antibody isotype, which is more effective and accurate than antigen based antibody detection methods.
Pronounced superiority of this Mouse Monoclonal Antibody Isotyping Kits is high specific, capable of identifying every subtype and isotype of antibodies existing in Hybridoma cell supernatant or purified forms through reacting with the Fc segments of target antibodies (Fig1). Comparison tests revealed much higher specifity of this Elisa kits than some other noteble companies produced .
Storage / Stability
The kit ships on wet ice and for continuous use, store at 2–8℃. For extended storage, the solutions may be frozen in working aliquots. Repeated freezing and thawing is not recommended. Storage in “frost-free” freezers is not recommended. If slight turbidity occurs upon prolonged storage, clarify the solution by centrifugation before use.
Procedure for Capture ELISA
1. Dilute the isotype specific antibodies in CBS (0.2 ml of each diluted antibody is needed for each sample to be tested). Dilution Ratio differs in detecting different isotypes. Ratio 1:1000 in PBS is appropriate for IgG1, IgG2b, and IgG3. 1:5000 in PBS is for IgG2a and IgM. HRP conjugated Rabbit anti-mouse IgG (Sell alone, 50323-RP02) should be diluted 1:5000~1:20000 before use. Immediately coat a 96-well microplate with 100ul per well of the diluted antibody. Incubate the plate (covered) overnight at 4°C or 2 hours at 37°C.
2. Aspirate each well and wash with at least 300μl wash buffer, repeating the process two times for a total of three washes. Complete removal of liquid at each step is essential for good performance. After the last wash, remove any remaining wash buffer by inverting the plate and blotting it against clean paper towels.
3.Block plates by adding 300μL of blocking buffer to each well. Incubate at room temperature for a minimum of 1hour.
4. Repeat the aspiration / wash as in step 2. The plates are now ready for sample addition.
5. Pipette 0.1 ml of the sample to be tested into each of the wells (use culture supernatant undiluted, dilute concentrated or purified samples diluted in PBS to 1–2 ug / ml). Incubate the plate at room temperature for 1 hour.
6. Repeat the aspiration / wash as in step 2.
7. Dilute the peroxidase labeled Rabbit Anti-Mouse IgG antibody 0.05ug / ml in dilution buffer. Add 0.1 ml of the enzyme conjugated antibody to each well. Incubate the plate at room temperature for 1 hour.
8. Repeat the aspiration / wash as in step 2.
9. Add 200μL of substrate solution to each well. Incubate for 10 minutes at room temperature (if substrate solution is not as requested, the incubation time should be optimized). Avoid placing the plate in direct light.
10. Add 50μL of stop solution to each well. Gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well immediately, using a microplate reader set to 450nm.
The antibody isotypes are visibly identified in ELISA applications. The high Optical Density (450nm) suggests the right antibody isotype or subtype. Nevertheless, given the nature of samples being evaluated, in many cases careful attention must be paid when the results are being interpreted.
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