|Rabbit Anti-Mouse IgG1||100 μL|
|Rabbit Anti-Mouse IgG2a||100 μL|
|Rabbit Anti-Mouse IgG2b||100 μL|
|Rabbit Anti-Mouse IgG3||100 μL|
|Rabbit Anti-Mouse IgM||100 μL|
|Detection Antibody (HRP conjugated Rabbit Anti-Mouse IgG)||20 μL|
|Positive Control: Mouse Ig isotype control mixture||25 μL|
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 notable companies produced .
Storage / Stability
Shipped at ambient temperature. Store at –20℃ upon receiving, which are stable for twelve months from date of receipt. Avoid repeated freeze-thaw cycles.
Procedure for Capture ELISA
1. Dilute the isotype specific antibodies in PBS. Dilution ratio 1:200 in PBS for IgG1, IgG2a, IgG2b, IgG3, IgM. Immediately coat a 96-well microplate with 100 μL per well of the diluted antibody. Incubate the plate (covered) overnight at 4℃ or 2 hours at 37℃.
2. Aspirate each well and wash with at least 300 μL 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 300 μL 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. Add 100 μL/well of your samples to the wells (use culture supernatant undiluted, dilute concentrated or purified samples diluted in Sample dilution buffer to 1-2 μg/mL). Add 100 μL/well of the Positive Control and Negative Control into the corresponding wells. Incubate the plate at room temperature for 1 hour.
6. Repeat the aspiration / wash as in step 2.
7. Dilute the HRP conjugated Rabbit Anti-Mouse IgG antibody 1:5000~1:10000 in Sample dilution buffer. Add 100 μL 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 200 μL 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 50 μL 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.
Engvall E, et al. (1971) Enzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulin
G. Immunochemistry. 8 (9): 871–4.
Leng S, et al. (2008) Elisa and Multiplex Technologies for Cytokine Measurement in Inflammation and Aging
Research. J Gerontol a Biol Sci Med Sci. 63 (8): 879–84.
MedLinePlus. (2007) HIV ELISA/western blot. U.S. National Library of Medicine.
Lequin R. (2005) Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA). Clin Chem. .
51 (12): 2415–8.