Isotyping Kit for Mouse Monoclonal Antibody

Temporarily not available outside of China.

Materials provided:

Items 10 Plates
 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

Note:

  • • Sufficient reagents are provided to perform 160 Clones by ELISA.
  • • Please quick-spin vial before opening, for maximal recovery of contents.
  • • IgG1, IgG2a, IgG2b, IgG3, IgM should be diluted 1:200 in PBS before use.
  • • Detection Antibody should be diluted 1:5000~1:10000 in Sample dilution buffer before use.
  • • Positive Control should be diluted 1:200 in Sample dilution buffer before use.

 

To determine the subclass of mouse monoclonal antibodies (IgG1, IgG2a, IgG2b, IgG3, IgM) derived from hybridoma supernatant or purified forms.

  • Guaranteed 100% Accuracy
  • No background
  • Higher specificity
  • Rabbit mAb
  • Optimized Antigen
  • Stable capture ELISA format
1. ULTRA-HIGH ACCURACY
Sino Biological mouse Mab isotyping kit is composed of the super specific rabbit monoclonal antibodies derived from specially optimized Ig antigens. It can determine each single Igisotype from an antibody mixture of several Ig types with almost 100% accuracy, compared with a US leading brand kit. and significantly reduces the false-positives caused by interfering types of Igs in samples.

 

 

2. NO BACKGROUND(NO MIS-MATCH)
Sino Biological isotyping reagents give no background and lower deviation results comparing to the US leading brand.

Description

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.

specificity

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.

Notes:

  • • The Detection Antibody (HRP conjugated Rabbit Anti-Mouse IgG) should been protected from prolonged exposure to light.
  • • 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 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.

Results

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.

References

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.

 

Citation List

  • Generation and purification of monoclonal antibodies against Der f 2, a major allergen from Dermatophagoides farinae
    Author
    Chen, H;Zhang, K;Wang, S;Xu, C;Zou, Z;Tao, A;
    Year
    2016
    Journal
    Drug Discov Ther
    Application
    ELISA
  • A novel enzyme-linked immunosorbent assay based on anti-lipovitellin monoclonal antibodies for quantification of zebrafish (Danio rerio) vitellogenin
    Author
    Wang, J;Wang, W;Tian, H;Zhang, X;Ru, S;
    Year
    2017
    Journal
    Ecotoxicol. Environ. Saf.
    Application
    ELISA
  • Development of an immunochromatographic strip for the rapid detection of maduramicin in chicken and egg samples
    Author
    Guo, L;Xu, L;Song, S;Liu, L;Kuang, H;
    Year
    2017
    Journal
    Food and Agricultural Immunology
    Application
    ELISA
  • Generation and characterization of a IgG monoclonal antibody specific for GM3 (NeuGc) ganglioside by immunizing β3Gn-T5 knockout mice
    Author
    He, D;Fan, X;Liu, B;Tian, Y;Zhang, X;Kang, L;Tai, Y;Liu, S;Wang, Q;Li, Q;Cai, J;
    Year
    2018
    Journal
    Sci Rep
    Application
    ELISA
  • Development of immunochromatographic assays for the detection of imidacloprid in soil chemical barrier
    Author
    Yang, J;Yang, Q;Deng, J;Tao, Z;Hua, X;Wang, M;
    Year
    2018
    Journal
    Environ Sci Pollut Res Int
    Application
    ELISA
  • A Novel and Efficient Approach for Screening Cancer Cell Specific Monoclonal Antibodies
    Author
    Pei, XH;
    Year
    2019
    Journal
    bioRxiv
    Application
    ELISA
  • Semisynthetic, self-adjuvanting vaccine development: Efficient, site-specific sortase A-mediated conjugation of Toll-like receptor 2 ligand FSL-1 to recombinant protein antigens under native conditions and application to a model group A streptococcal vaccine
    Author
    Xu, Z;Rivera-Hernandez, T;Chatterjee, O;Walker, MJ;Moyle, PM;
    Year
    2019
    Journal
    J Control Release
    Application
    ELISA
  • Development of an Enzyme-Mediated, Site-Specific Method to Conjugate Toll-Like Receptor 2 Agonists onto Protein Antigens: Toward a Broadly Protective, Four Component, Group A Streptococcal Self-Adjuvanting Lipoprotein-Fusion Combination Vaccine
    Author
    Xu, Z;Rivera-Hernandez, T;Moyle, PM;
    Year
    2020
    Journal
    ACS Infect Dis
    Application
    Antibody Isotyping
  • Determination of Dehydroepiandrosterone in Dietary Supplements and Pharmaceutical Products by a Competitive Chemiluminescent Enzyme Immunoassay
    Author
    Xu, L;Liu, X;Chen, C;Dias, A;Zhang, X;
    Year
    2020
    Journal
    Analytical Letters
    Application
    identification of antibody isotype
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