Description

Nori Equine IgM ELISA Kit Summary

Alternative names for IgM: Immunoglobulin M

Alternative names for equine: horse

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background: 

Immunoglobulin M (IgM) is a basic antibody that is produced by B cells. IgM is by far the physically largest antibody in the human circulatory system. It is the first antibody to appear in response to initial exposure to an antigen. The spleen is the major site of specific IgM production. IgM forms polymers where multiple immunoglobulins are covalently linked together with disulfide bonds, mostly as a pentamer but also as a hexamer. The J chain is found in pentameric IgM but not in the hexameric form.^[1] IgM has a molecular mass of approximately 970 kDa (in its pentamer form). Because each monomer has two antigen binding sites, a pentameric IgM has 10 binding sites. However, IgM cannot bind 10 antigens at the same time because the large size of most antigens hinders binding to nearby sites. Because IgM is a large molecule, it cannot diffuse well, and is found in the interstitium only in very low quantities. IgM is primarily found in serum; however, because of the J chain, it is also important as a secretory immunoglobulin. Due to its polymeric nature, IgM possesses high avidity, and is particularly effective at complement activation. By itself, IgM is an ineffective opsonin; however it contributes greatly to opsonization by activating complement and causing C3b to bind to the antigen.^[2] IgM antibodies appear early in the course of an infection and usually reappear, to a lesser extent, after further exposure. IgM antibodies do not pass across the human placenta (only isotype IgG). These two biological properties of IgM make it useful in the diagnosis of infectious diseases. Demonstrating IgM antibodies in a patient’s serum indicates recent infection, or in a neonate’s serum indicates intrauterine infection (e.g. congenital rubella). The development of anti-donor IgM after organ transplantation is not associated with graft rejection but it may have a protective effect.^[3]

References

1. Erik J. et al, J. Immunol., Jun 1998; 160: 5979 – 5989.
2. Wellek, B, et al. (1976). Agents and Actions 6 (1–3): 260–262.
3. Charpak, Y, et al. (2004). Liver Transpl 10 (2): 315–319.

Product Citations