SPECIAL CONSIDERATIONS:
IgM Serologic Assays IgM serologic assays commonly encounter two types of interference. The first stems from high levels of patient IgG antibodies, which can compete with IgM for antigen binding sites on the solid phase. Because IgG makes up approximately 75% of total serum antibodies, it can easily outcompete IgM due to its abundance. The second type of interference is caused by rheumatoid factor (RF), which can bind to the Fc region of immunoglobulins causing false-positive results. To address these issues, IgM serologic assays typically require two types of blockers: a HAMA/ RF blocker to reduce RF interference, and an IgG absorbent to prevent interference from the more abundant and potentially cross-reactive IgG antibodies.
RECOMMENDED BLOCKERS FOR IgM ASSAYS
IgG Absorbent
IgM Assay Diluent
L15406G
8120
Goat Anti-Human IgG Fc
ELISA IgM Assay Diluent I
IgG Absorbent is a purified goat anti-human IgG (GAH IgG Fc) Fc fragment designed for the removal of human IgG and IgG/rheumatoid factor (RF) complexes from serum prior to testing for specific IgM antibodies in ELISA or other immunoassays. Removal of IgG interference has been demonstrated to significantly increase the sensitivity of IgM detection in immunoassays. Meridian’s IgG Absorbent is tested by immunoelectrophoresis to ensure specificity for IgG and no cross-reactivity to IgM or IgA.
IgM Assay Diluent is intended for use in qualitative and quantitative assays that detect IgM antibodies. This diluent is formulated to reduce assay interference from a patient’s IgG antibodies, rheumatoid factor, heterophilic antibodies, and other non-specific proteins that can affect the immunoassay results. Blocking proteins within the IgM Assay Diluent are in high excess and will bind to any open binding site on the assay solid phase to prevent binding from interfering factors. When included as a part of a test kit, IgM Assay Diluent can increase the sensitivity of an IgM detection assay. In a separate tube, dilute the patient serum sample in the IgM Assay Diluent at a 1:21 dilution or greater (mix well). The diluent must be standardized with the other assay components.
RECOMMENDED PROTOCOLS
Dilute prior to adding to patient sample. Recommend diluting 1:10 in PBS. Add in a ratio of 1:10 to patient sample and allow to incubate 5-30 minutes.
TIPS TO ENHANCE ASSAY PERFORMANCE Blocker Use Guidelines ELISA
CLIA Add to the reaction mix during the assay setup
Lateral Flow Assay Add to the conjugate pad, sample buffer, or pre-treatment buffer, or apply directly to the membrane as a blocking stripe ahead of the test stripe
Add to the sample or conjugate diluent, but ideally should be in contact with the patient sample before incubation with the assay capture antibody
PRODUCT
RECOMMENDED CONCENTRATION
Mouse Serum
5-20% of the sample or conjugate diluent (v/v)
Mouse IgG
0.1-2 mg/mL or 10x the concentration of the conjugate MAb used in the assay
TRU Block TM Series Diluted samples: 0.5-20 μ g/mL, undiluted samples: 5-200 μ g/mL K-BLOCK TM 0.2-200 μ g/mL IgG Absorbent Dilute prior to adding patient sample (1:10 in PBS) in a ratio of 1:10 IgM Assay Diluent Warm diluent to room temp. before use and add to patient sample at a 1:21 ratio or greater
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