ToRCH & Childhood Diseases

Increasing Assay Sensitivity

SOLID PHASE BLOCKING BUFFERS

Solid phase blocking buffers are designed to efficiently prevent non-specific binding, reduce background noise, and stabilize coated proteins to enable more sensitive immunoassays. They work by blocking unoccupied spaces on the surface to prevent non-specific binding to this surface by other proteins or biomolecules.

Recommended Reagents: J82100B Blocking Buffer for ELISA J82300B Blocking Buffer for Lateral Flow, PBS Based J16430D Coating Stabilizer and Blocking Buffer

IgG ABSORBANTS AND RF

The sensitivity and specificity of IgM detection can be compromised by the presence of IgG in the patient sample. There are two major mechanisms by which IgG can interfere with assays for IgM and cause a false result: 1. by competing with specific IgM for substrate binding sites 2. by forming immune complexes with Rheumatoid Factor (RF) which can compete with specific IgM for substrate binding sites

Recommended Reagents: L15406G Goat anti-human IgG FC (GAHG)

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. IgM Diluent 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 as a unit with the other assay components.

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Removal of IgG and RF-IgM can be accomplished by treating the patient specimen with goat anti-human IgG.

HOW TO USE BLOCKING BUFFER AND GAH IgG

General Protocol

STEP 2 Add blocking buffer A. Add the blocking solution directly to the wells, blotting membrane or nitrocellulose membrane depending on the assay type being used. Use at 1X concentration or with further dilution ELISA Blocking Buffer: Cat# J82100B Lateral Flow Blocking Buffer: Cat# J82300B Coating Stabilizer & Blocking Buffer: Cat# J16430D B. Incubate at room temperature for 30 minutes to 2 hours C. Continue with

STEP 4 Add patient sample mixture to the reaction well A. Incubate patient sample with the antigen B. Perform wash steps: remove non-bound reagents

STEP 1 Optimize the plate/nitrocellulose-coating conditions for the antigen A. Coat the plate with antigen: 2-10 μg/mL solution of protein dissolved in an alkaline buffer such as phosphate-buffered saline (pH 7.4) or carbonate- bicarbonate buffer (pH 9.4) B. Incubate plate for several hours to overnight at 4-37°C C. Remove coating solution, perform wash steps

STEP 3 Pre-incubate GAHG with the patient’s serum A. Dilute GAH IgG 1:10 in PBS B. Add diluted GAH IgG to patient sample 1:10 and mix C. Incubate for 3-5 min and proceed with sample testing

If patient IgM is present it binds to the antigen.

Goat anti-human IgG (GAHG)

Patient’s serum with both IgG and IgM antibodies

Patient sample

your process and reagents according to the assay protocol

Patient’s serum and GAHG are pre-incubated prior to adding to plate

The GAHG complexes

with the patient’s IgG

STEP 5 Detection by direct or indirect methods

Antigen is coated to the plate