Concept of sample-specific correction of immunoassay results for precise and accurate IgG quantification in horse plasma.

This research focuses on a new in vitro method for accurate and precise quantification of Immunoglobulin G (IgG) in hyperimmune horse plasma (HHP). HHP is often used for antitoxin production and accurate IgG estimation is crucial for efficient purification processes. The study introduces caprylic acid precipitation for IgG isolation and a sample-specific correction of immunoassay results to enhance accuracy and precision in quantifying plasma IgG.

Hyperimmune Horse Plasma and IgG

• Hyperimmune horse plasma (HHP) is often attained through the active immunisation of horses with an antigen, largely serving as the primary source material in the creation of antitoxin therapeutics which are primarily animal antibody-based.
• The accurate and precise quantification of Immunoglobulin G (IgG) content, a type of antibody, within HHP is vital to correctly assess and calculate the efficiency of the purification process.

Limitations of Current IgG Quantification Methods

• Despite over a century’s worth of purification processes concerning immunoglobulins derived from HHP, there’s still no existing in vitro (outside the body) method that ensures the accurate and precise determination of the IgG content in HHP.
• Hence, most purification processes are still primarily assessed through antibody activity measurements, which are mostly undertaken in vivo (in the body).

Developing More Accurate In Vitro Methods

• This study outlines the development of an accurate and precise in vitro immunoassay (biochemical test that measures the presence or concentration of a substance in a solution using an immune reaction) for quantifying IgG in HHP effectively.
• The research asserts that any compositional variability within IgG population between the measuring standard and the sample, in terms of both IgG subclass distributions and antigen-specific IgG content, can influence and result in inaccurate IgG quantification.

Caprylic Acid Precipitation and Sample-Specific Correction

• Caprylic acid precipitation has been demonstrated to be an effective method for isolating IgG from horse plasma without altering population composition.
• This method is highly efficient, speedy, simple, and cost-effective, and enables the creation of an internal, sample-specific reference IgG for each plasma sample, tested simultaneously to a respective plasma sample.
• Subsequently, any deviation in IgG quantity as determined by Enzyme-Linked Immunosorbent Assay (ELISA), an often-utilized method for detecting and measuring antibodies in blood, for each sample-specific reference from its nominal value was used to correct the results of the corresponding plasma sample. This resulted in an accurate and precise IgG quantification as shown by method validation.

Widespread Applicability

• This novel concept of sample-specific correction of immunoassay results could have broad applicability and be beneficial to incorporate in various immunoassays to enhance the accuracy and precision of plasma IgG quantification.