Soluble CD4 antigen reactivity in intravenous immunoglobulin preparations: is it specific?
1Department of Biomedical Sciences and Human Oncology (DIMO), University of Bari Medical School, Italy.
Insights
The presence of soluble CD4 antigen (sCD4) in intravenous immunoglobulin (IVIg) preparations is questionable. Human anti-mouse antibodies (HAMA) in IVIg interfere with assays, falsely indicating sCD4 presence.
Area of Science:
- Immunology
- Biochemistry
- Analytical Chemistry
Background:
- Intravenous immunoglobulin (IVIg) is a therapeutic product derived from human plasma.
- Soluble CD4 antigen (sCD4) is a protein involved in immune responses.
- Accurate quantification of sCD4 in IVIg is crucial for product characterization and safety.
Purpose of the Study:
- To investigate the presence and origin of soluble CD4 antigen (sCD4) detected in commercial intravenous immunoglobulin (IVIg) preparations.
- To determine if the detected sCD4 signal is a true analyte or an artifact of the immunoassay.
- To characterize the interfering substances within IVIg that affect sCD4 measurement.
Main Methods:
- Double determinant immunoassay (DDIA) using monoclonal antibodies (MoAbs) against distinct CD4 epitopes.
- Neutralization assays using animal-derived serum and purified immunoglobulins.
- Absorption studies with insolubilized mouse IgG (mIgG).
- Western blot analysis to identify antibody components.
- Testing with a commercial 'CD4-specific Test Kit'.
Main Results:
- Six out of seven IVIg preparations showed apparent sCD4 reactivity via DDIA.
- Preincubation with mouse or bovine serum/immunoglobulins neutralized sCD4 reactivity in most IVIg.
- Absorption with mIgG completely abolished sCD4 reactivity in affected IVIg.
- Eluted human anti-mouse antibodies (HAMA) from IVIg demonstrated dose-dependent binding.
- Western blot confirmed no CD4 protein, but HAMA, in IVIg.
Conclusions:
- The detected sCD4 signal in IVIg preparations is primarily an artifact caused by human anti-mouse antibodies (HAMA).
- HAMA present in IVIg interfere with immunoassays, leading to false-positive results for sCD4.
- The actual occurrence of sCD4 in IVIg preparations is doubtful, necessitating critical evaluation of current assay methodologies.
Abstract:
Soluble CD4 antigen (sCD4) was measured in seven commercially available intravenous immunoglobulin preparations (IVIg) by means of a double determinant immunoassay (DDIA), whereby two MoAbs recognizing two distinct and spatially distant epitopes on CD4 were used to capture and detect the antigen, respectively. Preincubation of six out of seven IVIg, which were found to be apparently positive for sCD4, with mouse- and bovine-derived serum or purified immunoglobulins completely neutralized DDIA reactivity for sCD4. The inhibition was specific since it was not or only partially observed when IVIg were mixed with whole serum or purified IgG from rabbit. Extensive absorption of six IVIg on insolubilized mouse IgG (mIgG) resulted in a complete loss of reactivity. Eluted human anti-mouse antibodies (HAMA) from any of the IVIg displayed a dose-dependent binding in a DDIA, though its extent varied from one preparation to another. Western blot analysis showed that HAMA from all IVIg contained no component with a molecular weight identical with or close to that of recombinant CD4. Purified mIgG markedly influenced the sCD4 reactivity of two IVIg (Sandoglobulin and Globuman I.V.) when sCD4 was measured with a purchased 'CD4-specific Test Kit', thus suggesting that HAMA can exceed the absorbing capacity of the sample diluent. Taken as a whole, these data indicate that sCD4-based DDIA signal is mostly, if not completely, generated by the presence of human immunoglobulin with anti-mouse immunoglobulin reactivity, thus casting doubts on the actual occurrence of sCD4 in IVIg.
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