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Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

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Published on: September 6, 2017

HLA antibody specification using single-antigen beads--a technical solution for the prozone effect.

Martina Schnaidt¹, Christof Weinstock, Marija Jurisic

¹Center for Clinical Transfusion Medicine, University Hospital of Tübingen, Otfried-Müller-Strasse 4/1, Tübingen, Germany. martina.schnaidt@med.uni-tuebingen.de

Transplantation

|August 27, 2011

Summary

The prozone effect in Luminex single-antigen bead assays, causing false negatives, is linked to complement component 1 (C1). Adding EDTA to serum effectively prevents this issue in high antibody titer testing.

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Personalized Peptide Arrays for Detection of HLA Alloantibodies in Organ Transplantation

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Area of Science:

Immunology
Transplantation Immunology

Background:

Luminex single-antigen bead (SAB) assays advanced human leukocyte antigen antibody specification.
A prozone effect causing false-negative results in high antibody titers has impaired SAB assay progress.
The prozone effect was observed in serum but not EDTA plasma samples.

Purpose of the Study:

To investigate the cause of the prozone effect in SAB testing.
To identify methods to overcome the prozone effect.

Main Methods:

Sera exhibiting a prozone effect were tested using SAB assays with various pretreatment methods.
Parameters influencing the prozone effect were explored through serum pretreatment.

Main Results:

The prozone effect was absent in EDTA plasma and serum with added EDTA.
Dithiothreitol, C1 inhibitor, or heat inactivation abolished the prozone effect.
C1q was found on beads showing a prozone effect, and adding fresh serum restored the prozone.

Conclusions:

Complement component 1 (C1) dissociation or destruction eliminates the prozone effect.
Adding EDTA to serum is a simple method to prevent false-negative SAB testing results due to the prozone effect.