# Interrogation of Interfering Factors in ELISA Detecting Angiotensin Receptor Antibodies and Specificity Validation Using the Adsorption Elution Crossmatch (AXE) Technique

## TL;DR

This study investigates factors affecting ELISA detection of antibodies against the angiotensin receptor and validates a new method for improving specificity in transplant rejection monitoring.

## Contribution

The study identifies bovine serum albumin as a key interfering factor in ELISA detection and validates the AXE technique for improving antibody detection specificity.

## Key findings

- Bovine serum albumin in the AOB buffer suppresses ELISA signal even at 10−6 dilution.
- The AXE technique achieved 30% median elution efficiency for AT1R antibodies using the CellTrend ELISA platform.
- The CellTrend ELISA kit accurately detects anti-AT1R antibodies binding to the active receptor form.

## Abstract

Accurate detection of HLAs and non‐HLA antigens is critical for managing long‐term allograft transplantation, particularly in the context of hyperacute, acute, and chronic allograft rejection. Recent studies have identified the role of non‐HLA antibodies, such as those against Angiotensin II Type 1 receptor (AT1R) in transplant rejection. The enzyme‐linked immunosorbent assay (ELISA) is the primary method for measuring AT1R‐specific antibodies (AT1R‐Ab), offering high specificity and reasonable sensitivity. Despite its widespread use in clinical settings, some reports have suggested that pre‐treating the samples with latex beads can eliminate the detection signal in the CellTrend AT1R ELISA assay, potentially raising concerns over false reactivity in the assay. In this study, we demonstrate that the bovine serum albumin (BSA) present in the adsorb out beads (AOB) buffer, even at a dilution of 10−6, plays a key role in signal elimination in the CellTrend AT1R‐Ab detection kit. Additionally, we evaluated the performance of the CellTrend kit and an in‐house affinity‐purified AT1R ELISA in detecting eluted AT1R‐Abs from live cells using the adsorption crossmatch and elution (AXE) technique, which achieved a median elution efficiency of 30% when tested on the CellTrend ELISA platform. Our findings support that the CellTrend ELISA kit accurately detects anti‐AT1R antibodies that bind to the active form of AT1R. However, serum treatments containing BSA interfere with the antibody–antigen capture interface, leading to signal suppression.