Antibody Monitoring Education

Antibody-Mediated Rejection (ABMR) is one of the leading causes of immune-mediated allograft failure¹. The presence of donor-specific antibodies (DSAs) is associated with significantly lower graft survival and possible organ rejection demonstrating the need for post-transplant DSA monitoring. The ability to detect DSAs can provide early intervention or prevention of rejection and graft failure. Antibody monitoring is just one component of the post-transplant assessment for patients and their new organ.

HLA-specific antibodies are the most clinically significant in transplant immunology, making it important to detect pre-existing antibodies before transplantation and monitor for de novo antibody development post-transplantation.

There is also emerging significance of antibodies to other antigen systems involved in transplantation such as endothelial cell antigens² , MHC class I–related chain A (MICA)³, angiotensin II type 1 receptor (AT1R) and endothelin A receptor (ETAR)⁴. Such antibodies may explain ABMR episodes in the absence of HLA-specific antibodies.

Post transplant antibodies often emerge in the context of tissue injury, ischemia-reperfusion, or inflammatory episodes. These immune responses can mediate acute and chronic rejection, contribute to HLA-specific and non-HLA antibody-mediated injury, and promote graft fibrosis and dysfunction, ultimately affecting short or long-term graft survival.

Antibody monitoring involves regularly checking the recipient's blood for the presence and levels of DSAs after transplantation. Table 1 shows common reasons for post-transplant monitoring. This monitoring is crucial to detect the development of DSAs that can signal an immune response against the transplanted organ, thus helping ensure the long-term success and function of the transplanted organ. Regular antibody monitoring helps in managing immunosuppressive therapy and making informed clinical decisions to improve patient outcomes. Antibody monitoring shows the effectiveness of treatments, such as plasmapheresis, to remove DSAs in highly sensitized patients allowing for transplantation to occur and measuring the success of DSA removal post transplantation for patients undergoing ABMR. 

The importance of post-transplant monitoring was highlighted by Lefaucheur⁵, who demonstrated that de novo donor-specific antibody (DSA) formation is often one of the earliest indicators of an immune response leading to antibody-mediated rejection (ABMR). DSAs can frequently be detected before clinical signs such as peritubular capillaritis, C4d deposition, or elevated serum creatinine become apparent.

Table 1. Common reasons for post-transplant monitoring.

The methods used for antibody detection need to be of high sensitivity to detect emerging de-novo DSAs post transplant.

The most sensitive methods are solid phase techniques such as microbead multiplexed assay and ELISA, where antigens are immobilized on a solid phase and patient sera are tested for antibody binding. Table 2 describes commonly used solid phase assays used in antibody detection.

Table 2. Commonly used solid phase assays used in antibody detection.

Single Antigen Bead (SAB) Assays

Purpose: Detects antibodies with high sensitivity and specificity.

Target: HLA Class I and II antigens and autoantigens. See table 3 for example of autoantigen SAB panels.

Potential clinical use: 

• Pre-transplant crossmatch risk assessment.
• Post-transplant monitoring for DSA emergence, especially after graft dysfunction.
• Guides immunosuppression adjustments.

Complement-Binding Assays (e.g., C1q or C3d assays) 

These may be used in conjunction with SAB assays to potentially risk-stratify patients.

Purpose: May identify complement-fixing antibodies in SAB assays.

Target: Antibodies with the ability to activate complement.

Potential clinical use: Helps differentiate complement-fixing from non-complement fixing antibodies.

    a. C1q assays

• Detects antibodies that bind C1q, the first component of the classical complement pathway.
• Indicates the complement-fixing potential of antibodies.

    b. C3d assays 

• Similar to C1q but measures binding to C3d, a later complement split product.
• Provides complementary data to C1q; may correlate better with injury.

ELISA-based Assays for Non-HLA Antibodies

Purpose: Detects autoantibodies or antibodies to non-HLA antigens.

Targets include:

• Angiotensin II Type 1 Receptor (AT1R) 
• Endothelin Receptor A (ETAR)

Potential Clinical Use: Investigational and increasingly used to assess chronic rejection or antibody-mediated injury in the absence of DSAs.

 Microbead Multiplexed Assay

Use of testing such as bead-based assays with a secondary detection antibody allow for evaluation of the patient’s antibody architecture. This allows for tracking of changes in the patient's antibody profile over time including the time prior to transplant. This type of testing includes various methodologies:

• Microbead multiplexed  Single Antigen Bead HLA antigen assay: The use of Single Antigen Bead Assays as part of a comprehensive monitoring program identifies changes in existing HLA-specific antibodies or the appearance of new specificities which may reveal an early rejection process. Single antigen beads allow for a precise, highly sensitive determination of a patient’s HLA antibody profile.

• Microbead multiplexed autoantigen  assay. Single Antigen Bead technology can also be used to detect antibodies to autoantigens that are not usually exposed to the immune system, but may become exposed or upregulated during biological stress, such as inflammation or rejection episodes. Autoantibodies to these antigens may often appear after transplant, especially during tissue injury, ischemia-reperfusion, or inflammatory episodes.

ELISA non-HLA

There is also the possibility of other non-HLA antibodies that can be involved in the development of antibodies leading to rejection. One example, Angiotensin II type 1 receptor antibodies (AT1R), are one of many non-HLA antibodies that can be utilized in the detection of rejection and loss of graft function. The ETAR assay is another ELISA based assay designed to detect antibodies that form against endothelin receptor type A (anti-ETAR), a G protein-coupled receptor.

Table 3. Autoantigens by researched organs.

1. El-Zoghby, Z. M., Stegall, M. D., Lager, D. J., Kremers, W. K., Amer, H., Gloor, J. M., & Jordan, S. C. (2009). Identifying specific causes of kidney allograft loss. American Journal of Transplantation, 9(3), 527–535. https://doi.org/10.1111/j.1600-6143.2008.02519.x
2. Delville M, Charreau B, Rabant M, Legendre C, Anglicheau D. Pathogenesis of non-HLA antibodies in solid organ transplantation: Where do we stand? Hum Immunol. 2016 Nov;77(11):1055-1062. doi: 10.1016/j.humimm.2016.05.021.
3. Carapito, R., Aouadi, I., Verniquet, M. et al. The MHC class I MICA gene is a histocompatibility antigen in kidney transplantation. Nat Med 28, 989–998 (2022). https://doi.org/10.1038/s41591-022-01725-2
4. Philogene, M. C., Johnson, T., Vaught, A. J., Zakaria, S., & Fedarko, N. (2019). Antibodies against angiotensin II type 1 and endothelin A receptors: Relevance and pathogenicity. Human Immunology, 80(8), 561–567. https://doi.org/10.1016/j.humimm.2019.04.012 
5. Lefaucheur, C., Viglietti, D., Bentlejewski, C., Duong van Huyen, J. P., Vernerey, D., Aubert, O., Verine, J., Jouven, X., Legendre, C., Glotz, D., Loupy, A., & Zeevi, A. (2016). IgG donor-specific anti-human HLA antibody subclasses and kidney allograft antibody-mediated injury. Journal of the American Society of Nephrology, 27(1), 293–304. https://doi.org/10.1681/ASN.2014111120