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fbtwitterlinkedinvimeoflicker grey 14rssslideshare1
Daga, Sunil
Languages: English
Types: Doctoral thesis
Subjects: RC0254
Abstract\ud \ud Following five decades of kidney transplantation, increasingly high risk immunological kidney transplantation (which previously was considered as sub-optimal) are carried out. The risk stratification with the current available assays have allowed safe transplantation in low risk non-sensitised patients and direct transplantation in high risk highly sensitised patients by removal of circulating donor specific antibodies (DSA) with reasonable outcomes. However, a large number of patients with chronic kidney disease and with low or intermediate antibody levels measured by current assay, the best way forward is uncertain resulting in denial of transplantation in some cases. Whilst in other cases, the solid phase Luminex assay may under or overestimate the risks of rejection and graft failure following direct kidney transplantation.\ud \ud Currently only IgG-class of DSA is considered immunologically important and routinely measured in clinical laboratories. Other bio-physiological characteristics such as class, sub-class and binding kinetics of DSA may be more specific for risk stratification of immunological risks. In this thesis, we studied effect of de novo IgM class of HLA-specific antibodies on outcome of kidney transplantation and characterised binding kinetics and strength of HLA-specific antibodies.\ud \ud De novo IgM or IgG HLA-specific responses alone were not associated with adverse outcomes following kidney transplantation. Presence of both IgM and IgG responses, however, was associated with poor graft function at 36 months. There was no temporal relationship of antibody response and episodes of rejections. De novo Donor specific responses were less frequent compared to non-specific responses. A shorter follow-up and use of modern triple immunosuppressant therapy (Tacrolimus, Mycophenolate and Steroid) may explain this.\ud \ud Binding kinetics measured by biosensor assay- surface plasmon resonance (SPR) on purified monoclonal HLA-specific antibodies showed binding kinetics and strength differed between HLA alleles despite same epitope and paratope interactions. There was a tendency towards higher affinity and faster association rate for HLA protein that was the initial immunizing antigen for the corresponding monoclonal HLA-specific antibodies. The dissociation constant (KD) of human monoclonal HLA-specific antibodies range between 10-8 to 10-10 M. Thermodynamic analysis showed higher Gibbs free energy released for interactions with higher binding strength. The binding strength of mixed monoclonal HLA-specific antibodies is generally average of the strength of individual monoclonal HLA-specific antibodies.\ud \ud Enriched polyclonal HLA-specific antibodies from clinical sample gave distinct binding response on bio-sensor based on SPR assay. Quantification of polyclonal HLA-specific antibodies using sandwich ELISA and SPR allowed quantitative measurement of binding kinetics and strengths. A range of binding strength was observed between patients and within same patient antibodies of different affinities was observed. Thus the antibodies could be grouped in four groups based on the strength of binding and this can serve as additional biomarker for risk stratifications.\ud
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    • affinity .................................................................................................................................................256
    • Purification of polyclonal HLA-specific antibodies..............................................................260
    • Quantification of HLA-specific antibodies ..........................................................................292
    • (rejection and graft loss).................................................................................................................321
    • Appendix A - Kidney Advisory Group KAG(13)34................................................................363
    • Appendix B - KIDNEY ADVISORY GROUP - KAG(14)45a ......................................................364
    • Appendix C - Publications / Abstracts.................................................................................368
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