Unopened kits can be stored stably for 12 months at 2-8°C. Do not use the kit beyond the expiration date.
Precision
Assay precision was determined by both intra (n=5 assays) and inter assay (n=5 assays), CV of the ELISA kit all less than 15%. While actual precision may vary from laboratory to laboratory and technician to technician, it is recommended that all operators achieve precision below these design goals before reporting results.
Sensitivity
The analytical sensitivity of the Sirolimus ELISA was found to be 0.31 ppb.
General Description
Sirolimus, also known as rapamycin, is a potent macrolide immunosuppressant with low toxicity and is commonly used in kidney transplants. The mechanism of action of sirolimus is different from that of other immunosuppressants and can be used to treat and reverse acute rejection and prevent chronic rejection. However, sirolimus has a long half-life, a narrow therapeutic window, and large individual variations in the pharmacokinetics and therefore, its improper use may cause adverse reactions, such as immune rejection, thrombocytopenia, leucopenia, or hyperlipidemia. Immunosuppressive drugs in the body are usually measured once or twice a day before the patient is given a new dosage and it is, therefore, important to monitor their blood levels for the duration of any treatment. In addition, a rapid and efficient monitoring method for sirolimus should allow its quantification in human whole blood, because it is mainly distributed in erythrocytes.
Citations
Publication ()
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Hypoimmune FK binding protein 12-knockout CD19 CAR T cells achieve deep tissue B-cell depletion and control Nalm6 tumors in immunosuppressed humanized mice
Xiaomeng Hu, Andrew McAlister, Jeremy Kinder, Adam Johnson, Kathy White, Carolin B Caruso, Chenyan Wang, Ron Basco, Corie Gattis, Annabelle Friera, Tobias Deuse, Sonja Schrepfer
Applications: ELISA Reactive species: Unspecified reactive species
"Abstract: Autologous chimeric antigen receptor (CAR) T-cell therapy in solid-organ transplant recipients on life-long systemic immunosuppression has so far been disappointing. The mTOR inhibitors tacrolimus (Tac) and rapamycin (Rapa) are among the most common immunosuppressive drugs. While they prevent organ allograft rejection, they also suppress the efficacy and persistence of the CAR T-cell product. A reduction in immunosuppression is usually recommended before leukapheresis and after infusion of the final CAR therapeutic to facilitate CAR T-cell activity. However, this reduction jeopardizes allograft survival and multiple graft failures have been reported. Here, we report the engineering of allogeneic, human CD19 CAR T cells that are resistant to mTOR inhibitors through the knockout of FK-binding protein 12 (FKBP-KO). Both HLA-replete WT-FKBP-KO and hypoimmune (HLA class I- and II-depleted and CD47-overexpressing, HIP) CD19 CAR T cells maintained effective in vitro killing capacity of benign human B cells and malignant Nalm6 tumor cells when incubated with high Tac and Rapa concentrations. CAR T cells without the FKBP-KO failed to kill both targets in the presence of Tac or Rapa. HIP-FKBP-KO CAR T cells were able to fully evade cytotoxicity from primed allogeneic T cells, natural killer (NK cells), and macrophages. When injected into immunosuppressed Nalm6-bearing humanized mice, only the HIP-FKBP-KO CAR T cells achieved persistence and deep tissue depletion of CD19+ cells over a 25-day study period, whereas the WT-FKBP-KO CAR T cells diminished in number and efficacy. Allogeneic HIP-FKBP-KO CAR T cells may treat post-transplant lymphoproliferative disease effectively in solid organ transplant recipients maintained on immunosuppression." Article snippet: Tacrolimus (*) and rapamycin (Creative Diagnostics, Shirley, NY, USA) enzyme-linked immunoassay (ELISA) kits were used to measure immunosuppressant levels using a microplate reader (*).
Figure 1. Only HIP-FKBPKO CAR T cells can control Nalm6 tumors in immunosuppressed humanized mice.
Background
Sirolimus is a potent inhibitor of antigen-induced T- and B-cell proliferation and antibody production. Demonstration of the potent immunosuppressive activity of sirolimus in animal models of organ transplantation led to clinical trials and subsequent regulatory approval for prophylaxis of renal allograft rejection. Interest in sirolimus as an immunosuppressive therapy in organ transplantation stems from its unique mechanism of action, its unique side-effect profile and its ability to synergize with other immunosuppressive agents.
Figure 1. Chemical structure of Sirolimus.
Sirolimus offers a new therapeutic option for many conditions where conventional therapies are either ineffective or intolerable. In addition, the new and emerging evidence for topical therapy further broadens the scope of use. Sirolimus acts on the mammalian target of rapamycin (MTOR) pathway, forming a complex that inhibits the activation of MTOR complexes 1 and 2. activation of MTOR complexes 1 and 2. This results in reduced protein synthesis, induction of apoptosis, inhibition of cell migration and reduced expression of vascular endothelial growth factor (VEGF). It also downregulates T-cell proliferation and activation and reduces B-cell antibody production.
In some areas, such as TSC, there is strong evidence to support its safety and use. However, for many of the other conditions reviewed, the evidence is limited to case reports and series. As the conditions reviewed are often very rare, especially the severe cases, the evidence needs to be considered carefully. needs to be considered carefully.
References
1. Andrew W Swarbrick. et al., Systematic review of sirolimus in dermatological conditions. Australasian Journal of Dermatology. 2021, doi: 10.1111/ajd.13671
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References
A gold-based immunochromatographic strip for the detection of sirolimus in human whole blood
Sirolimus is a potent macrolide immunosuppressant, with a long half-life, a narrow therapeutic window, and large individual variations in pharmacokinetics. In this study, an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold-based immunochromatographic strip were developed to establish a rapid and efficient clinical screening of sirolimus. The hapten for sirolimus was synthesized by the oximation reaction and then coupled to keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) to prepare the immunogen and coating antigen. Based on highly specific monoclonal antibodies (mAbs), the 50% inhibitory concentration (IC50) of the optimized ic-ELISA was 1.54 ng mL?1 and the limit of detection (LOD) was 0.19 ng mL?1 in whole blood samples. Good specificity was revealed by its low cross-reactivity (CR) with several analogs of sirolimus. The visual limit of detection (vLOD) and cut-off values of the strip were 20 and 100 ng mL?1 in whole blood samples, as assessed by the naked eye. Therefore, both methods could be used as potentially rapid monitoring approaches for the detection of sirolimus in human whole blood.
COA
Publication 
Figure 1. Chemical structure of Sirolimus.
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