Autoradiography Core Facility - our offer

Autoradiography is a quantitative in vitro or ex vivo method employed to visualize and quantify the binding of radioactive isotope-labelled compounds (normally called “radioligand” or “tracer”) to specific targets (e.g. receptors, transporters, enzymes, protein aggregates) in post-mortem or excised tissue from human donors or animal models. [Whole body autoradiography is only currently possible with mouse models.]

This method is a critical component in drug development, for initial characterization of a compound’s biodistribution, binding kinetics and pharmacodynamic properties to predict the suitability of its application in vivo. It is also a useful tool for validation of novel radioligands for PET or theranostics since it provides high-resolution images that help characterize the region and target specificity of the tracer.

Besides traditional radioligands, autoradiography can also be done with radiolabeled monoclonal antibodies or other bioconjugates. The use of such radiolabeled bioconjugates in PET and theranostics studies has seen a surge in recent years, particularly in development of next-generation cancer therapies and precision medicine.

The types of autoradiography assays that can be done are largely the same as those which can be done in tissue or cell homogenates (see radioligand binding section below), with the advantage of being able to visualize the anatomical location of the binding in situ, and the disadvantage that using tissue sections limits the throughput of the experimental design and larger volumes of tracer are required.

For validation of tracer specificity, the radioligand binding signal can be compared with immunohistochemical staining of the target protein.

Autoradiograms obtained by exposure and scanning of phosphor storage imaging plates reach a pixel resolution of 50 µm. Higher resolution (<10 µm) can be achieved by applying a thin coat of nuclear emulsion followed by exposure for subsequent examination using dark-field microscopy, where white silver grains represent the bound radioligand. The crystal diameter of the emulsion is smaller than that used to coat the phosphor storage imaging plates. The drawback with emulsion autoradiography is the higher cost and lower throughput.

Radioligand binding is a highly accurate, quantitative, and high-throughput method used to study a range of pharmacological properties (binding kinetics and pharmacodynamics) of a radioligand or tracer to a specific target protein. This method uses tissue or cultured cell homogenates, normalized to a defined protein concentration per reaction, which helps reach very high reproducibility and quantitative value. 

There are several types of radioligand binding assays that can done, e.g.: 

• Saturation binding assays, where samples are incubated, to equilibrium, with increasing concentrations of radioligand to determine of the maximum density of target available to the radioligand (Bmax) and the affinity (i.e. the equilibrium dissociation constant, Kd). Specificity is measured by adding a high concentration of non-labeled compound that is expected to fully displace the radioligand.
• Competition (displacement) binding assays, where samples are co-incubated, to equilibrium, with a fixed concentration of a target-specific radioligand and increasing concentrations of other unlabeled compounds expected to compete for binding to the same target. These assays are mainly used for:
  • High throughput screening of (up to hundreds) of compounds for characterizing their affinity to the target.
  • Confirming the pharmacological binding site of a candidate tracer / radioligand. This is done by running displacement assays using known drugs (unlabeled) whose specificity and affinity for the target of interest are already known. If these known drugs displace the radioligand, we can determine whether the radioligand specifically binds to that target.
• Binding Kinetics assays, to measure the ligand association (K_on) and dissociation (K_off) rates on a specific target:
  • With association binding assays, a fixed concentration of radioligand is added and specific binding is measured at increasing incubation times. This is useful to characterize the interaction of the ligand with the receptor and measure the time it takes for the radioligand to reach binding equilibrium with the target. 
  • With dissociation binding assays, the radioligand is first allowed to bind the target to equilibrium, then dissociated either by adding a very high concentration of unlabeled competitor or by adding a very large volume of buffer to dilute the concentration of the radioligand to an extremely low and negligible concentration. 
• Functional [³⁵S]GTPγS binding assays: If you are studying G protein coupled receptors (GPCRs), the level of G protein activation following agonist binding can be measured with this assay.
  • When occupied by an agonist, GPCRs become activated and directly bind to heterotrimeric Gαβγ proteins, which initiates the guanine nucleotide exchange on the Gα subunit, where the dissociation of GDP from Gα allows the binding of GTP in its place. GTP binding leads to the dissociation of the G protein from the receptor and of Gα-GTP from Gβγ, each of which can go on to activate downstream effectors such as e.g. adenylyl cyclase, phospholipase C, and ion channels. Finally, GTPase hydrolysis of GTP to GDP induces the binding of Gα-GDP back to Gβγ, thus inactivating the G protein.

This guanine nucleotide exchange process can be monitored in vitro by measuring the binding of [³⁵S]GTPγS, which is resistant to hydrolysis by GTPase and therefore accumulates in cellular membranes upon GPCR activation. The advantage of this method is that it targets the earliest event in the intracellular signal transduction cascade, so measures are not confounded by downstream signaling amplification or other modulation that other assays can’t exclude.

Studies can be conducted in cell lines and post-mortem tissue samples from animal or human donors. With this assay you can for example:

• Measure potency and efficacy of a drug candidate for a GPCR.
• Characterize phenotype differences of GPCR activity in your model of choice.
• Accurately quantify GPCR desensitization in different conditions, treatments, etc.