General Considerations

While the core component of each of the procedures described in this guide is a radiolabeled immunoglobulin, we will only provide protocols for the immunoreactivity assays themselves. Thankfully, there are several excellent extant reviews and protocols describing the synthesis and purification of radioimmunoconjugates [9,10,11,12]. It is recommended that each of these assays is performed at least in triplicate, as multiple replicates will help identify random experimental error and allow for the calculation of standard deviations. To provide representative data, we have performed each of the assays using a 89Zr-labeled variant of huA33 — [89Zr]Zr-DFO-huA33 — a humanized mAb that binds the transmembrane glycoprotein A33 that is expressed in > 95% of colorectal cancers. For the assays requiring cells, we have paired the radioimmunoconjugate with A33 antigen-expressing SW1222 human colorectal carcinoma cells acquired from American Type Culture Collection [13,14,15]. A list of required materials for each assay is available in the Supplemental Information.

The Linear Extrapolation Assay (“The Lindmo Assay”)

In 1984, Lindmo et al. first reported an assay for measuring immunoreactivity by determining the fraction of radioimmunoconjugate that binds to several concentrations of cells and then using a modified Lineweaver–Burk plot to extrapolate these results to a theoretical environment with infinite excess antigen (Fig. 1) [16]. A protocol is described below, our sample data is shown in Table 1, and a spreadsheet that can be used for this assay is available at https://doi.org/10.5061/dryad.mcvdnck6j. It is important to note that the cell numbers we describe below represent benchmarks and/or starting points but need not be the same in all experiments. Indeed, it may be necessary to adjust the number of cells based on the expression level of the target antigen (see “Discussion” for more).

1.

In microcentrifuge tubes, prepare six aliquots of 5 × 106 antigen-expressing cells in 1 mL PBS (pH 7.4) containing 1% BSA (PBS-BSA) (n = 3 for both the experimental and blocking cohorts).

2.

Perform five 1:2 serial dilutions of the cell suspensions in PBS-BSA for each of the aliquots resulting in final volumes of 1 mL. The dilutions will contain 2.5 × 106, 1.25 × 106, 6.25 × 105, 3.125 × 105, and 1.5625 × 105 cells.

3.

Add 50 µg (3.33 × 10−10 mol) of non-radioactive immunoconjugate to the aliquots in the blocking series to saturate the antigens on the cells. Add the same volume of PBS to the experimental cohort to ensure that all the samples have the same volume.

4.

Incubate all samples on ice for 30 min and manually agitate the tubes every 10 min via gentle inversion to prevent the formation of a cell pellet.

5.

Prepare the radioimmunoconjugate to a final concentration of 40 ng/mL in PBS-BSA and add 500 µL to each sample.

6.

At 1 h, remove a replicate sample (e.g., 75 µL) from each aliquot in both the experimental and blocking series to serve as the total radioactivity aliquots.

7.

Centrifuge the experimental and blocking samples at 650 rcf and remove the remaining liquid to isolate the cells.

8.

Wash the cells 3 × with 1 mL PBS via centrifugation for 2.5 min at 650 rcf. Discard the supernatants.

9.

Determine the counts-per-minute (CPM) of radioactivity in each sample using a gamma counter.

Fig. 1

Schematic of the linear extrapolation assay

Table 1 Immunoreactive fraction values for [89Zr]Zr-DFO-huA33. Each experiment was performed in triplicate.

The count data collected via the protocol above should first be used to calculate the ratio of the cell-associated radioactivity to the total radioactivity for each sample. These ratios should then be plotted as a function of increasing cell concentration, and the plateau of the resulting curve denotes the immunoreactive fraction of the radioimmunoconjugate by approaching a value of 1. Alternatively — and more commonly — the same data can be graphed on a double-inverse plot. In this case, the ratio of the total radioactivity to the cell-associated radioactivity is plotted against the inverse of the cell concentration. Linear regression analysis can then be used to fit a straight line to these data. The inverse of the y-intercept represents the immunoreactive fraction (1/r).

In the event that the assay provides suboptimal immunoreactivity values — i.e., values under 40–50% — it is (of course) possible that the antigen binding domains of the radioimmunoconjugate have been irreparably altered. However, this result could also be explained by a failure to include data from samples in which the radioimmunoconjugate is incubated with a sufficient excess of antigen. In this case, we recommend increasing the number of samples in the serial dilutions to provide more data points with high cell concentrations, a change that should result in a more distinct plateau and an improved linear extrapolation.

The Saturation Assay

The cell-based saturation assay is the simpler, more straightforward cousin of the linear extrapolation assay described above. While the latter is predicated on the linear extrapolation of data to a theoretical condition of infinite antigen excess, the former relies upon single experimental samples that provide the radioimmunoconjugate in question with a vast excess of antigen (Fig. 2). This assay’s roots lie in a 1986 paper by Beaumier et al. in which the investigators sought to interrogate the binding of a radiolabeled monoclonal antibody to antigen-expressing melanoma cells [17]. A protocol is described below, our sample data is shown in Table 1, and a spreadsheet that can be used for this assay is available at https://doi.org/10.5061/dryad.mcvdnck6j. As we mention above, the cell numbers we describe represent benchmarks and/or starting points but need not be the same in all experiments. It may be necessary to adjust the number of cells based on the expression level of the target antigen (see “Discussion” for more).

1.

Prepare six aliquots of 2 × 107 cells (n = 3 for both the experimental and blocking cohorts) in microcentrifuge tubes.

2.

Centrifuge the cells for 5 min at 650 rcf and remove the supernatants without disturbing the cell pellet.

3.

Resuspend the cells in 200 µL of appropriate media.

4.

Add 1 ng (6.67 × 10−15 mol) of the radiolabeled immunoconjugate to the experimental samples. Add 1 ng of the radioimmunoconjugate and 5 µg (3.33 × 10−11 mol) of unlabeled antibody to the blocking samples. The relative 5000-fold molar excess of non-radioactive antibody is necessary to saturate antigens on the cells.

5.

Incubate the cells on ice for 1 h, manually agitating the tubes every 15 min via gentle inversion to prevent the formation of a cell pellet.

6.

Prepare 18 additional microcentrifuge tubes: 6 labeled as supernatant, 6 labeled as first wash, and 6 labeled as second wash.

7.

Following the incubation, centrifuge the cells for 2.5 min at 650 rcf.

8.

Transfer the supernatants to the microcentrifuge tubes labeled as supernatant.

9.

Add 500 µL of ice-cold media to the cell pellets, resuspend the cells, and re-centrifuge the cells for 2.5 min at 650 rcf.

10.

Transfer the supernatants to the microcentrifuge tubes labeled as first wash.

11.

Add 500 µL of ice-cold media to the cell pellets, resuspend the cells, and re-centrifuge the cells for 2.5 min at 650 rcf.

12.

Transfer the supernatants to the microcentrifuge tubes labeled as second wash.

13.

Determine the counts-per-minute (CPM) of radioactivity in each sample using a gamma counter.

Fig. 2

Schematic of the cell-based saturation assay

The following equation may be used to determine the immunoreactive fraction (IF) of the radioimmunoconjugate:

$$\mathrm\mathrm=\frac}_\text}}_\text+}_\text+}_1}+}_2}}$$

As with the linear extrapolation assay, the most obvious source of poor immunoreactive fraction values is a damaged radioimmunoconjugate. However, it is also possible that the abundance of the antigen on the cells in question is not sufficient to provide a dramatic excess with only 2 × 107 cells. In this case, increasing the number of cells in the assay and/or decreasing the amount of radioimmunoconjugate employed may provide better results.

The Bead-Based Assay

In the first two assays, antigen-expressing cells are used as the means of interrogating the antigen-binding capability of the radioimmunoconjugate. While these assays are undoubtedly effective, cells are not always the most reliable, convenient, or cost-effective vehicle for the presentation of antigens. Indeed, even if we set aside the cost of cell culture and the persistent risk of cells dying, the expression of a given antigen by a cell line can vary significantly as a function of both passage number and growth conditions. Starting in the late 1990s and early 2000s, a handful of laboratories began reporting immunoreactivity assays in which the antigen in question was attached to a resin or bead rather than a cell [18,19,20]. More recently, Sharma et al. described a particularly well-designed assay predicated on attaching recombinant antigen bearing a His-tag to Ni–NTA-coated magnetic beads, though other adhesion methods — e.g., biotin/streptavidin — could be used as well (Fig. 3) [21]. Ultimately, bead-based assays offer more control over antigen density than their cell-based cousins but, as we will discuss below, are limited to recombinant antigens that can be purchased or produced reliably. A protocol is described below, our sample data is shown in Table 1, and a spreadsheet that can be used for this assay is available at https://doi.org/10.5061/dryad.mcvdnck6j.

1.

Prepare and label nine microcentrifuge tubes (n = 3 for the control, experimental, and blocking cohorts).

2.

Add 20 µL of magnetic beads (12.5 mg/mL in 20% ethanol) to each of the tubes.

3.

To wash the beads, add 380 µL of PBS (pH 7.4) with 0.05% Tween-20 and 50 mM Imidazole (PBS-T), vortex the tubes for 10 s, and quickly centrifuge the tubes to get any liquid off the underside of the lids.

4.

Place the tubes on a magnetic rack for 30 s to allow the beads to move towards the rack.

5.

While the tubes are still on the magnetic rack, remove and discard the supernatant from each of the tubes by pipetting.

6.

Remove the tubes from the rack and repeat steps 3–5 with 400 µL of PBS-T.

7.

Remove the tubes from the rack.

8.

Add 390 µL of PBS-T to the experimental and blocking tubes. Add 400 µL of PBS-T to the control tubes.

9.

Add 10 µL of a 0.1 mg/mL solution of antigen in PBS with 1% BSA to the experimental and blocking tubes.

10.

Place all of the tubes on a rotating stand mixer and turn it up to the minimum speed that ensures that the solutions are mixed with each revolution.

11.

Incubate the samples at room temperature for 30 min.

12.

After incubation, place all the tubes on a magnetic rack for 30 s and remove and discard the supernatants.

13.

Wash the beads by adding 400 µL of PBS-T, vortexing the tubes for 10 s, and then quickly centrifuging the tubes to get any liquid off the underside of the lids.

14.

Place the tubes on a magnetic rack for 30 s to allow the beads to move towards the rack.

15.

While the tubes are still on the magnetic rack, remove and discard the supernatant from each of the tubes.

16.

Remove the tubes from the rack and repeat steps 12–14 with 400 µL of PBS-T.

17.

Add 400 µL PBS-T and 1 ng (6.67 × 10−15 mol) of the radiolabeled antibody to the control and experimental tubes. Add 400 µL of PBS-T, 5 µg (3.33 × 10−11 mol) of unlabeled antibody and 1 ng of radiolabeled antibody to the blocking tubes.

18.

Thoroughly vortex all tubes and place them on a rotating stand mixer at the minimum speed that ensures that the solutions are mixed with each revolution.

19.

Incubate the samples while rotating at room temperature for 30 min.

20.

Prepare 27 additional microcentrifuge tubes: 9 labeled as supernatant, 9 labeled as first wash, and 9 labeled as second wash.

21.

After incubation, place all tubes on the magnetic rack for 30 s. Remove the supernatant from each tube, and pipette it into the appropriate supernatant tube.

22.

Wash the beads 2 × with 400 µL PBS-T and pipet the supernatants from each of the washes in either the first wash tubes or the second wash tubes.

23.

Determine the counts-per-minute (CPM) of radioactivity in each sample using a gamma counter.

Fig. 3

Schematic of the bead-based assay

The following equation may be used to determine the immunoreactive fraction (IF) of the radioimmunoconjugate:

$$\mathrm\mathrm=\frac}_\text}}_\text+}_\text+}_1}+}_2}}$$

In a manner very similar to the cell saturation assay described above, one possible cause for low immunoreactivity values — beyond a damaged mAb — is the failure to present the radioimmunoconjugate with a vast excess of antigen. If this is the case, the situation may be alleviated by increasing the amount of antigen loaded onto the beads, increasing the number of beads used in the assay, and/or decreasing the amount of radioimmunoconjugate used in the assay.