> ## Documentation Index
> Fetch the complete documentation index at: https://docs.adaptyvbio.com/llms.txt
> Use this file to discover all available pages before exploring further.

# SPR methods

> Standard methods for Adaptyv SPR binding characterization on the Carterra LSA XT

This page holds the standard methods for our [SPR](/wiki/technologies/SPR) binding characterization assay. The concentration series, cycle timings and capture format for your experiment can differ, so check your [Binding Data Package](/docs/experiment-setup/binding-data-package) for the values used in your run.

For the BLI equivalent, see [BLI methods](/wiki/methods/bli-methods).

## Cell-free expression of protein ligands

DNA constructs encoding the ligands, each fused to a C-terminal assay tag, were designed by reverse-translating the target protein sequences. The sequences were optimized for manufacturability and yield using codon optimization algorithms to maximize expression efficiency. The optimized DNA constructs of the variants and the 3′ fragment containing the linker and affinity tags were ordered as Gene Fragments from Twist Bioscience.

Constructs were assembled using the NEBuilder HiFi DNA Assembly Kit (NEB) in 2 μL reactions. The assembled products were characterized by capillary electrophoresis (Agilent ZAG DNA Analyzer, ZAG-135-5000 Kit, FA/ZAG 96-Capillary Array Short, 33 cm), and their concentrations were measured using the Qubit DNA Quantification Kit (Invitrogen). Ligand expression was carried out in 8 μL reactions using an optimized prokaryotic in vitro translation system and 4 nM of assembled gene fragment. Reactions were incubated at 37 °C for 8 hours. Post-expression, protein concentration and yield were normalized using an affinity-based quantification assay.

## Surface plasmon resonance (SPR) affinity characterization

Binding kinetics of the expressed ligands to their cognate targets were characterized using surface plasmon resonance (SPR) on a Carterra LSA XT instrument. Twin-Strep-tagged ligands were captured on a sensor surface functionalized with Strep-Tactin XT (IBA Lifesciences), followed by sequential injections of increasing antigen concentrations.

### Chip preparation (Strep-Tactin XT immobilization)

Surface functionalization was performed on a carboxymethyl dextran chip (Carterra CMDP planar or HC30M 30 nm hydrogel, chosen per run) following a multi-step chip preparation protocol:

1. **Conditioning:** the sensor surface was conditioned with 50 mM NaOH to remove residual contaminants and prepare the surface for activation.
2. **Activation:** the surface was activated using freshly prepared EDC/S-NHS (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/N-hydroxysulfosuccinimide) (Xantec) solution to form reactive esters.
3. **Capture:** Strep-Tactin XT was diluted to 50 μg/mL in 10 mM sodium acetate buffer (pH 4.5) and injected onto the activated surface for covalent coupling.
4. **Quenching:** excess reactive groups were quenched with 1 M ethanolamine hydrochloride (pH \~8.5).
5. **Wash:** the surface was washed with 0.1 M sodium borate, 1 M NaCl, pH 9.0 to remove loosely bound material.

Reference and capture sensors underwent identical treatment, allowing for accurate background subtraction.

### Ligand capture and kinetics

Following surface preparation, Twin-Strep-tagged ligands were captured on the immobilized Strep-Tactin XT using the multichannel head (96-channel array) with bidirectional flow for 750 s, followed by a 600 s baseline step in running buffer.

The analyte (for example, the SARS-CoV-2 antigen) was diluted in running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% Tween-20, pH 7.4) to 7 concentrations in a half-log dilution series (for example, 1000 nM to 1 nM). Cycles were run over increasing analyte concentrations as a single-cycle kinetic assay, without intermediate regeneration, at a flow rate of 50 μL/min. Each assay cycle consisted of:

* **Baseline:** 60 s of buffer-only flow
* **Association:** 300 s of analyte injection
* **Dissociation:** 600 s of buffer-only flow

After each full concentration series, the chip surface was regenerated with 10 mM glycine-HCl (pH 1.5) for 5 min, followed by a 20 min wash in running buffer.

### Data processing and analysis

Data were processed with Adaptyv fitting software. All sensorgrams underwent standardized preprocessing: trimming to the relevant phases (baseline, association, dissociation), correcting signal jumps at phase transitions, aligning the association and dissociation phases, and subtracting baseline and reference signals.

Each design was fitted as a group: all of its association and dissociation curves across the concentration series were fitted simultaneously, with k<sub>on</sub> and k<sub>off</sub> shared across concentrations and R<sub>max</sub> fitted per curve. The default model is a 1:1 Langmuir interaction, and K<sub>D</sub> was calculated as k<sub>off</sub> / k<sub>on</sub>, with 95% confidence intervals for each parameter. Alternative binding models (bivalent analyte and conformational change) were also fitted. Where one described the curves better than 1:1, it was selected and its apparent K<sub>D</sub> reported; the portal shows this value, and the API returns it as `kd_app` with the model name in `binding_model`.

Candidate fits were filtered on quality before selection. A fit was kept only if its residual error in both the association and dissociation phases, normalized to signal size, stayed below a fixed threshold, its R<sub>max</sub> was consistent across concentrations, and bulk refractive-index jumps were small relative to the binding signal. The remaining fits were ranked by residual error, then penalized when a rate constant sat at the edge of what the assay can resolve, then ranked by confidence-interval width.

For how to read the result, with examples of good and poor fits, see [Judging fit quality](/guides/binding-characterization#judging-fit-quality) and [Common curve shapes](/guides/binding-characterization#common-curve-shapes) in the binding characterization guide.

Ligands were classified as binders or non-binders based on the presence of quantifiable binding curves and calculated K<sub>D</sub> values. Where a ligand produced a significant signal shift during the association phase (≥300% over the negative control) but the signal could not be reliably fit, the binding label was assigned based on the magnitude of the observed shift.

## Related pages

* [SPR](/wiki/technologies/SPR)
* [BLI methods](/wiki/methods/bli-methods)
* [Binding](/docs/experiment-types/binding)
* [Binding Data Package](/docs/experiment-setup/binding-data-package)


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