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Mesa Labs
Efficient Gyolab anti drug antibody detection solution simplifies operation while improving accuracy
Date: 2024-12-10Read: 1
The Gyrolab platform uses automated injection to perform miniaturized immunoassay on microfluidic CDs. The CD microstructure is filled with streptavidin coated affinity microspheres, which are specifically bound to the analyte through biotinylated capture reagents, and then bound to fluorescently labeled detection reagents. The fluorescence signal on the affinity column is excited by laser and read by a detector. The Gyrolab immunoassay method hasAutomation, high throughput, time-saving, low reagent and sample usage, wide dynamic range, and low matrix effectsSuch advantages have been widely applied in the field of biopharmaceutical research and production.
Gyrolab uses a homogeneous bridging format to detect ADA, and drug molecules are simultaneously used as capture reagents (biotinylation) and detection reagents (Alexa Fluor) ® 647 fluorescent labeling), the sample and reagent are mixed and added to a streptavidin column with CD microstructure, and then ADA or positive control is detected to form bridging complexes between biotinylated drug molecules and fluorescently labeled drug molecules. This detection method can detect all subtypes of ADA (IgG, IgM, IgA, etc.) and is species independent, making it suitable for both preclinical and clinical research.
Gyrolab platformProvide a complete solution for ADA detectionSimplify the workflow from screening experiments to confirmation experiment development, including ADA analysis software modules, Rexxip ADA sample dilution buffer, and two different formats of CDs.
Gyrolab Mixing CD 96 是Microfluidic CD specifically designed for acid hydrolysis detection of ADA, which integrates the sample acid dissociation pretreatment step into the automated detection processEach Mixing CD contains 96 microstructures, each with a sample mixing chamber available for sample incubation and acid dissociation. When conducting ADA detection, the sample is first added to a volume defined channel in the microstructure. A 200 nL sample is introduced into the mixing chamber under CD centrifugal force, and then an acidic buffer is added to the mixing chamber. The ADA drug complex can dissociate in a low pH environment. Then add the Master Mix containing capture reagent and detection reagent into the mixing chamber, and the ADA molecules come into contact with the labeled reagent to form a new ADA complex. This ratio of labeled reagent to unlabeled free drug facilitates the formation of detectable labeled reagent ADA bridging complexes, which are then captured onto streptavidin coated microspheres and detected by a laser-induced fluorescence detector. This detection process achieves full automation from sample reagent addition, online acid dissociation, to signal detection. The automated program precisely controls the time and reaction volume of each step,Minimizing manual operations to the greatest extent possible not only significantly improves detection accuracy, but also greatly shortens analysis time.
GyrolabSupport two different sample pretreatment methods to overcome the interference of free drugs on ADA detectionThe appropriate method can be flexibly selected based on the characteristics and application of the drug. Using Gyrolab technology for ADA detectionmain advantagesincluding:
The upgraded reaction system requires less reagent and sample usage
Automatic online acid dissociation improves detection precision
Affinity column flow-through technology minimizes matrix effects, improves detection sensitivity and drug tolerance
High loading capacity of affinity columns is more conducive to competition between high concentration labeled reagents and free drugs, further improving drug tolerance through metrological advantages
Fast analysis speed, with a running time of about 1 hour per CD. Using Mixing CD 96 online acid dissociation detection, the running time is about 2 hours
Less hands-on operation, reducing human error
Specialized ADA software modules are developed according to regulatory standards to guide the screening and validation testing process
The key parameters of immunogenicity detection methods includeSensitivity, drug tolerance, and selectivity of detectionDrug tolerance refers to the ability to detect anti drug antibodies (ADA) in the presence of residual therapeutic drugs, and evaluating the binding of free drugs to ADA is to avoid the risk of false negatives. Selectivity refers to evaluating the robustness of a test in normal or diseased matrices, and is crucial for ensuring consistency in evaluating different samples in clinical studies. In addition, a major challenge in using bridging methods for ADA detection is target interference, as soluble targets in the form of dimers or oligomers present in clinical samples may lead toFalse positive results.
During the development of an ADA detection method for a therapeutic antibody drug (Novimab), researchers from Novimune SA used Gyrolab and ECL (Electochemiluminescence, ECL) technologies to detect samples processed in different ways（Target depletion samples, non target depletion samples, acidified samples, and non acidified samples）And provide key parameter examples for the two detection methodsSensitivity, drug tolerance, and target tolerance were evaluated and comparedTo obtain a clinical immunogenicity testing method that is fit for purpose [1].
Researchers useBiotinylated drugs as capture reagents， Alexa Fluor ® 647 labeled (Gyrolab) or Sulfo tag labeled (ECL) drugs were used as capture reagents, and purified polyclonal rabbit anti drug antibodies were used as ADA positive reference materials for ADA detection in a homogeneous bridging format on two platforms. The Gyrolab platform uses Bioaffy 200 CD for non acidic sample detection and Mixing CD 96 for acidic sample detection. The sample acid dissociation and neutralization are automatically completed on the Mixing CD.
Detect one negative control sample (NC) and nine positive control samples (PC, 2, 5, 20, 50, 100, 250, 500, 1000, and 10000 ng/mL ADA) within three days, and determine sensitivity based on the results of running between the three tests, that is, continuously returning low PC concentrations above the threshold signal.
Both methods have high sensitivity, below 100 ng/mL. For non acidic sample detection, the sensitivity of both the Gyrolab and ECL platforms is 2 ng/mL; For samples using acid dissociation steps,The sensitivity of Gyrolab detection is 5 ng/mL, which is higher than the sensitivity of ECL at 20 ng/mL.
Signal values of PC samples detected by Gyrolab and ECL platforms under non acid and acid dissociation conditions. The X-axis represents the concentration of ADA reference substance, and the Y-axis represents the signal-to-noise ratio (PC signal mean divided by NC signal mean)
A comparative study was conducted on the detection of acidified and non acidified samples using optimized optimal conditions on two platforms: nine PC samples (5, 20, 50, 100, 250, 500, 1000, 2500, and 10000 ng/mL ADA) were tested in the presence of 0, 20, and 100 µ g/mL monoclonal antibody drugs,Data shows that the Gyrolab platform exhibits higher drug toleranceFor the detection of non acidified samples, at drug concentrations of 20 µ g/mL and 100 µ g/mL, Gyrolab can detect ADA at 100 ng/mL and 500 ng/mL, respectively, while the ECL platform can detect ADA at 250 ng/mL and 1000 ng/mL, respectively. For acid dissociation sample detection, at a drug concentration of 20 µ g/mL,Gyrolab can detect 5 ng/mL ADA, The ECL platform is 50 ng/mL; When the drug concentration level is increased to 100 µ g/mL, the detection sensitivity of both platforms is 50 ng/mL ADA.
The detection sensitivity of the two platforms under drug levels of 0, 20, and 100 µ g/mL. Sensitivity is defined as the low PC concentration exceeding the threshold signal.
In this study, soluble targets may form dimers that interfere with detection, leading to false positive results. Additionally, in disease populations, the baseline levels of soluble dimer targets may be much higher than those measured in healthy human serum. ThereforeImprove the target tolerance of detection methodsIt is very important for clinical analysis.
To evaluate the efficiency of the target depletion step, four different concentrations of soluble dimer targets were included(5、20、50 和 200 ng/mL)The samples are divided into two groups, and one group is subjected toPre treatment of target exhaustionAnother groupDo not exhaust the targetThen, the detection of acid dissociation steps was conducted on two platforms separately. Data shows that in the absence of acid dissociation, both Gyrolab and ECL assays can only tolerate soluble dimer targets at a concentration of 5 ng/mL; By combining target depletion and acid dissociation steps, the target tolerance levels of both platforms were increased to 200 ng/mL.
Evaluate the tolerance of two platforms at different concentrations of soluble dimer targets under conditions of no acid dissociation or target depletion steps. The threshold represented by the dashed line is fixed at a signal-to-noise ratio of 1.25.
The detection results of two platforms at different concentrations of soluble dimer targets, with symbols'+'and' - 'representing sample signals above or below the threshold, respectively.
Samples containing only free ADA were used for analysisPrecision evaluation of the testing room, including the detection of acid dissociated samples and non acid dissociated samplesPerform 3 independent tests on nine different concentrations of PC (2, 5, 20, 50, 100, 250, 500, 1000, and 10000 ng/mL) using the ECL platform on different dates, and perform 5 independent tests on five different concentrations of PC (5, 20, 100, 500, and 10000 ng/mL) using the Gyrolab platform. Calculate the inter run precision (CV%) based on the signal values obtained for each PC level. The data shows that without the acid dissociation step, the precision of Gyrolab and ECL detection is high, and the CV of all PC samples is below 10%; However, in the acid dissociation detection, it was clearly observed that the CV of ECL detection increased from 10% to 26%, while the Gyrolab detection still maintained good precision, with CV values below 10% for all samples except for the 5 ng/mL PC sample. This is becauseGyrolab technology adopts fully automated acid dissociation treatment, reducing manual operation and errors, thereby reducing the variability of results.
Precision between detection rooms, 'NT' indicates no detection has been performed, “
The data from this study shows that Gyrolab and ECL technologies are effective in ADA detectionSensitivity, drug tolerance, target tolerance, and precisionwaitBoth detection methods demonstrate good performance in terms of parameters and are suitable for method validation and clinical sample analysis
For sample detection using acid dissociation steps,Gyrolab has higher detection sensitivity and drug tolerance
The Gyrolab platform integrates fully automatic sample acid dissociation,Reduced manual operation and errors, improved the repeatability of results between detections
The amount of reagents required for Gyrolab testing is less, and the testing runs smoothlyShorter time, higher throughput
References:
[1] Collet-Brose J, Couble PJ, Deehan MR, Nelson RJ, Ferlin WG, Lory S. Evaluation of Multiple Immunoassay Technology Platforms to Select the Anti-Drug Antibody Assay Exhibiting the Most Appropriate Drug and Target Tolerance. J Immunol Res. 2016; 2016:5069678. doi: 10.1155/2016/5069678. Epub 2016 May 3. PMID: 27243038; PMCID: PMC4868908.