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Validation of the Meso Scale Discovery (MSD®) V-PLEX Proinflammatory Biomarker Panel 1 in Normal Human Serum (NHS)M. Saxena, K.E. Jeltema, S.E. Henson, A.E. Lopez, C.L. Evans and J.F. Marcelletti

AbstractThe MSD human Proinfl ammatory Panel 1 V-PLEX®

assay system (Meso Scale Diagnostics, LLC) allows for

the simultaneous analysis of IFN-γ, IL-1β, IL-2, IL-4,

IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α. It has been

validated following fi t-for-purpose (FFP) principles by

MSD. Tandem Labs sought to corroborate MSD’s FFP

fi ndings and extend the analysis to include method

validation parameters using NHS as matrix for the

validation samples (VS).

V-PLEX kits were performed according to

manufacturer’s recommendations. NHS samples were

screened for optimal minimum required dilution and

endogenous levels of the listed cytokines. A pool of

NHS was used to generate VS for the 10 cytokines.

Multiple lower limit of quantitation (LLOQ) VS were

evaluated to defi ne LLOQ for the individual analytes in

this multiplex format. Calibration standards (CS) and

MSD controls were assayed according to instructions.

Validation parameters in this on-going study include

accuracy and precision, selectivity, specifi city, linearity

on dilution, and stability.

A 5-PL curve fi t for the CS yielded a mean R2 > 0.99

for all cytokines. Lower limits of detections (LLOD)

≤1.0 pg/mL were observed for the biomarker CS vs.

buffer blank. LLOQ values were defi ned using NHS

VS. There were two classes of cytokines based on

LLOQ, including 1) LLOQ <1.5 pg/mL (IL-1β, IL-4, IL-

6, IL-10, IL-12, TNF-α), and 2) LLOQ 2-7 pg/mL (IFN-γ,

IL-2, IL-8, IL-13). All VS and MSD controls exhibited

acceptable percent CV and bias. Linearity on dilution

was confi rmed using CS in NHS. A high degree of

correlation (R2 ~0.99) was observed between CS

concentrations in Diluent 2 vs. CS in NHS. Selectivity

was established by spiking NHS samples with the

cytokines between their respective LLOQ and LQC

levels. Testing for stability of the cytokines in NHS is

underway at the time of abstract submission. Typical

responses of NHS samples for the 10 cytokines in the

assay have been documented.

We conclude that these results: 1) corroborate the FFP

fi ndings obtained by MSD and 2) demonstrate the high

likelihood that the MSD V-PLEX Proinfl ammatory Panel

1 assay system can be adapted to fully validated status

in NHS.

Introduction Methods Results

FIGURE 4Comparison of CS in Buffer vs. CS in NHS

FIGURE 2Calibration Curve Characteristics

TABLE 1CS Limits of Quantitation & Inter-assay

Reproducibility of MSD Controls

FIGURE 1 Layout and Assay Format Specifi cs for the MSD®

MultiplexProinfl ammatory Panel I Sandwich Immunoassay

This work was designed to further validate the human Proinfl ammatory Panel 1 V-PLEX PLUS biomarker assay system launched by Meso Scale Discovery (MSD®). The kit allows for the simultaneous assay of human specimens for IFN-γ, IL-1β, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, and TNF-α. The kit includes calibration standards (CS) and three levels of controls: H/M/L. The system has been very well characterized by MSD using a fi t-for-purpose (FFP) approach. The validation included analyses for sensitivity, specifi city, accuracy and precision (A&P), matrix effects, stability and normal reference intervals for the cytokines. Many of the MSD conclusions, such as lower limits of quantitation (LLOQ), are based on results with CS in buffer. The controls (high, medium, low) are prepared in non-human matrix rather than human serum or plasma.

The FFP approach has been a widely accepted means to characterize biomarker assays in anticipation of preclinical and clinical trial sample analysis. The approach recognizes that not all aspects of bioanalytical method validation (BMV), as prescribed by guidance documents and industry standards, are necessarily feasible. Moreover, not all aspects of BMV are necessary depending on the intended use of the data derived. However, biomarkers are increasingly being used as drug co-diagnostics and clinical endpoints necessitating full BMV when possible. Signifi cantly, advances in preparation and characterization of biomarker assay reagents have made it possible to approach full validation of such assays in a manner consistent with drug pharmacokinetic (PK) ligand-binding assay (LBA) standards generally.

However, full BMV of biomarker assays in matrix such as NHS is complicated by at least three important considerations.

■ First, biomarkers are often present endogenously in normal bodily fl uids and tissues.

■ Second, factors able to inhibit or augment the assay signal can also be present in such specimens.

■ Finally, individual biomarkers can be heterogeneous in the host and model systems using recombinant proteins may not accurately refl ect that heterogeneity.

Nevertheless, we’ve undertaken to fully validate this multiplex assay to the extent possible using VS prepared in NHS. The validation analytical plan includes the full range of parameters expected for a regulated, clinical study involving human serum samples, including A&P and selectivity, among others. This poster presents our progress to-date in this endeavor.

Figure 4. Comparison of CS in Buffer vs. CS in NHS. CS blend was diluted in buffer and NHS pool. The high NHS CS solution was serially diluted 1:2 in matrix, before diluting 1:2 in assay diluent. Shown in Panel A are derived concentrations obtained using the respective cytokine standard curve in buffer. The values in Panel A depict total cytokines observed, endogenous plus exogenously added. Shown in Panel B are the values after subtracting baseline endogenous concentrations for the cytokines.Table 1. CS Limits of Quantitation & Inter-assay Reproducibility of MSD

Controls. LLOQ is lowest respective concentration of CS capable of reproducibly exhibiting a %CV <20% and %AR between 80-120%. The upper limit of quantitation (ULOQ) is the highest respective concentration of cytokine tested in the standard curves. The ULOQ values shown exhibited %CV and %AR <20% and between 80-120%, respectively. MSD PLUS kit controls were tested according to manufacturer’s instructions in three independent runs by two analysts. Inter-assay statistics are shown. Intra-assay statistics were similar: CV < 20% and %AR between 80-120% (not shown).

Figure 2. Characterization of Calibration Standard (CS) in Assay Diluent. CS blend was reconstituted in Diluent 2 and added into wells of the multiplex 96-well plate. Data are expressed as ECLU vs. nominal concentration (Panel A) and derived back calculated cytokine concentrations vs. nominal (Panel B). These latter data points were derived by regression using a 5-PL curve fi tting model and Watson LIMS™ software.

Figure 3. Proinfl ammatory Panel 1 Cytokines Detected in NHS Specimens. NHS samples were screened in the multiplex assay at the suggested minimum required dilution of 1:2. Shown are mean ECLU obtained from duplicate determinations per sample. Also shown are the proportions of specimens (N= 14) that exhibited a signal above the LLOD. LLOD (black symbol) was defi ned as the mean of the blank control values (red symbol) plus three standard deviations of that mean.

Conclusions

Principle of the Method. The method follows closely to that of the manufacturer, except as required to incorporate NHS matrix. In this method, the amounts of the ten listed biomarkers are measured by a sandwich immunoassay. The kit provides a 96-well plate pre-coated with capture antibodies on independent and well-defi ned spots (Figure 1). Each well of the 96-well MSD plate is coated with ten specifi c antibodies that can each capture one of the ten desired biomarkers. Samples and a solution containing ten detection antibodies conjugated with SULFO-TAG™ are added over the course of sequential incubation and wash steps. Analytes in the sample bind to the capture antibodies immobilized on the working electrode surface (spots) and also bind the corresponding detection antibodies, completing the typical sandwich immunoassay confi guration. MSD read buffer is added and the plate is loaded into the MSD instrument where a voltage is applied to the plate electrodes, causing the capture labels to emit light as electrochemiluminescence units (ECLU). The instrument measures the intensity of emitted light (ECLU) to provide a quantitative measure of analytes in the sample. A regression of the ECLU of the standard curve samples versus concentrations is performed using Watson LIMS™ software (Thermo Fisher Scientifi c Inc.) and the concentrations of the analytes in the samples determined.

TABLE 2Verifi ed Limits of Quantitation Using NHS Validation Samples

TABLE 3Validation Summary

MSD V-PLEX Proinfl ammatory Panel 1 in NHS

Table 2. Verifi ed Limits of Quantitation Using NHS Validation Samples. VS were made at the concentrations shown using NHS as matrix. The VS were diluted 1:2 in assay diluent and assayed for cytokine levels. All VS were subjected to six accuracy and precision runs. The data presented indicate those concentrations that passed acceptance criteria indicated in the blue boxes and those that did not pass as represented by red boxes.

Table 3. Validation Summary for the MSD V-PLEX Proinfl ammatory Panel 1 in NHS. The results from this bioanalytical method validation are presented in Table 3 and summarized as follows:

1. The validation parameters were defi ned using VS made with ≥95% by content NHS pool.

2. The upper and lower limits of quantitation are based on accuracy and precision results of VS against the buffer standard curve.

3. The respective LLOQ and ULOQ were defi ned by meeting acceptable intra- and inter-assay %CV and %AR. The assay ranges are suited for detection and quantitation of the studied cytokines in disease serum samples.

4. The calibration standard blend serially diluted in NHS exhibited highly signifi cant dilutional linearity for all cytokines tested (R2 ≥0.99).

5. The assays displayed acceptable selectivity as shown by spike and recovery of known low concentrations of cytokines in 10 individual NHS specimens (5 male, 5 female).

6. The proinfl ammatory cytokines exhibited the anticipated stability as spiked into NHS, with acceptable recovery observed with:

a. minus 70°C storage for 60 days, b. four freeze-thaw (F/T) cycles, and c. fi ve hours incubation at 4°C.

7. We conclude that this MSD V-PLEX Proinfl ammatory Panel 1 has been successfully validated in NHS by our laboratory and is suitable for use in regulated, clinical sample analysis.

1. MSD calibration standards and controls functioned as specifi ed in the certifi cate of analysis and as illustrated in the kit insert.

2. NHS exhibit baseline endogenous levels for many of the tested cytokines, some of which were evident by elevated ECLU only and others by quantifi able levels of biomarker.

3. The CS in NHS displayed standard curve characteristics very similar to CS in assay diluent, confi rming the suitability of this assay system to measure these cytokines in human specimens.

4. The VS in NHS displayed accuracy and precision, selectivity, and stability well within the limits of acceptability.

5. We have fully validated the MSD V-PLEX Proinfl ammatory Panel 1 in NHS.

Preparation and assay of CS, VS, and MSD® controls. The lyophilized calibrator blend provided with the kit was reconstituted with MSD buffer (Diluent 2) and then serially diluted in Diluent 2 to obtain the desired concentrations for the standard curves. NHS samples were purchased from Bioreclamation and screened for suitability for use in a NHS matrix pool. Seven levels of VS were prepared by spiking this pool of NHS with the MSD calibrator reference material. All VS contain at least 95% NHS. MSD controls were reconstituted with Diluent 2. Diluted calibrators were added directly to the plate without further dilution. VS, MSD controls, and NHS samples were diluted 1:2 in Diluent 2 for a fi nal minimum required dilution (MRD) of 50% when added to the wells. All determinations are assayed in duplicate (2 wells).

FIGURE 3Proinfl ammatory Panel 1 Cytokines

Detected in NHS Specimens

Ref: MSD® Multi-spot Assay System