7
Rapid antigen assays are highly scalable, can be stored at room temperature, require minimal training, and can provide a result in less than 10-15 minutes. However, immunoassays have challenges with multiplex testing due to the overall variability in the levels of targets present in the patient sample. Compared to singleplex immunoassays, multiplex versions must have a wider dynamic range in order to detect targets that are present at radically different concentrations. Although multiplex ELISA assays are reported to maintain linearity better than singleplex ELISA assays (over three or even five orders of magnitude), a key component to an assay’s performance in the selection of antibodies. In order to detect both low and high-abundance targets, antibodies selected for a multiplex assay must be highly sensitive and they must be very specific so that the antibodies do not cross-react with each other or with other proteins in the assay mixture or patient sample. In addition, the antibody binding affinity is also a critical factor, especially in lateral flow assays where the association (k on ) and dissociation rate (k off ) can directly influence the sensitivity, specificity, and overall performance of an assay. A high antibody on-rate is desirable as the analyte has only a limited amount of time for interacting with the immobilized capture antibody as the sample diffuses along the test device. A fast on-rate and a slow off-rate will enhance the analyte binding to antibody which correlates to enhanced performance in assay sensitivity, specificity, signal intensity, dynamic range and limit of detection. 5
Flu A mAb Pairs – Nucleoprotein (NP) Capture Detection BN1072 BN1071 BN1073 BN1071 BN1074 BN1071 C01736M C01731M C01760M C01731M Antigens BN1119 – Recombinant Ag to NP Flu B mAb Pairs – Nucleoprotein (NP) Capture Detection BN1075 BN1076 C01744M C01747M C01744M C01897M C01746M C01747M C01746M C01897M C01897M C01747M Antigens BN1120 – Recombinant Ag to NP RSV mAb Pairs Capture Detection C01770M C01773M C65063M C65065M C01769M C01773M pAb – Goat anti RSV B65860G SARS-CoV-2 mAb to SARS-CoV-2 Nucleocapsid Capture Detection 9547 9548 ^ BN1060 BN1061 * mAb to SARS-CoV-2 Nucleocapsid Capture Detection C01769M C01773M C65063M C65065M Antigens BN1006 | BN1011 ^Pair detects Omicron down to 1.31 x 10 2 PFU/mL, and 1.88 x 10 2 PFU/mL for other variants. *New Product Meridian Top Performing Pairs.
Antibody
K D (M)
K on (M
-1 s -1 )
k
off (s
-1 )
X 2
R 2
MAb to Flu A (BN1069)
2.60x10 -11
4.80x10 5
1.25x10 -5
0.0110 0.9992
MAb to Flu A (BN1070)
6.10x10 -11
6.90x10 5
4.21x10 -5
0.0074
0.9979
MAb to Flu B (BN1224)
<1.00x10 -12
2.83x10 5
<1.00x10 -5
0.0133
0.9993
MAb to Flu B (BN1222)
<1.00x10 -12
2.15x10 5
<1.00x10 -5
0.0146
0.9996
MAb to Flu B (BN1223)
2.23x10 -11
3.29x10 5
7.34x10 -6
0.0176
0.9992
Figure 6. Binding kinetics of interactions between soluble Influenza A/B and immobilized Influenza antibodies from Meridian, as measured by bio-layer interferometry. Binding kinetics and affinity for the interactions of Influenza antibodies with Influenza A/B antigens were measured using an Octet QK384 instrument (ForteBio). All kinetic experiments were conducted in 69-well black plates (Greiner, 655209). Each purified Influenza antibody was diluted to 10 μ g/mL in buffer [1X PBS, containing 0.09% Tween 20] and directly immobilized onto AR2G sensors (ForteBio). The association of the antigen was measured for 600 s, followed by a dissociation step for 500 s. The association (k on ) and dissociation rate (k off ) constants as well as the equilibrium dissociation constant (KD) were determined by fitting to sensorgrams via the 1:1 binding model with a correlation coefficient (R2) value greater than 0.99.
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