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Comparison of surface plasmon resonance, resonant waveguide grating biosensing and enzyme linked immunosorbent assay (ELISA) in the evaluation of a dengue virus immunoassay.

Hu D, Fry SR, Huang JX, Ding X, Qiu L, Pan Y, Chen Y, Jin J, McElnea C, Buechler J, Che X, Cooper MA - Biosensors (Basel) (2013)

Bottom Line: However, there is a significant scope to improve both the sensitivity and the specificity of those tests.The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD = 1-0.1 μM) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR.Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, 4072 Australia. zjyyhdm@163.com.

ABSTRACT
Two label-free biosensor platforms, Resonance Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR), were used to rank a large panel of anti-dengue virus NS1 antibodies. Dengue non-structural 1 (NS1) protein is an established serological marker for the early detection of dengue infection. A variety of commercial dengue NS1 antigen capture immunoassays are available in both ELISA and lateral flow format. However, there is a significant scope to improve both the sensitivity and the specificity of those tests. The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD = 1-0.1 μM) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR. There were significant differences in the absolute affinities determined by the two technologies, and there was a poor correlation between antibodies best ranked by RWG and the lower limit of detection (LLOD) found by ELISA. Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats.

No MeSH data available.


Related in: MedlinePlus

On-off rate plots of mAbs for DENV NS1 determined using Surface Plasmon Resonance (SPR).(A)DENV-1 NS1reactive mABs, (B) DENV-2 NS1 reactive mABs, (C) DENV-4 NS1 reactive mABs, and (D) DENV-4 NS1 reactive mABs. The dotted diagonal lines represent affinities (kdiss/kass). Open circles represent mAbs reactive with all four dengue NS1 serotypes. Open squares represent the commercial Alere mAb used for comparison.
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biosensors-03-00297-f004: On-off rate plots of mAbs for DENV NS1 determined using Surface Plasmon Resonance (SPR).(A)DENV-1 NS1reactive mABs, (B) DENV-2 NS1 reactive mABs, (C) DENV-4 NS1 reactive mABs, and (D) DENV-4 NS1 reactive mABs. The dotted diagonal lines represent affinities (kdiss/kass). Open circles represent mAbs reactive with all four dengue NS1 serotypes. Open squares represent the commercial Alere mAb used for comparison.

Mentions: The recombinant NS1 protein was in a predominantly hexameric structure, which was indicated by a Native-PAGE (data not shown). In addition, the mAbs have two binding sites against the NS1 antigen. As a result, a bivalent binding model should be more appropriate for data analysis. However, because of the two-step binding, the bivalent binding model will generate two KD values [20], which make the ranking very complicated and almost impossible. Figure 3 shows an example (mAb Dv3 M7 binds to DENV-3 NS1) of the fitting results of the same sensorgrams using both 1:1 Langmuir binding model and bivalent binding model. The KD values were calculated by kdiss/kass. In the 1:1 binding model, the KD was 5 nM; whereas in the bivalent binding model, two sets of kdiss and kass were acquired, resulting in two KD values (18 nM and 7.9 M). Considering the complication of bivalent model, we optimized the experimental conditions of SPR to promote the 1:1 binding in the flow system. These were achieved by capturing the lowest possible amount of mAb on the sensor chip surface and increasing the flow rate of sample injection to minimize avidity effects and mass-transport promoted re-binding. Therefore, a reasonable fitting was obtained (Figure 3(A)). The optimized SPR experiment hence measures the real kinetics of the interaction. The affinities of mAbs identified by SPR ranged from 1 nM to 200 nM (Figure 4). The kass ranged from 103 to 105 M−1·s−1, and kdiss ranged from 10−7 to 10−2 s−1.


Comparison of surface plasmon resonance, resonant waveguide grating biosensing and enzyme linked immunosorbent assay (ELISA) in the evaluation of a dengue virus immunoassay.

Hu D, Fry SR, Huang JX, Ding X, Qiu L, Pan Y, Chen Y, Jin J, McElnea C, Buechler J, Che X, Cooper MA - Biosensors (Basel) (2013)

On-off rate plots of mAbs for DENV NS1 determined using Surface Plasmon Resonance (SPR).(A)DENV-1 NS1reactive mABs, (B) DENV-2 NS1 reactive mABs, (C) DENV-4 NS1 reactive mABs, and (D) DENV-4 NS1 reactive mABs. The dotted diagonal lines represent affinities (kdiss/kass). Open circles represent mAbs reactive with all four dengue NS1 serotypes. Open squares represent the commercial Alere mAb used for comparison.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263579&req=5

biosensors-03-00297-f004: On-off rate plots of mAbs for DENV NS1 determined using Surface Plasmon Resonance (SPR).(A)DENV-1 NS1reactive mABs, (B) DENV-2 NS1 reactive mABs, (C) DENV-4 NS1 reactive mABs, and (D) DENV-4 NS1 reactive mABs. The dotted diagonal lines represent affinities (kdiss/kass). Open circles represent mAbs reactive with all four dengue NS1 serotypes. Open squares represent the commercial Alere mAb used for comparison.
Mentions: The recombinant NS1 protein was in a predominantly hexameric structure, which was indicated by a Native-PAGE (data not shown). In addition, the mAbs have two binding sites against the NS1 antigen. As a result, a bivalent binding model should be more appropriate for data analysis. However, because of the two-step binding, the bivalent binding model will generate two KD values [20], which make the ranking very complicated and almost impossible. Figure 3 shows an example (mAb Dv3 M7 binds to DENV-3 NS1) of the fitting results of the same sensorgrams using both 1:1 Langmuir binding model and bivalent binding model. The KD values were calculated by kdiss/kass. In the 1:1 binding model, the KD was 5 nM; whereas in the bivalent binding model, two sets of kdiss and kass were acquired, resulting in two KD values (18 nM and 7.9 M). Considering the complication of bivalent model, we optimized the experimental conditions of SPR to promote the 1:1 binding in the flow system. These were achieved by capturing the lowest possible amount of mAb on the sensor chip surface and increasing the flow rate of sample injection to minimize avidity effects and mass-transport promoted re-binding. Therefore, a reasonable fitting was obtained (Figure 3(A)). The optimized SPR experiment hence measures the real kinetics of the interaction. The affinities of mAbs identified by SPR ranged from 1 nM to 200 nM (Figure 4). The kass ranged from 103 to 105 M−1·s−1, and kdiss ranged from 10−7 to 10−2 s−1.

Bottom Line: However, there is a significant scope to improve both the sensitivity and the specificity of those tests.The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD = 1-0.1 μM) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR.Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats.

View Article: PubMed Central - PubMed

Affiliation: Division of Chemistry and Structural Biology, Institute for Molecular Bioscience, University of Queensland, Brisbane, 4072 Australia. zjyyhdm@163.com.

ABSTRACT
Two label-free biosensor platforms, Resonance Waveguide Grating (RWG) and Surface Plasmon Resonance (SPR), were used to rank a large panel of anti-dengue virus NS1 antibodies. Dengue non-structural 1 (NS1) protein is an established serological marker for the early detection of dengue infection. A variety of commercial dengue NS1 antigen capture immunoassays are available in both ELISA and lateral flow format. However, there is a significant scope to improve both the sensitivity and the specificity of those tests. The interactions of antibody (Ab)-antigen (Ag) were profiled, with weak interactions (KD = 1-0.1 μM) able to be detected under static equilibrium conditions by RWG, but not observed to under more rigorous flow conditions using SPR. There were significant differences in the absolute affinities determined by the two technologies, and there was a poor correlation between antibodies best ranked by RWG and the lower limit of detection (LLOD) found by ELISA. Hence, whilst high-throughput RWG can be useful as preliminary screening for higher affinity antibodies, care should be exercised in the assignation of quantitative values for affinity between different assay formats.

No MeSH data available.


Related in: MedlinePlus