A lateral flow test detecting SARS-CoV-2 neutralizing antibodies - … · 2020. 11. 5. · Eighty...

A lateral flow test detecting SARS-CoV-2 neutralizing antibodies

Nan Zhang, Shuo Chen, Jin V. Wu, Xinhai Yang and Jianfu J. Wang

Novodiax Inc. Hayward, CA 94587

Send correspondence to Jianfu J. Wang: [email protected]

Abstract

It is of critical importance for COVID-19 survivors, vaccine recipients, and public to know

whether they have developed neutralizing antibodies or immunity. Here, we describe a 15

minutes lateral flow test for rapid detection of neutralizing antibodies against SARS-CoV-2. All

other currently available neutralization tests require hours or days to complete and have to be

performed in a well-equipped laboratory. This lateral flow test is the first of its kind and will

serve as a convenient diagnostic tool in management of COVID-19 disease.

Main

Over 40 million confirmed COVID-19 cases and more than one million deaths caused by this

disease have been reported as of October 2020.1 To combat this threat, more than 300 vaccines

are under development2, with more than 30 of them in various clinical stages.3 The majority of

these vaccines aim to target SARS-CoV-2’s spike (S) protein or the S protein’s receptor binding

domain (RBD) in order to induce neutralizing antibodies (NAbs) that block the interaction of

RBD with its receptor angiotensin-converting enzyme 2(ACE2) on host cells.4

Unlike other binding antibodies (BAbs), NAbs represent a type of humoral immunity capable of

neutralizing or blocking viruses from entering host cells, preventing them from reproducing and

causing severe damage. However, NAbs are not equally developed among the COVID-19

convalescent patients5,6 and IgG antibodies specific to RBD developed in patients with mild

COVID-19 decay rapidly, with a half-life of approximately 36 days7. Multiple COVID-19 re-

infection cases have been reported.8-10 Evaluation of SARS-CoV-2 NAbs is critical for both

better understanding of immunity to COVID-19 and monitoring levels of protective immunity

among the recipients of COVID-19 vaccines.

Various assay methods for SARS-CoV-2 NAbs are available. Conventional virus neutralization

tests use live ACE2-expressing cells and live SARS-CoV-211 and need to be performed in a

. CC-BY-NC-ND 4.0 International licenseIt is made available under a

is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity.(which was not certified by peer review)preprint The copyright holder for thisthis version posted November 10, 2020. ; https://doi.org/10.1101/2020.11.05.20222596doi: medRxiv preprint

NOTE: This preprint reports new research that has not been certified by peer review and should not be used to guide clinical practice.

mailto:[email protected]://doi.org/10.1101/2020.11.05.20222596http://creativecommons.org/licenses/by-nc-nd/4.0/

biosafety level 3 laboratory. Several virus neutralization tests using pseudovirus12-17 have also

been developed. These tests only need a biosafety level 2 facility and usually take several days to

complete. A simpler and faster enzyme-linked immunosorbent assay (ELISA) that detects

SARS-CoV-2 NAbs based on ACE2-RBD interaction has been recently reported.18 This new test

mimics the virus-host interaction in an ELISA test which can be completed in a safety level 2

laboratory within a few hours. The lateral flow test (LFT) is the fastest and most convenient test

among the popular immunoassays, which typically takes only 15 minutes to complete. It can be

performed either in a professional laboratory or by an individual, thus if available should be a

necessary complement to all available neutralizing tests. Here we report the development of the

first COVID-19 NAb LFT, or Point of Care Test (POCT), along with several noteworthy insights

that we’ve gleaned from its development.

Eighty patients’ plasmas were purchased from a Bio-Bank company, 50 of these were PCR

positive for SARS-CoV-2 (named P1-P50) and 30 were PCR negative (N1-N30). Twenty normal

plasmas (NP1-NP20) were randomly picked from an in-house collection dated between 2014 and

2016, before the COVID-19 pandemic, to establish negative baselines. With all of this in place,

we developed an ELISA to evaluate all 100 plasmas. We then set up a pseudovirus based

neutralization test and developed a Nab LFT. The latter test, the first of its kind to our

knowledge, was compared with and evaluated against the other two popular assays.

Using the ELISA, we measured IgG and IgM BAbs specific to the spike (S) protein, envelop (E)

protein and nucleocapsid (N) protein of SARS-CoV-2 for all 100 plasmas, and measured NAbs

for the 80 patients’ plasmas. In line with previous findings, patients’ humoral immune response

to SARS-CoV-2 varied widely, from completely negative to high titer of both BAbs and NAbs

(Fig 1A-1C). Initial data analyses revealed that positive antibody response rates to N and E

proteins were far lower than the response rates to S protein, and no patient responded to N or E

protein without raising IgG or IgM against S protein. We thus focused only on S protein in the

following development. Based on the relative sensitivity of the assay (patients’ plasmas vs the 20

normal plasmas), signals above average plus 2SD of that of the 20 normal plasmas at 1:200

dilution were chosen as the positive cutoff for both IgG and IgM. In general, PCR positive

patients developed more detectable anti-S1 IgG than the PCR negative patients (78% versus

53%, p=0.0006) (Fig 1D-1F), and similar level of anti-S1 IgM (70% versus 66.7%, p=0.19) (Fig

1D-1F).



https://doi.org/10.1101/2020.11.05.20222596http://creativecommons.org/licenses/by-nc-nd/4.0/

Our NAb LFT was designed to have three test lines and one control line (Fig 2A), using 60 nm

Gold Nanoparticle (GNP)-conjugated RBD or S1 protein to display signal, and using ACE2 in

strip (T1 line in Fig 2A) to capture GNP if the interaction between RBD and ACE2 was not

completely blocked by neutralizing antibodies from a plasma. Two additional test lines showing

anti-S1 protein or anti-RBD IgG (T2) and IgM (T3) were included as a positive reading

reference for positive specimen.

Using the above 4-line strip, we evaluated NAbs from all 80 patients’ plasmas. A single dilution

of 1:12 was used for the purpose to match the common practice in the field. Typical in the field,

with aid of a lancet, 10 µl of finger blood was taken and transferred into the sample pad of a LFT

device, and 50-70ul of buffer was then added to drive the blood sample through the LFT device.

Partial results are demonstrated in Figure 2B. The images of the test results on strips were

captured with a smartphone camera and analyzed with Image J software. The intensity of the test

line was computed against that of a known negative plasma and presented as inhibition

percentage (Supplementary Table 1). A good correlation was observed between the inhibition

percentage at 1:12 dilution of LFT and that of inhibition ELISA (r=0.79, 95% CI: 0.79-0.86,

p

contain neutralizing antibody by both inhibition ELISA and NAb LFT, thus should be considered

false PCR negatives. One (plasma # N9) of those three has been chosen to demonstrate

application of our NAb LFT (Fig. 2D). In contrast, IgG and IgM against S1 protein showed very

poor negative percent agreement with PCR and NAb data (Fig. 1F, Supplementary Table 1, 4

and 5). 53.3% of the 30 PCR negative patients showed S1 IgG and 66.7% showed S1 IgM. It is

known that the S and N proteins among all seven human infecting coronaviruses share a

significant amount of sequence homology and can induce cross reactive antibody responses.21 To

determine the contributing factor to these high negative percent disagreements, we tested 12 of

the 30 PCR negative plasmas against S and N proteins of the 4 common cold coronaviruses,

using the same ELISA protocol we used for SARS-CoV-2 proteins. Surprisingly, a higher IgG or

IgM level against S or N proteins of at least one of the 4 common cold coronaviruses was

observed, compared to that of SARS-CoV-2 among all 12 of the plasmas that were tested.

Representative expression profiles by number 1 and 2 PCR negative plasmas (N1 and N2) are

shown in Supplementary Figures 1A to 1D. Based on all of the available data, we believe that a

certain percentage of PCR negative results were true false negatives and may be improved by

testing patients’ neutralizing antibody rather than their binding antibody. Still, we cannot rule out

the possibility that some of the patients in this group actually suffered severe infection caused by

one of the 4 common cold coronaviruses rather than by SARS-CoV-2.

Using the strip with Positive Control and T1 lines, we demonstrated IC50 measurement of two

monoclonal NAbs and three plasmas. In this experiment, a series of dilutions of each NAb or

plasma were tested. The results were computed with Image J software and then analyzed and

graphed with GraphPad Prism (Fig 2C and 2D). We also used the same set of patients’ plasmas

to compare this procedure with inhibition ELISA and a pseudovirus neutralization test (Fig 2E).

In general, we observed quite comparable IC50 results. Among the three methods, the

pseudovirus neutralization test is the most complicated, expensive and lengthy. We used a

commercially available pseudovirus and a one-step luciferase detection system, but still needed

days of cell culture work before and after ACE2 gene transfection. Inhibition ELISA, on the

other hand, has been a routine and standard method in our laboratory, like many other

laboratories. It therefore provides a straightforward technical process. But even so, it requires

several hours of experimental work and needs to be performed in a laboratory setting. In

contrast, the NAb LFT that we’ve established works in the same way as most other lateral flow




tests and can be completed within 15 minutes. Besides its convenience and short turnaround

time, though, its most prominent advantage might be its nature of flexibility. In other

neutralization tests, a negative specimen has to be tested for full dilution range in duplicate or

triplicate in order to know the result. Using our LFT, we were able to eliminate all negative

specimens after testing a single concentration and then continuously testing specimens with a

higher neutralizing antibody titer until their IC50s were captured.

Theoretically, any neutralization assays using live cell and live virus simultaneously measure

neutralizing antibodies targeting three regions, N terminal domain (NTD) and RBD in S1and cell

fusion domain in S2. LFT and ELISA, without live cell involved and depending on which

protein fragment is used, can be used to evaluate NAbs specific to one or two domains in S1

protein. We compared performance of GNP labeled RBD with GNP labeled S1 and indeed found

higher NAb titer by using GNP labeled S1 (Supplementary Figure 2). However, we did not see

higher NAb titer in the pseudovirus neutralization test than the other two tests in a small but

direct comparison (Fig 2E).

To combat COVID-19 pandemic, our NAb LFT is continuously improved toward an IVD

product. Since a neutralizing antibody result has to be quantitative or semi-quantitative, we

anticipate using a smartphone and web-based solution for data capturing and processing. This not

only offers convenience, but also necessary quality control for the product.

Materials and Methods: All materials and methods are available as Supplementary information.

Acknowledgments: We acknowledge the excellent proofreading and editing by David Beglin,

PhD and Stephanie Wang, MD.

Author Contributions: J.J.W. designed the NAb LFT experiments; N.Z. prepared GNP and

strips; N.Z. and J.J.W. performed LFT and ELISA tests; X.Y. set-up and performed pseudovirus

neutralization assays; J.V.W., S.C., N.Z. and J.J.W. contributed to data analyses; all authors

contributed to the writing, editing, and completion of the manuscript.

Competing interests All authors are full time employee of Novodiax Inc. N.Z., S.C. J.V.W and

J.J.W. are author and inventor of a patent application that covers the NAb measurement by LFT

described in this article.




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Figure 1. Serological evaluation of 80 plasmas from COVID-19 patients by ELISA.

A. Antibody expression profile of PCR negative patient plasma N6 shows no SARS-Cov-2

specific binding antibody and neutralizing antibody developed; B. Antibody expression profile of

PCR positive patient plasma P42 represents specimens with moderate level of SARS-Cov-2

antibodies; C. Plasma P7 profile represents a group of specimens with high level of antibody

expression against S, N and E proteins and with strong neutralizing activity; D. Distribution of

IgG and IgM against S1 protein among different specimen groups, IgG is significantly higher in

PCR positive plasmas than in PCR negative plasmas but no difference was seen for IgM. E.

Distributions of S1 IgG and S1 IgM of the 50 PCR positive patients sorted first by IgG and then

IgM expression level, only displayed are the those signals above the average plus 2SD of the 20

normal plasmas; F. The same distributions as in Fig. 1E but for PCR negative group, the scale

has been adjusted to be the same as Fig. 1E. A few representative specimens were indicated with

label and arrow.

Figure 2. A Lateral Flow Test based on ACE2-RBD interaction

A. Schematic structure of the LFT strip constructed in this report and projected results. One

representing format used a 60nm GNP labeled RBD with a rabbit Fc tag and accordingly an anti-

rabbit IgG gamma chain antibody in control line. The strip contains three test lines, one for

neutralization activity (T1) and the other two for measurement of RBD-specific IgG and IgM

respectively (T2 and T3). Another 60nm GNP labeled with S1 protein was also used through the

project. B. A manually prepared strip (using adjustable pipette to dispense/coat) demonstrating

various levels of inhibition effect by different patient’s plasmas. C. Measurement of inhibition

concentration of two neutralizing antibodies using 60nm GNP labeled RBD with a rabbit Fc tag

and a workflow from 15 min test to IC50 calculation. D. Half-Strip demonstration of 3 plasma

samples and the workflow to obtain their IC50. A GNP labeled S1 protein was used in this test.

Inhibitory effects on some control lines were caused by the use of a neutralizing antibody in the

control line. E. A comparison of three different inhibition tests, LFT, ELISA and pseudovirus

based neutralization test, using one moderate and two strong inhibitory plasma specimens.




1A

0 2 4 60

20000

40000

60000

80000

100000

0

20

40

60

80

100

PCR positive patient P7

Titer (LogX)

Bind

ing

antib

ody

(RFU

)

Anti-S IgG

Anti-S IgM

Anti-E IgG

Anti-E IgM

Anti-N IgG

Anti-N IgM

Neutralizing Antibody

1:5

1:20

1:20

0

1:2,

000

1:20

,000

1:20

0,00

0

1:2,

000,

000

Dilution of Plasma

NA

bs (Inhibition %)

0 2 4 60

20000

40000

60000

80000

100000

0

20

40

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100

PCR positive patient P42

Titer (LogX)

Bind

ing

antib

ody

(RFU

) NA

bs (Inhibition %)

1:5

1:20

1:20

0

1:2,

000

1:20

,000

1:20

0,00

0

1:2,

000,

000

Dilution of Plasma

1B 1C

0 2 4 60

20000

40000

60000

80000

100000

0

20

40

60

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100

PCR negative patient N6

Titer (LogX)

Bind

ing

antib

ody

(RFU

) NA

bs (Inhibition %)

1:5

1:20

1:20

0

1:2,

000

1:20

,000

1:20

0,00

0

1:2,

000,

000

Dilution of Plasma

1D 1F1E

20 no

rmal

plasm

a IgG

50 PC

R+ pl

asma

IgG

30 PC

R- pl

asma

s IgG

20 no

rmal

plasm

a IgM

50 PC

R+ pl

asma

IgM

30 PC

R- pl

asma

IgM

0

20000

40000

60000

80000

100000

120000

Fluo

resc

ent U

nits

Antibodies against S1 proteinS1-specific IgG and IgM among 30 PCR negative patients

N9 →P7 →

P1 → N18 →

N28 →




FIG. 2A

SARS-CoV-2 NAbs in sample 1st test line (T1) 2nd test line (T2) 3rd test line (T3) Control line (C)

NAbs+ Vary Vary Vary +

NAbs- + Vary Vary +

Nitrocellulosemembrane

T1

Backing card

T2Samplepad

Conjugate pad

Absorptionpad

60nm gold-bead labeled S1 or RBD-tag

ACE2

Anti-hIgG

C

Anti-tag

T3

Anti-hIgM




FIG. 2B

Control

T3

T2

T1

Cont

rol

PCR+

P1

PCR+

P2

PCR+

P3

PCR+

P4

PCR+

P5

PCR+

P6

PCR+

P7




Smartphonecamera

Image J

NAb Conc µg/ml

Inhibition % vs “0” conc

Mouse NAb Rabbit NAb

0 0.00% 0.00%0.033 10.97%

0.1 2.93% 77.35%0.33 20.87% 98.62%

1 52.10% 96.80%3.3 54.20%10 93.76%33 98.50%

Excel

GraphPad

FIG. 2C

Rabbit NAb (µg/ml)

0 0.03

3

0.1

0.33

1

0 0.1

0.33

1 3.3

10 33

Mouse NAb (µg/ml)

1 2 3 4 50

20

40

60

80

100

rNAb & mNAb inhibition %

Log10 Antibody Conc. (ng/ml)

Inhi

bitio

n %

Mouse NAb IC50: 1,402 ng/ml

Rabbit NAb IC50: 66 ng/ml




Smartphonecamera

Image J

Plasma dilution

Inhibition vs normal plasma NP1

P1 P7 N9

12,288 -0.2565 -0.0531 0.2668

3,072 -0.2515 0.3475 0.2391

768 -0.0768 0.5439 0.4839

192 0.0321 0.9147 0.7464

48 0.5426 0.9788 0.9349

12 0.8605 1.0019 0.9999

Excel

GraphPad

FIG. 2D

1 2 3 40

20

40

60

80

100

Nonlinear regression curve fit

Log10 Dilution

Inhi

bitio

n %

P1(S1-GNP) IC50: 49

P7 (S1-GNP) IC50: 1,138N9 (S1-GNP) IC50: 897

P1

P7

N9

C

T1

1:12

1:12

Plasma dilution

1:12

1:48

1:19

21:

768

1:3,

072

1:12

,288

NP1




1 2 3 40

20

40

60

80

100

Comparison of Inhibition effect among different methods

Log10 Dilution

Inhi

bitio

n %

P1 LFT (S1-GNP) IC50: 49

P7 LFT (S1-GNP) IC50: 1,138

N9 LFT (S1-GNP) IC50: 897

N9 Pseudovirus IC50: 467

N9 ELISA IC50: 1,165

P7 Pseudovirus IC50: 1,400

P7 ELISA IC50: 1,324

P1 Pseudovirus IC50: 185

P1 ELISA IC50: 658

FIG. 2E




A lateral flow test detecting SARS-CoV-2 neutralizing antibodiesDRAFT FIGURES - Lateral flow 10-25-2020-Jin 2DRAFT FIGURES - Lateral flow 10-25-2020-Jin 3DRAFT FIGURES - Lateral flow 10-25-2020-Jin 4DRAFT FIGURES - Lateral flow 10-25-2020-Jin 5DRAFT FIGURES - Lateral flow 10-25-2020-Jin 6DRAFT FIGURES - Lateral flow 10-25-2020-Jin 7

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