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Covid-19 Rapid Test - BLUE PAPER

Preprint · March 2020

DOI: 10.31219/osf.io/jpukc

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 Mikael Franzén | mikael.franzen@alten.se
Alten Life Science | Knarrarnäsgatan 7, 164 40 Kista

BLUE PAPER: Covid-19 quick-test

Keywords: COVID-19, Rapid test, immunoassay, POC, Point-of-Care

Introduction
In the light of the current global situation, a new inexpensive rapid test for Covid-19 has been
conceptually developed and the details are outlined in this document. Given the lack of approved
vaccines and anti-viral drugs, the importance of quick diagnoses cannot be understated.
Rationale
Research suggest [1] [2] that antigen material should be readily available in serological samples from
the onset until the end of infection. Hence, a direct antigen rapid test, such as the one to be
presented here, has the advantage of not being dependent on immune response and the availability
of specific immunoglobulin antibodies. Diagnosis should therefore be possible from the first day of
symptoms. This is very significant and means that acute diagnosis is theoretically possible.

Proposal
Two alternatives are suggested. Both rely on eye-sight-detectable color changes and are based on
the Sandwich principle [3] .
Alternative #1
The first alternative presented below (fig 1) is a pregnancy-test-inspired lateral flow version of the
Sandwich principle.

Fig 1 – Alternative # 1: Overview.

The strip in fig 1 is a paper suitable for chromatographic applications (e.g. nitrocellulose) [3] [4] . The
sample (blood/saliva/mucus/other) is loaded on to the strip and moves from left to right by capillary
action. At area A it passes the loosely bound monoclonal antibodies (mAbs), and then at area B, the
immobilized polyclonal antibodies (pAbs). Finally, at area C the sample encounters the secondary pAbs.

It is suggested that COVID-19 Nucleocapsid Protein (NP) mAbs and pAbs are used at area A and B
respectively. For area C, anti-COVID-19 NP pAbs can be used. Alternatively, at area C, anti-COVID-19 Spike
protein or other secondary pAbs, such as the envelope protein, may also be possible to use [5] [6] .

All antibodies (Abs) are immobilized together with their respective substrate. Hence, Abs at area A
will be bonded to HRP-conjugated streptavidin but without the biotin substrate. At area B and C, Abs
are biotinylated but not HRP-linked. As you probably know, at area A, mAbs binds the antigen and
the bound entity then travels to B, where the antigen is subsequently sandwiched between mAbs
and pAbs. C is a control that binds the leftover mAbs, which is standard practice. The above

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description is for a positive test. In contrast, a negative test does not bind at A and as a result there is no
color change at area B. At area C, as Ab-to-Ab binding occurs, there is always a color change regardless.
In the case that C does not undergo color change, the test must be considered invalid [3] [4] .

Fig 2 below is a biochemical illustration of the process:

Fig 2 – Alternative # 1: Biochemical overview

There are standard protocols for immobilization on nitrocellulose and on other porous cellulose-
based materials as well as on plastics, such as polyvinylidene fluoride and polyethersulfone [3] [4] .
In order to determine the optimal parameters in this particular case, some testing will be needed.
The strip itself may also need to include a sample- and adsorbent-pad to ensure uniform distribution
and collection.
Alternative #2
The second alternative (fig 3) works according to the same principle but is reliant on sample and
reagent transferring between test tubes:

A B C1 C2
Fig 3 Alternative #2: Overview.

Reagent tubes A and B contain COVID-19 NP mAbs and pAbs. The mAbs in A are not immobilized
while the pAbs in B are. C1 and C2 contain the anti-COVID-19 NP pAbs (or similar, see page 1, §5).
The assay is performed according the numbering (fig 3):

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0. Transfer a given amount of A into C2 and vortex/shake: A color change should be observed.
1. Transfer the sample to A. Vortex/shake (no color change).
2. Transfer a given amount from A to B. Vortex/shake (color change if positive).
3. Transfer a given amount from B to C. A color change should be observed.

The biochemical reaction scheme is the same as in Alternative #1 with the exception of C. Since
there are no free flowing unbound mAbs, they are added at step 0 as an initial control. The third and
final step may not be needed, but since there is more manual handling in Alternative #2 it ensures a
valid result as well as correctly executed test [3] [6] .

Discussion
It is suggested that both alternatives are developed. Alternative #1 is completely self-contained and
can be tweaked a lot with regard to mechanical design and cost effectiveness if needed. The
downside is that Alternative # 1 will require some more time to develop fully. Alternative #2 is more
straightforward and is probably even more cost effective. The downside is that the kit contains many
parts. Nevertheless, it is believed that factors such as blocking, buffering and immobilization can be
optimized and developed quite easily [3] . In terms of advantages, both alternatives use the
Sandwich principle as specificity and sensitivity are very high due to the antigen binding to two
antibodies. Furthermore, the enzyme has a greater surface area available for conjugation and hence
signal strength is further increased. Both alternatives, when fully developed, will be easy to use and
will not rely on expensive and/or bulky machinery for detection. Additionally, it must be mentioned
that a variation of antibody types should be tested and evaluated, including recombinant options.
Likewise, a number of detection-enhancing methods, such as colloidal gold, can be similarly
evaluated [9] [10] .

It is anticipated that a working prototype will be available, for at least one alternative, quite quickly
and that mass production can begin within a month or two from the start of development. Of course,
this is dependent on available resources and team size, but it is likely to require less than many other
similar efforts that are not as important in terms of public health.
Unknowns
There are a few unknown factors that need to be addressed (for both alternatives):

▪ Incubation time; ideally it will be zero or relatively short, but this is not known.
▪ Quality of commercial antibodies may be poor; it is not known [7] [8] [10] .
▪ Dilution factors for samples and reagents are unknown.
▪ Regulatory compliance time-lines dependent on state institutions are not known.

Given a week of literature search and experimentation it is estimated that all of the above will be
known.

Furthermore, the effort required and extent to which Abs can be engineered is not known. This
something that may be necessary if overall test accuracy is low. The World Health Organization
report highly variable sensitivity scores for similar types of tests in the past [9] .
Combinations
Antibody tests using immunoglobulin detection can reveal those in late stages of infection as well as
those who recently had an infection [12] . As such, for a more complete picture, the rapid test
alternatives described here can be used in conjunction with the latter.

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 Mikael Franzén | mikael.franzen@alten.se
Alten Life Science | Knarrarnäsgatan 7, 164 40 Kista

Conclusion
This blue paper has presented a concept that may be developed further into working test kits to be
used in a point-of-care environment. It is likely achievable without requiring an unrealistic amount of
resource. From a public health perspective, it is something that is worth taking further.
This method has the potential to provide rapid diagnosis across the spectrum; from asymptomatic to
acute, and is therefore highly relevant as a first line of defense, either on its own, or in combination
with other techniques.

References
[1] L. Liu, W. Liu, S. Wang, S. Zheng, et.al, medRxiv 20031856, (2020)
doi: https://doi.org/10.1101/2020.03.06.20031856

[2] S. Khan, R. Nakajima, A. Jain, R. Ramiro de Assis, et.al, BioRxiv 006544, (2020)
doi: https://doi.org/10.1101/2020.03.24.006544

[3] R. Gerbers, M.S. thesis, University of Rhode Island, 2013. Retrieved from
https://digitalcommons.uri.edu/theses/123

[4] R. Nezlin, J. of Immun.Methods, 23, 1 (2000)

doi: https://doi.org/10.1016/s0022-1759(00)00139-3

[5] M. Li, R. Jin, Y. Peng, C. Wang, W. Ren, F. Lv, et.al, medRxiv 20025999, (2020)
doi: https://doi.org/10.1101/2020.02.20.20025999

[6] A.R. Fehr, S. Perlman Coronaviruses: An Overview of Their Replication and Pathogenesis, in H. Maier, E.
Bickerton, P. Britton (eds) Coronaviruses, Methods in Molecular Biology 1282 (Humana Press 2015, New York,
NY), pp. 1-23
doi: https://doi.org/10.1007/978-1-4939-2438-7_1

[7] ProSci. COVID-19 Antibodies.

https://www.prosci-inc.com/covid-19/
(Accessed: 2020-03-01)
[8] Creative Diagnostics. SARS-CoV-2 NP.
https://www.creative-diagnostics.com/search.aspx?pageid=1&keys=SARS-CoV-2%2bNP&status=0&fl=SARS-
CoV-2%2bNP%257e&flt=1,&cid=4
(Accessed: 2020-03-01)

[9] NanoComposix. Lateral Flow Immunochromatographic Assays. https://nanocomposix.com/pages/lateral-flow-

immunochromatographic-assays?gclid=CjwKCAiAzJLzBRAZEiwAmZb0agJH249Nm9mUgd5u0Cn1ZJ1vXpTkY-
MPVd6Es4w6JJ1Bn1sWFDUCfRoC54cQAvD_BwE
(Accessed: 2020-03-07)

[10] Life Tein. Proteins.

https://www.lifetein.com/peptide-product/proteins-c-19.html
(Accessed: 2020-03-07)

[11] World Health Organization, Scientific Brief: Advice on the use of point-of-care immunodiagnostic tests for
COVID-19 (World Health Organization 2020)
https://www.who.int/news-room/commentaries/detail/advice-on-the-use-of-point-of-care-immunodiagnostic-
tests-for-covid-19
(Accessed: 2020-04-08)
[12] R. Racine, G.M. Winslow, Immunol Lett., 125, 79 (2009)
doi: https://doi.org/10.1016/j.imlet.2009.06.003

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