23-25 March Serology on antigens and antibodies

Serology 1st part

I wondered what is known about serology, but, obviously, I found only two papers on SARS-Cov-2.  However, there is a very instructive review on the former pathogenic corona’s from the Bonn group and an inspiring review of SARS viruses by Jiabao Xu.

1. The review from Bonn of 2014 mainly focuses on SARS-Cov (the one from 2003) and starts with the warning that cross reactivity with other Coronaviruses is likely in most formats (ELISA, indirect immunofluorescene assay or IFA) and they stick to the WHO recommendation to use virus neutralization assays as the gold standard (although also in that format cross-reactivity has been noted with Flaviviruses for instance).  In Western blot, ELISA and IFA, they s recombinant Nucleoprotein (N) of Spike antigens (S), how evidence that the latter is more specific, but also less sensitive. They conclude that S-based IFA is the best choice. This can be explained, because S proein has less homology between Corona’s and they indicate that focusing on three particular regions of S (instead of the whole protein could further improve specificity.
2. The next review by Jiabao is very broad, but Table 2 provides interesting information for those who would like to develop assays that distinguish between the “2005” SARS-CoV and the “2019” SARS-CoV-2.  Whereas overall homology between both is high (85 %), there are proteins with much lower homology such as nsp2, nsp3 and S. There are also two open reading frames (orf 8 and orf 10), which are unique to SARS-CoV-2 and therefore might be ideal to differentiate between both.  At this moment, I guess the question is whether this unique or low-homology sequences give rise to sufficient protein expression to elicit measurable antibody responses to be used in serology.
3. An interesting short paper by Wei Zang et al. on molecular and serological evolution in 11 patients, apparently with a home-made ELISA, focusing on N protein
4. Finally an overview of the available molecular and serological tests in China by Loeffelholz and Tang, nicely listed in Table 3, but without a really critical evaluation.  I guess it is too early for that. 

Serology 2nd part 25 March

SARS-CoV-2 Antigen detection kits

1. Sino Biological: S or N specific: pg/ml sensitivity

https://www.sinobiological.com/research/virus/2019-ncov-antigen

2. CD Creative diagnostics: N specific ng/ml sensitivity

http://img2.creative-diagnostics.com/pdf/DEIA2020.pdf

3. Coris Biocencept, a Gembloux-based biotech, announced just yesterday that they have a point-of care antigen test via RTBF. I could not find more info….

https://www.rtbf.be/info/societe/detail_coronavirus-un-test-belge-en-15-minutes-recoit-la-certification?id=10466576

4. FIND website shows a wealth of molecular and serological (Ag and AB) tests under development

https://www.finddx.org/covid-19/pipeline/

Clearly, there are no scientific publications yet about the validity of the antigen assays.  It is also not clear if SARS-CoV antigens will be detectable in biological samples.  NOP swabs may be difficult to use  for this purpose. Remember that RT-PCR in blood are rather low. Could saliva be suitable? 

The experience of antigen tests in other viruses (on serum) is mixed. It works fine with Flaviviruses (e.g. Dengue NS1) and detection of HIV p24, combined with antibody detection is the standard for modern HIV diagnosis.  However detection of p24 alone is restricted to a narrow “window” period (when PCR is already positive, but antibodies are still absent), once Ab are present, p24 is complexed and difficult to measure.  So we will see what the diagnostic application of COVID antigen test will be.

SARS-Cov-2 Antibody tests

Publications on the validation of antibody tests are emerging.

1.  In the Amanat “American” paper, the focus is on the spike protein, from which several constructs, expressed in mammalian or insect cells were used as the antigen (nicely presented in Fig 1).  A very limited set of positive samples and control sera (only one with a history of common CoV infection). Data in Fig 2 and 3 shows results.

2.  In the Okba “European” paper, in house and commercial (EUROIMMUN) ELISA using S1, S2, receptor binding domain (RBD) and N protein as the antigens were validated against the “gold standard” PRNT (plaque reduction neutralization tests), using 3 SARS-CoV-2 seroncenverters, SARS-CoV, MERS and subjects with positive serology for common corona’s.   Clearly the in house test were better than the commercial test, in terms of specificity and sensitivity (see Fig 1-4).  There was cross-reactivity with SARS and MERS (as could be expected, but not a big issue), but much less or none with the common CoV. 

In my opinion, the “European in-house assay” is the better option.