Screening of Omicron Virus Strain by Quantifying the Spike Protein Content
Round 1
Reviewer 1 Report
This is an excellent contribution to faster creation and testing of vaccines. The methods are clearly described.
All my comments are designed so that the authors can clarify points for which other virus researchers will be curious to know their answers.
Methods
“BALB/c mice weighing 18–20 g”
[How many mice did you use and how did you decide the number?]
How variable were the results between the mice?]
“Omicron strain HK-OM-P0 was isolated from a patient’s pharyngeal swab”
[please explain why you chose this sample]
“The membrane was sealed in 5% skim milk in PBST for 2 hours at 37 °C, followed by overnight incubation with anti-S protein rabbit polyclonal antibody (Sino-Biological, 1:1000) at 4 °C. The membrane was incubated with the secondary antibody goat anti-rabbit IgG (HRP) (GE, 1:4000) for 1 hour at room temperature and exposed to the Image Quant TL system for imaging analysis.
2.6. Antigen analysis based on multiclonal antibody ELISA
The antigenic content was measured based on the ELISA method using a multiclonal antibody. The “homemade” polyclonal antibody was created using the following steps. First, guinea pigs and rabbits were immunized against the SARS-CoV-2 B.1.1.529 (Omicron) S1+S2 trimer protein (Sino Biological, 40589-V08H26), respectively, and the serum was collected for titer detection. The serum with high titer was purified to obtain the pol-yclonal antibody, and the guinea pig polyclonal antibody was the capture antibody, while the rabbit polyclonal antibody was the detection antibody.’”
[three types of animals were used here. For the benefit of other researchers please explain you reasons why you chose each animal for the different steps]
“homemade rabbit anti-S protein polyclonal antibodies, which were replaced by Omicron-specific antibodies (Acro SPD-M415,1:20000), and donkey anti-rabbit IgG-Pe- roxidase antibodies, which were replaced by goat anti-mouse IgG peroxidase antibodies from another manufacturer”
[please explain the reasons for these substitutions]
[Line 177 the legends under the figures are barely legible. Please increase the font size]
Results
“the equilibrium dissociation constant (KD) of Omicron-specific anti-RBD antibody and virus strain 4 is 14 nM, and that of this anti-body and virus strain 3 is 572 nM, which is a 40-fold increase in affinity, reinforcing the fact that virus strain 4 is more suitable as a candidate for vaccine development”
[this is an enormous and commendable difference. Do you have any explanations for this?]
Figure 4. We now discover the sample size of the mice. How did you arrive at this number? Did you perform a power computation and did you have enough data to perform a power computation?]
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
1. The manuscript needs extensive English editing.
2. It is not an inactivated vaccine; it is an inactivated virus vaccine.
3. Table 3 shows comparable content for variant #3; explain in more detail why it was considered the “worst”
4. The concentration in Table 1 cannot be differentiated since most values are likely within the variability of the test method; to decide, it should be elaborated.
5. While the authors are presenting a faster method, it is not clear, given the experimental details, how it can be completed in 24 hours, such as including various incubation steps.
6. It is indeed a different approach if the conclusions can be substantiated; also, the comparative accuracy with other established methods should be discussed.
See abobe
Author Response
Please see the attachment.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Your response answers most of my questions; it will be helpful if you could go through a grammar check, as well as
reference formatting.
Your response answers most of my questions; it will be helpful if you could go through a grammar check, as well as
reference formatting.
