The fifth wave of A(H7N9) virus infection in China from 2016 to 2017 caused 
great concern due to the large number of individuals infected, the isolation of 
drug-resistant viruses, and the emergence of highly pathogenic strains. 
Antibodies against neuraminidase (NA) provide added benefit to 
hemagglutinin-specific immunity and may be important contributors to the 
effectiveness of A(H7N9) vaccines. We generated a panel of mouse monoclonal 
antibodies (MAbs) to identify antigenic domains on NA of the novel A(H7N9) virus 
and compared their functional properties. The loop formed in the region of 
residue 250 (250 loop) and the domain formed by the loops containing residues 
370, 400, and 430 were identified as major antigenic regions. MAbs 1E8, 2F6, 
10F4, and 11B2, which recognize these two antigenic domains, were characterized 
in depth. These four MAbs differ in their abilities to inhibit cleavage of small 
and large substrates (methyl-umbelliferyl-acetyl neuraminic acid [MU-NANA] and 
fetuin, respectively) in NA inhibition assays. 1E8 and 11B2 did not inhibit NA 
cleavage of either MU-NANA or fetuin, and 2F6 inhibited cleavage of fetuin 
alone, whereas 10F4 inhibited cleavage of both substrates. All four MAbs reduced 
the in vitro spread of viruses carrying either the wild-type N9 or N9 with 
antiviral-resistant mutations but to different degrees. These MAbs have 
different in vivo levels of effectiveness: 10F4 was the most effective in 
protecting mice against challenge with A(H7N9) virus, 2F6 was less effective, 
and 11B2 failed to protect BALB/c mice at the doses tested. Our study confirms 
that NA-specific antibodies can protect against A(H7N9) infection and suggests 
that in vitro properties can be used to rank antibodies with therapeutic 
potential.IMPORTANCE The novel A(H7N9) viruses that emerged in China in 2013 
continue to infect humans, with a high fatality rate. The most recent outbreak 
resulted in a larger number of human cases than previous epidemic waves. Due to 
the absence of a licensed vaccine and the emergence of drug-resistant viruses, 
there is a need to develop alternative approaches to prevent or treat A(H7N9) 
infection. We have made a panel of mouse monoclonal antibodies (MAbs) specific 
for neuraminidase (NA) of A(H7N9) viruses; some of these MAbs are effective in 
inhibiting viruses that are resistant to antivirals used to treat A(H7N9) 
patients. Binding avidity, inhibition of NA activity, and plaque formation 
correlated with the effectiveness of these MAbs to protect mice against lethal 
A(H7N9) virus challenge. This study identifies in vitro measures that can be 
used to predict the in vivo efficacy of NA-specific antibodies, providing a way 
to select MAbs for further therapeutic development.