Table 4.

Comparison between serological assays used in antigenic characterization of influenza viruses^a

	Hemagglutination inhibition (HI)	Neutralization assays		Neuraminidase inhibition		Polyclonal serum–based proximity ligation assay (PolyPLA)
		Micro-neutralization (MN)	Focus reduction neutralization test	Micro-neuraminidase inhibition	Enzyme-linked lectin assay (ELLA)
Principle	Antibodies inhibiting binding between virus and erythrocytes	Antibodies inhibiting viral cell entry and viral replication		Antibodies inhibiting the NA activity		Direct binding between virus and antibody
Primary epitopes	HA heads, particularly those close to RBD	HA and NA		NA		HA and NA
Standardization before assays	Yes, to determine 4 HAU	Yes, to determine 100 TCID50	Yes, to determine 20–85% ICP	Yes, to determine an OD549 of 0.45 to 0.85	Yes, to determine 90% maximum OD490	Not needed
Sera/reaction	25 uL	50 uL	50 uL	50 uL	50 uL	0.5–1 uL
Virus/reaction	4 HAU, and 1 HAU represents ~104 virion/25 uL [220]	100 TCID50/50 uL	20–85% ICP/50 uL	OD549 of 0.45 to 0.85/50 uL	90% of maximum OD490/50 uL	~103 TCID/mL in a volume of 0.5 uL
Terminal titer determination	Agglutination	≥50% absorbance readout reduction compared to virus control	≥80% ICP reduction compared to virus control	50% inhibition concentration		PolyPLA units derived from normalized ΔCt

^aHAU hemagglutinating unit; TCID50 50% tissue culture infectious dose; ICP infected cell population; OD optical density; ΔCt the change between threshold cycle of negative control and that of sample