Some studies have suggested that differences in antigenicity exist among different genotypes of EV71 strains, though no difference had been seen between different subtypes within same genotype [23]. In a cross-neutralization study by Sanden et al., B0, B1, B2, C1, and C2 strains were used to cross-react with B2- and C1-immunized rabbit sera, it was shown that B2 sera could not neutralize C strain, but

C1 sera could neutralize B strain [26]. Different genotype strains were tested in neutralizing assays with marmoset sera immunized with EV71 type A attenuated strain [27]. The neutralizing activity was found to be as follows: BrCr-TR(A) > Nagoya(B1) > 75-Yamagata-2003(C4) > 1530-Yamagata-2003(C4) and 2399-Yamagata-2003(C4) > C7-Osaka(B4) and 1095 (C2). Neutralization titers of B4 and C2 were only 1.6% (1/64) those of type A. Six subtypes of strains B and C were tested with guinea pig sera immunized with B2 and C1 [28]. Results showed that the differences Regorafenib between the neutralizing titers of various subtypes could reach a factor of ten. The above finding suggested that strains with different genotypes and strains with same genotype but different origins could affect the results of NTAb analysis. Standards for EV71–NTAb MK2206 need to be developed to ensure

the accuracy and comparability of assay data. For the representativity of NTAb reference standards, we collected plasma from healthy adults who were naturally infected by EV71 as the source of NTAb reference standards. Then, eight candidate standards with different EV71 neutralizing titers were selected by screening from fifty plasma Rutecarpine samples, aliquoted and lyophilized. Collaborative calibration was carried out in four labs. A first ever EV71–NTAb standard was established. Each parameter met WHO and Chinese Pharmacopoeia requirements. Based on collaborative calibration results, the EV71–NTAb titer of the N12 standard was defined as 1000 U/ml. One negative standard, J10, one weakly positive standard, N3, and one strongly positive standard, N12, made up a QC serum panel

for antibody analysis. This panel was adapted from that used in polio virus standard antibody analysis [29]. QC antisera repeats were performed for each strain. The upper and lower limits of the detection ranges were defined using the median and four times the deviation of the antibody GMTs of each strain. In practice, assuming that all three QC sera were valid, NTAb GMTs were converted to U/ml from titers based on defined standards (N12). In initial applications, a common strain distributed by Lab 1 was used in three different labs. Seventeen serum samples from healthy people were tested with standards and QC sera. The results showed that the average of CV and Max–Min deviation were reduced 11.0% and 3.2 times after standardization. This suggests that the application of defined standards could reduce discrepancies between analyses performed in different labs.