Comparison of influenza-specific neutralizing antibody titers determined using different assay readouts and hemagglutination inhibition titers: good correlation but poor agreement
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Determination of influenza-specific antibody titers is commonly done using the hemagglutination inhibition assay (HAI) and the viral microneutralization assay (MN). Both assays are characterized by high intra- and inter-laboratory variability. The HAI assay offers little opportunity for standardization. For the MN assay, variability might be due to the use of different assay protocols employing different readouts. We therefore aimed at investigating which of the MN assay readout methods currently in use would be the most suitable choice for a standardized MN assay that could serve as a substitute for the HAI assay. For this purpose, human serum samples were tested for the presence of influenza specific neutralizing antibodies against A/California/7/09 H1N1 (49 sera) or A/Hong Kong/4801/2014 (50 sera) using four different infection readout methods for the MN assay (cytopathic effect, hemagglutination, ELISA, RT qPCR) and using the HAI assay. The results were compared by correlation analysis and by determining the level of agreement before and after normalization to a standard serum. Titers as measured by the 4 MN assay readouts showed good correlation, with high Person's r for most comparisons. However, agreement between nominal titers varied with readouts compared and virus strain used. In addition, Pearson's correlation of MN titers with HAI titers was high but agreement of nominal titers was moderate and the average difference between the readings of two assays (bias) was virus strain-dependent. Normalization to a standard serum did not result in better agreement of assay results. Our study demonstrates that different MN readouts result in nominally different antibody titers. Accordingly, the use of a common and standardized MN assay protocol will be crucial to minimize inter-laboratory variability. Based on reproducibility, cost effectiveness and unbiased assessment of results we elected the MN assay with ELISA readout as most suitable for a possible replacement of the HAI assay.Keywords:
Hemagglutination assay
Antibody titer
Hemagglutmation has been demonstrated with the prototype Huie strain and two nonprototype strains of adenovirus type 12. This viral type resembles those classified in Group 3 in that hemagglutination occurred with rat cells in the presence of hetero-typic immune serum. Hemagglutination was specifically inhibited by immune serum to adenovirus type 12, but not immune serum to other adenoviruses. The identity of the hemagglutinating virus as adenovirus type 12 was confirmed by both hemagglutination–inhibition and neutralization tests with reference immune sera prepared in other laboratories.
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To evaluate the accuracy of hemagglutination inhibition (HI) test as the index of feline panleukopenia virus (FPV)-protective ability, sera from 153 FPV-vaccinated cats aged ≥7 months with HI titer of <1:10-1:40, were examined for serum neutralizing (SN) antibody. SN antibody was detected (≥1:10) in 33 (62.3%) of 53 HI antibody-negative cats, and ranged <1:10-1:160. This suggests that FPV-antibody detection sensitivity of HI test is lower than SN test, and SN test is more suitable for the assessment of FPV-vaccine effect than HI test especially in cats with negative or low HI titer. SN titer was 1:32, FPV-protective threshold, or higher in all cats with HI titers of ≥1:20, suggesting it may be appropriate to set protective HI threshold at 1:20.
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An ELISA-based method to estimate hemagglutination-inhibition (HI) titer was developed. Subtype specificity was obtained by using purified H1 and H3 hemagglutinin antigens. Using the linear relation that exists between ELISA and HI methods, regression lines for H1N1- and H3N2-monospecific porcine antisera were constructed. Approximation of actual HI titer could be obtained from insertion of ELISA values into the appropriate regression line. The HI estimations were within 50% of the actual measured HI value 84% of the time. In young pigs that had suckled immune sows, use of this ELISA revealed estimated HI titer > 320 at 2 and 4 weeks of age. After a typical farm outbreak of influenza A/swine (H1N1), estimated HI titers remain high for 4 to 6 months. Sub-type-specific estimation of the distribution frequency of positive influenza A (H1N1 or H3N2) results for sera from swine in regional herds indicated that 31.3 and 7.4% of the swine tested were positive (HI > 41) for H1N1 and H3N2, respectively. From these observations, we conclude that in many circumstances, an ELISA-based HI estimation method could be used as a substitute for the HI test.
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I. Of 91 cases of active tuberculosis 50 yielded haemagglutination titres of I: 16-1 : 128, 14 gave a titre of 1: 8, and the remaining 30 yielded titres of 0-1: 4. 2. Of 119 normal persons, 101 yielded titres of 0-1 :4,14 gave a titre of I: 8, 3 a titre of I: 32, and I a titre of 1: 128. 3. It would appear that a positive haemagglutination result (a titre above I: 8) is significant, and a titre of 1: 8 or below may occur in a high percentage of cases of active tuberculosis. 4. There appears to be little correlation between the erythrocyte sedimentation rate (Wintrobe) and the haemagglutination titre in individual cases of active tuberculosis. Work is in progress on the correlation of the haemagglutination titre and erythrocyte sedimentation rate in cases under treatment.
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Three assays for detection of rubella antibodies, Rubella G (fluorescence immunoassay [FIA]), Rubacell (passive hemagglutination), and Rubaquick (passive hemagglutination with rotation), were compared with hemagglutination inhibition. A total of 100 serum specimens were selected, 68 of which had an FIA value of less than or equal to 25. On initial testing, among the four tests, there was agreement for 88 specimens for assignment of rubella immune status. On repeat testing, all the results agreed by the hemagglutination inhibition, passive hemagglutination, and passive hemagglutination rotation methods, and only one discrepant specimen remained by FIA.
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The rate of decline of antibody titers to influenza following infection can affect results of serological surveys, and may explain re-infection and recurrent epidemics by the same strain. We followed up a cohort who seroconverted on hemagglutination inhibition (HI) antibody titers (≥4-fold increase) to pandemic influenza A(H1N1)pdm09 during a seroincidence study in 2009. Along with the pre-epidemic sample, and the sample from 2009 with the highest HI titer between August and October 2009 (A), two additional blood samples obtained in April 2010 and September 2010 (B and C) were assayed for antibodies to A(H1N1)pdm09 by both HI and virus microneutralization (MN) assays. We analyzed pair-wise mean-fold change in titers and the proportion with HI titers ≥ 40 and MN ≥ 160 (which correlated with a HI titer of 40 in our assays) at the 3 time-points following seroconversion. A total of 67 participants contributed 3 samples each. From the highest HI titer in 2009 to the last sample in 2010, 2 participants showed increase in titers (by HI and MN), while 63 (94%) and 49 (73%) had reduction in HI and MN titers, respectively. Titers by both assays decreased significantly; while 70.8% and 72.3% of subjects had titers of ≥ 40 and ≥ 160 by HI and MN in 2009, these percentages decreased to 13.9% and 36.9% by September 2010. In 6 participants aged 55 years and older, the decrease was significantly greater than in those aged below 55, so that none of the elderly had HI titers ≥ 40 nor MN titers ≥ 160 by the final sample. Due to this decline in titers, only 23 (35%) of the 65 participants who seroconverted on HI in sample A were found to seroconvert between the pre-epidemic sample and sample C, compared to 53 (90%) of the 59 who seroconverted on MN on Sample A. We observed marked reduction in titers 1 year after seroconversion by HI, and to a lesser extent by MN. Our findings have implications for re-infections, recurrent epidemics, vaccination strategies, and for cohort studies measuring infection rates by seroconversion.
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Objectives To prepare a mask from extracts of Isatis leaf and root, filter a solution of influenza virus through it, and then detect viral activity in the filtrate in order to evaluate the mask's effectiveness against the influenza virus. Methods A solution of H1N1 influenza virus with a hemagglutination titer of 2-12 was filtered through the drug mask. After the filtrate was collected, the hemagglutination titer of the viral solution was determined by performing a hemagglutination test. Then the filtrate was inoculated into chick embryos. The hemagglutination test was performed with allantoic fluid collected after 48 h. Viral activity in the filtrate was analyzed via the hemagglutination titer and EID50. Results Compared to the unfiltered solution of influenza virus with a hemagglutination titer of 2-12, the hemagglutination titer of the filtrate was 2-3 (P0.01). The hemagglutination titer of allantoic liquid was 2-6 after inoculation with filtrate (P0.01). Compared to the unfiltered H1N1 solution with an EID50 of 10-4.95/0.1ml, the filtrate had an EID50 of 10-2.10/0.1ml (P0.01). Conclusion The influenza virus lost more than 93% of its infectivity after it was filtered through a mask made from extracts of Isatis leaf and root. This suggests that the drug mask could be used to prevent infection with the influenza virus.
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OBJECTIVE To investigate the anti-influenza A3 effect of Qing Re He Ji(QRHJ)mixture.METHODS The techniques of chick embryo culture were used for evaluating antiviral activity of serum from the Wistar rats dosed with QRHJ by hemagglutination assay with the influenza virus hemagglutination titer as index.RESULTS The first,second,third and fouth hour's serum of the Wistar rats after oral administration of QRHJ by 24 g·kg-1 dose had antiviral effect.The inhibition rates(%)of hemagglutination titer were 86.37,82.32,80.72 and 72.74,respectively.The first,second and third hour's serum of the Wiistar rats after oral administration of QRHJ by 12 g·kg-1dose had antiviral effect.The inhibition rates(%)of hemagglutination titer were 71.53,67.58 and 66.14.QRHJ was found to be an inhibitor of influenza A3 virus in a concentration-dependent manner.CONCLUSION QRHJ has significant effect on influenza virus type A3 in the chick embryo.
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Determination of influenza-specific antibody titers is commonly done using the hemagglutination inhibition assay (HAI) and the viral microneutralization assay (MN). Both assays are characterized by high intra- and inter-laboratory variability. The HAI assay offers little opportunity for standardization. For the MN assay, variability might be due to the use of different assay protocols employing different readouts. We therefore aimed at investigating which of the MN assay readout methods currently in use would be the most suitable choice for a standardized MN assay that could serve as a substitute for the HAI assay. For this purpose, human serum samples were tested for the presence of influenza specific neutralizing antibodies against A/California/7/09 H1N1 (49 sera) or A/Hong Kong/4801/2014 (50 sera) using four different infection readout methods for the MN assay (cytopathic effect, hemagglutination, ELISA, RT qPCR) and using the HAI assay. The results were compared by correlation analysis and by determining the level of agreement before and after normalization to a standard serum. Titers as measured by the 4 MN assay readouts showed good correlation, with high Person's r for most comparisons. However, agreement between nominal titers varied with readouts compared and virus strain used. In addition, Pearson's correlation of MN titers with HAI titers was high but agreement of nominal titers was moderate and the average difference between the readings of two assays (bias) was virus strain-dependent. Normalization to a standard serum did not result in better agreement of assay results. Our study demonstrates that different MN readouts result in nominally different antibody titers. Accordingly, the use of a common and standardized MN assay protocol will be crucial to minimize inter-laboratory variability. Based on reproducibility, cost effectiveness and unbiased assessment of results we elected the MN assay with ELISA readout as most suitable for a possible replacement of the HAI assay.
Hemagglutination assay
Antibody titer
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Citations (27)