Assay Harmonization and Use of Biological Standards To Improve the Reproducibility of the Hemagglutination Inhibition Assay: a FLUCOP Collaborative Study.

The hemagglutination inhibition (HAI) assay is an established technique for assessing influenza immunity, through measurement of antihemagglutinin antibodies. Improved reproducibility of this assay is required to provide meaningful data across different testing laboratories. This study assessed the impact of harmonizing the HAI assay protocol/reagents and using standards on interlaboratory variability. Human pre- and postvaccination sera from individuals (n = 30) vaccinated against influenza were tested across six laboratories. We used a design of experiment (DOE) method to evaluate the impact of assay parameters on interlaboratory HAI assay variability. Statistical and mathematical approaches were used for data analysis. We developed a consensus protocol and assessed its performance against in-house HAI testing. We additionally tested the performance of several potential biological standards. In-house testing with four reassortant viruses showed considerable interlaboratory variation (geometric coefficient of variation [GCV] range of 50% to 117%). The age, concentration of turkey red blood cells, incubation duration, and temperature were key assay parameters affecting variability. Use of a consensus protocol with common reagents, including viruses, significantly reduced GCV between laboratories to 22% to 54%. Pooled postvaccination human sera from different vaccination campaigns were effective as biological standards. Our results demonstrate that the harmonization of protocols and critical reagents is effective in reducing interlaboratory variability in HAI assay results and that pools of postvaccination human sera have potential as biological standards that can be used over multiple vaccination campaigns. Moreover, the use of standards together with in-house protocols is as potent as the use of common protocols and reagents in reducing interlaboratory variability. IMPORTANCE The hemagglutination inhibition (HAI) assay is the most commonly used serology assay to detect antibodies from influenza vaccination or influenza virus infection. This assay has been used for decades but requires improved standardization of procedures to provide meaningful data. We designed a large study to assess selected parameters for their contribution to assay variability and developed a standard protocol to promote consistent HAI testing methods across laboratories. The use of this protocol and common reagents resulted in lower levels of variability in results between participating laboratories than achieved using in-house HAI testing. Human sera sourced from vaccination campaigns over several years, and thus including antibody to different influenza vaccine strains, served as effective assay standards. Based on our findings, we recommend the use of a common protocol and/or human serum standards, if available, for testing human sera for the presence of antibodies against seasonal influenza using turkey red blood cells.

Comparison of hemagglutination inhibition tests, immunoperoxidase monolayer assays, and serum neutralizing tests in detecting antibodies against blue eye disease in pigs.

Blue eye disease (BED) of pigs was identified in the early 1980s in La Piedad, Michoacan, Mexico. The causal agent is Porcine orthorubulavirus (PRV), which affects pigs of all ages, producing nervous, respiratory, and reproductive disorders. BED is geographically endemic to the center of Mexico, where 75% of the country's swine industry is concentrated. Due to its adverse effects on the swine industry and the risk of dissemination to other countries, it is essential to have reliable diagnostic methods for BED. The objective of this study was to establish the optimal conditions for three serological tests, hemagglutination inhibition (HI), immunoperoxidase monolayer assay (IPMA), and serum neutralization (SN), and to compare their sensitivity, specificity, kappa coefficient, and predictive values. Twelve different HI protocols (9408 tests), one SN protocol and one IPMA protocol (784 tests, each) were evaluated. Forty-nine sera were analyzed, and thirty-seven sera showed true positive results, while twelve showed true negative results. The kappa coefficient was used to assess the variation in each test. The best HI protocol registered a sensitivity and specificity of 89 and 100%, respectively, the IPMA test showed values of 85 and 100%, and the SN test registered a sensitivity of 91% and a specificity of 96%. One of the disadvantages of the HI test is that when chicken red blood cells (RBCs) are used, elution occurs in a short incubation time, which would decrease the specificity. The use of bovine RBCs increases the specificity of the testy and makes it more stable, but it decreases the sensitivity. The results of HI and SN revealed the importance of eliminating the complement system of the serum and removing other inhibitors to avoid test nonspecificity. The IPMA test does not use an active virus; hence, it is considered safe and does not present any risk of disseminating PRV.

A Rapid and Facile Purification Method for Glycan-Binding Proteins and Glycoproteins.

Glycosylated proteins, namely glycoproteins and proteoglycans (collectively called glycoconjugates), are indispensable in a variety of biological processes. The functions of many glycoconjugates are regulated by their interactions with another group of proteins known as lectins. In order to understand the biological functions of lectins and their glycosylated binding partners, one must obtain these proteins in pure form. The conventional protein purification methods often require long times, elaborate infrastructure, costly reagents, and large sample volumes. To minimize some of these problems, we recently developed and validated a new method termed capture and release (CaRe). This method is time-saving, precise, inexpensive, and it needs a relatively small sample volume. In this approach, targets (lectins and glycoproteins) are captured in solution by multivalent ligands called target capturing agents (TCAs). The captured targets are then released and separated from their TCAs to obtain purified targets. Application of the CaRe method could play an important role in discovering new lectins and glycoconjugates. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Preparation of crude extracts containing the target proteins from soybean flour Alternate Protocol 1: Preparation of crude extracts from Jack bean meal Alternate Protocol 2: Preparation of crude extracts from the corms of Colocasia esculenta, Xanthosoma sagittifolium, and from the bulbs of Allium sativum Alternate Protocol 3: Preparation of Escherichia coli cell lysates containing human galectin-3 Alternate Protocol 4: Preparation of crude extracts from chicken egg whites (source of ovalbumin) Basic Protocol 2: Preparation of 2% (v/v) red blood cell suspension Basic Protocol 3: Detection of lectin activity of the crude extracts Basic Protocol 4: Identification of multivalent inhibitors as target capturing agents by hemagglutination inhibition assays Basic Protocol 5: Testing the capturing abilities of target capturing agents by precipitation/turbidity assays Basic Protocol 6: Capturing of targets (lectins and glycoproteins) in the crude extracts by target capturing agents and separation of the target-TCA complex from other components of the crude extracts Basic Protocol 7: Releasing the captured targets (lectins and glycoproteins) by dissolving the complex Basic Protocol 8: Separation of the targets (lectins and glycoproteins) from their respective target capturing agents Basic Protocol 9: Verification of the purity of the isolated targets (lectins or glycoproteins).

Comparison of influenza-specific neutralizing antibody titers determined using different assay readouts and hemagglutination inhibition titers: good correlation but poor agreement.

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.

A comparison of viral microneutralization and haemagglutination inhibition assays as measures of seasonal inactivated influenza vaccine immunogenicity in the first year after reduced intensity conditioning, lymphocyte depleted allogeneic haematopoietic stem cell transplant.

Traditionally, immune response to influenza vaccines has been measured using the haemagglutination inhibition (HAI) assay. A broader repertoire of techniques including the sensitive viral microneutralization (VMN) assay is now recommended by the European Medicines Agency (EMA). Comparing HAI and VMN, we determined immune response to a trivalent 2015-2016 seasonal inactivated influenza vaccine (SIIV) administered to 28 recipients of allogeneic haematopoietic stem cell transplant (HSCT). Vaccination was within the first-year post-transplant at a median of 78.5 (24-363) days. The proportion of patients with baseline and post-vaccination HAI titres ≥ 1:40 were 28.6% and 25% for A(H1N1)pdm09, 14.3% at both timepoints for A(H3N2), and 32.1% and 25% for B(Phuket). Pre and Post-vaccination geometric mean titres(GMT) were higher by VMN than HAI for A(H1N1)pdm09 and A(H3N2), but lower for B(Phuket)(p=<0.05). Geometric mean ratios(GMR) of baseline and post-vaccination titres were similar by HAI and VMN(p > 0.05) for all components. A single seroconversion to A(H1N1) was detected by ELISA-VMN. None of patient age, lymphocyte count, days from transplant to vaccination, donor type, or graft-versus-host disease (GVHD) or immunosuppressive therapy (IST) at vaccination correlated with baseline or post-vaccination titres by either assay. This absence of seroresponse to SIIV in the first-year post HSCT highlights the need for novel immunogenic vaccination formulations and schedules in this high-risk population.

Determination of current reference viruses for serological study of swine influenza viruses after the introduction of pandemic 2009 H1N1 (pdmH1N1) in Thailand.

Since the introduction of pandemic H1N1 2009 virus (pdmH1N1) in pigs, the status of Thai swine influenza virus (SIV) has changed. The pdmH1N1 and its reassortant viruses have become the predominant strain circulating in the Thai swine population based on the surveillance data from 2012 to 2014. For this reason, the reference viruses for serological assays using the hemagglutination inhibition (HI) test needed to be updated. Six anti-sera against reference viruses from 2006 to 2009 (enH1N1-06, enH1N1-09, enH1N2-09, pdmH1N1-09, enH3N2-07 and enH3N2-09) were used for the HI test with four contemporary viruses (enH1N1-10, pdmH1N1-10, rH1N2 and rH3N2) and the selected reference viruses were tested with sera collected from the field to determine the current SIV status. The results showed that anti-sera of swH1N1-06 had the highest titers against enH1N1-10. Anti-sera of pdmH1N1-09 had the highest titers against pdmH1N1-10 and rH1N2, whereas, anti-sera of enH3N2-09 had the highest titers against rH3N2. The results demonstrated that enH1N1-06, pdmH1N1-09 and enH3N2-09 should be selected as reference viruses for contemporary serological studies and HI tests. The seroprevalence results from 410 samples revealed enH1N1 (37.79%), pdmH1N1 (37.32%) and H3N2 (35.86%), respectively. The present study indicated that pdmH1N1 was widespread and commonly found in the Thai pig population increasing the risk of novel reassortant viruses and should be added as a reference virus for HI test. SIV surveillance program and serological studies should be conducted for the benefits of SIV control and prevention as well as monitoring for zoonotic potential.

Serological diagnosis of avian influenza in poultry: is the haemagglutination inhibition test really the 'gold standard'?

BACKGROUND: The serological diagnosis of avian influenza (AI) can be performed using different methods, yet the haemagglutination inhibition (HI) test is considered the 'gold standard' for AI antibody subtyping. Although alternative diagnostic assays have been developed, in most cases, their accuracy has been evaluated in comparison with HI test results, whose performance for poultry has not been properly evaluated. OBJECTIVE: The objective of this study was to estimate the diagnostic sensitivity (Se) and specificity (Sp) of the HI test and six other diagnostic assays for the detection of AI antibodies without assuming a gold standard. METHODS: We applied a Bayesian version of latent class analysis to compare the results of multiple tests from different study settings reported in the literature. RESULTS: The results showed that the HI test has nearly perfect accuracy (i.e. 98·8% sensitivity and 99·5% specificity). It performed well in both chickens and turkeys and yet was less accurate in experimentally infected poultry, compared to naturally infected. Blocking ELISA and the indirect immunofluorescence assay also performed very well. CONCLUSIONS: Given its very high Se and Sp, the HI test may be effectively considered a gold standard. In the framework of LPAI surveillance, where large numbers of samples have to be processed, the blocking ELISA could be a valid alternative to the HI test, in that it is almost as sensitive and specific as the HI test yet quicker and easier to automate.

Collaborative study on influenza vaccine clinical trial serology - part 1: CHMP compliance study.

The Quality of Medicines & HealthCare (EDQM, Council of Europe) and the European Union (EU) Commission to evaluate the reproducibility of clinical serology results for seasonal influenza vaccines and to assess the impact of technical differences between laboratories on the compliance with the Committee for Human Medicinal Products (CHMP) criteria set by the European Medicines Agency (EMA). The study was run in 2 phases. The present article reports the 1st phase of the study, which aimed at evaluating the variability of the results obtained by 11 laboratories (5 national control laboratories and 6 influenza vaccine manufacturers) using their routine haemagglutination inhibition (HI) assay to test a common panel of clinical trial sera. The results confirmed the limited inter-laboratory reproducibility of the HI testing of influenza vaccine clinical trial samples. In some cases a good agreement was found between laboratories, while a systematic bias or a random scatter of results was observed in other cases. Analysis of estimated systematic bias confirmed that differences between laboratories can be significant (up to 16-fold) in some cases. Correction for this bias resulted in limited improvement. Differences between laboratories were found to result in discrepant decisions on marketing acceptance of vaccines or to decisions based on compliance to different criteria. The study showed that the seroconversion (SC) and mean fold increase (MFI) criteria are more robust against systematic over- or under-estimation of titres whereas the protection rate (PR) is very sensitive to this effect. The fundamental issues with the PR criteria are discussed.

Collaborative study on influenza vaccine clinical trial serology - part 2: reproducibility study.

A collaborative study was run by the Biological Standardisation Programme (BSP) under the aegis of the European Directorate for the Quality of Medicines & HealthCare (EDQM, Council of Europe) and the European Union (EU) Commission, to address the issue of the poor standardisation of serological assays used for the evaluation of seasonal influenza vaccines in Europe. The Phase 1 of the study focused on the compliance to Committee for Human Medicinal Products (CHMP) criteria by 6 manufacturers and 5 public laboratories. It confirmed the poor inter-laboratory correlation of haemagglutination inhibition (HI) test results. Phase 2 consisted in a reproducibility study examining the impact of extended method standardisation and the use of reference sera on inter-laboratory variation. Six manufacturers and 5 public laboratories contributed HI results, while the 5 public laboratories also performed single radial haemolysis (SRH) tests on the same sample panels. Results showed that method standardisation failed to significantly improve the inter-laboratory variation. Correction for pre-vaccination titres (Beyer correction) was found to have limited effect to improve the bias constituted by the Protection Rate (PR) criterion. The reasons underlying the difficulty in standardisation of HI and SRH tests are discussed and improved approaches for the compliance testing to CHMP criteria are suggested.

Qualification of the hemagglutination inhibition assay in support of pandemic influenza vaccine licensure.

Continued outbreaks of highly pathogenic avian influenza over the past decade have spurred global efforts to develop antivirals and vaccines. As part of vaccine development, standard methods are needed for determining serum antibody titers in response to vaccination. Hemagglutination inhibition (HAI) assays are appropriate for assessing the immunogenicity of pandemic influenza vaccines in support of license approval. We demonstrate that a rigorous qualification of the HAI assay for H5N1 influenza virus, evaluating for precision, intermediate precision, linearity, range, specificity, and robustness, satisfies the intent of regulatory guidance for assay validation despite the lack of availability of specific reference standard antigens and antisera.

The influence of oseltamivir carboxylate and oseltamivir on hemagglutinin inhibition and microneutralization test.

The administration of oseltamivir in humans is suggested to affect the results of hemagglutinin-inhibition test. To investigate this phenomenon, the concentrations of oseltamivir and oseltamivir carboxylate (OC) in sera obtained from oseltamivir-administered individuals were quantified by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The analysis revealed that the concentrations of OC in sera obtained at 4 and 7h after administration were greater than those at 24h after administration. Flow cytometry analyses revealed that OC or oseltamivir added in the sera affects the expression level of sialic acid alpha2,3-Gal linkages on horse erythrocytes; however, no effect was observed on the expression level of these linkages on chicken erythrocytes. Moreover, the addition of oseltamivir or OC may yield pseudopositive results in hemagglutinin-inhibition assays. These results suggest that the pseudopositive results obtained in hemagglutinin-inhibition assays occurred by the presence of OC, and that it is very important to take care of the patients in the prescription of oseltamivir when anti-influenza investigations are performed.

Evaluation of eight anti-rubella virus immunoglobulin g immunoassays that report results in international units per milliliter.

An evaluation of anti-rubella virus immunoglobulin G (IgG) immunoassays that report in international units per milliliter (IU/ml) was performed to determine their analytical performance and the degree of correlation of the test results. A total of 321 samples were characterized based on results from a hemagglutination inhibition assay. The 48 negative and 273 positive samples were used to determine the sensitivity and specificity of the assays. When equivocal results were interpreted as reactive, the sensitivity of the immunoassays ranged from 98.9 to 99.9% and the specificity ranged from 77.1 to 95.8%. All assays had positive and negative delta values of less than 2. A significant difference between the mean results of all assays was demonstrated by analysis of variance. However, post hoc analysis showed there was good correlation in the mean results expressed in IU/ml between some of the assays. Our results show the level of standardization between anti-rubella virus IgG immunoassays reporting results expressed as IU/ml has improved since a previous study in 1992, but further improvement is required.

Comparison of neutralising antibody assays for detection of antibody to influenza A/H3N2 viruses: an international collaborative study.

A study was performed to investigate the reproducibility of haemagglutinin-inhibition (HI) and virus neutralising (VN) assays for detection of anti-influenza antibody. Participants in 11 laboratories from eight countries measured antibody to egg-grown A/Japan/434/2003, cell-grown A/Japan/434/2003 and A/Panama/2007/99 (H3N2) viruses in 18 human and two post-infection ferret sera. There was significant intra-laboratory assay variability for VN compared to HI. For replicate assays within laboratories, 14/410 (3%) and 130/631 (21%) titres differed by >2-fold (p<0.0001), and 0/410 (0%) and 35/631 (6%) titres differed by >5-fold (p<0.0001) by HI and VN, respectively. Although both assays showed inter-laboratory variation, VN assays were significantly more variable than HI. Median geometric coefficients of variation (GCV) for VN assays with each virus were 256%, 323% and 359% compared to 138%, 155% and 261% with HI. A serum standard improved inter-laboratory agreement and reduced median GCVs. This study raises concern about comparability of serology results from H5N1 vaccine trials and it is proposed that an International Standard for influenza H5N1 antibody is developed.

Utility of the Determine Syphilis TP rapid test in commercial sex venues in Peru.

OBJECTIVES: This study sought to evaluate the utility of the Determine Syphilis TP test performed in Peruvian commercial sex venues for the detection of active syphilis; and determine the feasibility of integrating rapid syphilis testing for female sex workers (FSW) into existing health outreach services. METHODS: We tested 3586 female sex workers for syphilis by Determine in the field using whole blood fingerstick, and by rapid plasma reagin (RPR) and Treponema pallidum haemagglutination assay (TPHA) in a central laboratory in Lima using sera. RESULTS: 97.4% of the FSW offered rapid syphilis testing participated; and among those who tested positive, 87% visited the local health centre for treatment. More than twice as many specimens were RPR reactive using serum in Lima (5.7%) than tested positive by whole blood Determine in the field (2.8%), and although most were confirmed by TPHA, only a small proportion (0.7%) were RPR reactive at >or=1:8 dilutions, and likely indicating active syphilis. Sensitivity, specificity and positive predictive value of the Determine Syphilis TP test in whole blood when compared to serum RPR reactivity at any dilution confirmed by TPHA as the gold standard were 39.3%, 99.2% and 71.4%, respectively. Sensitivity improved to 64.0% when using serum RPR >or=1:8 confirmed by TPHA. Invalid tests were rare (0.3%). CONCLUSIONS: Rapid syphilis testing in sex work venues proved feasible, but Determine using whole blood obtained by fingerstick was substantially less sensitive than reported in previous laboratory-based studies using serum. Although easy to perform in outreach venues, the utility of this rapid syphilis test was relatively low in settings where a large proportion of the targeted population has been previously tested and treated.

Statistical analysis of influenza vaccine lot consistency studies.

This paper presents an integrated statistical approach to the analysis of influenza vaccine lot consistency studies in which three lots are compared. The approach ensures that the overall Type I error rate (i.e., the probability of wrongly concluding that the lots are similar) is controlled. It is argued that the optimum efficacy measure is the geometric mean titer. The approach is demonstrated using data from a randomized, double-blind lot consistency study in which three consecutive production lots of Solvay Pharmaceuticals' new, virosomal subunit influenza vaccine Invivac were compared.

On the bias in HI titers and how to reduce it.

HI titers measure anti-HA antibody concentrations in sera. By the way they are defined standard titers underestimate the true amounts of antibody. A new definition is proposed, the mid-value definition. Under mild conditions mid-value titers are on average closer to the true value than standard titers.

Inability of kaolin treatment to remove nonspecific inhibitors from equine serum for the hemagglutination inhibition test against equine H7N7 influenza virus.

The hemagglutination inhibition test is used by many diagnostic and surveillance laboratories for detection of antibodies to influenza viruses. It is well known that the hemagglutination inhibition test is affected by nonspecific inhibitors present in equine serum. Several serum treatments are in use to remove these inhibitors, including treatment with kaolin. Discrepant results were observed in the authors' laboratories when using kaolin treatment before testing equine sera for antibodies against equine influenza virus (EIV) subtype-1 (H7N7). It is demonstrated here that kaolin treatment leads to false positive results when testing for antibodies against EIV subtype-1, as compared to other standard serum treatments (trypsin-periodate, receptor-destroying enzyme). Against EIV subtype-2 (H3N8), however, false positive results were not evident. Trypsinperiodate and receptor-destroying enzyme (RDE) treatments appear to be superior to kaolin for removal of nonspecific inhibitors from equine serum and should be used for serological diagnosis and surveillance of equine influenza virus.