Titer Estimation Research Articles

Evaluation of immunoglobulin M and immunoglobulin G titers of ABO blood group system in blood donors at a blood center of a tertiary care hospital

Abstract BACKGROUND AND OBJECTIVES: ABO antibodies have implications in platelet transfusions, solid organ, and bone marrow transplants. The study aimed at measuring the immunoglobulin G (IgG) and immunoglobulin M (IgM) ABO antibody titers in A, B, and O blood group blood donors and correlating the titers with age, gender, and blood group of donors. SETTINGS AND DESIGN: A prospective observational study was performed over 3 years at a tertiary care blood center. MATERIALS AND METHODS: Equal number of A, B, and O group blood donor samples chosen by random sampling were evaluated for titers by doubling dilution using tube method. IgM titers were determined at room temperature. IgG titers were tested at 37°C with anti-IgG antiglobulin reagent after inactivating IgM antibodies by dithiothreitol. The titers were correlated with age, gender, and blood group. STATISTICAL ANALYSIS: Mann–Whitney U-test, Kruskal–Wallis test, and Bonferroni correction were employed for various correlations using IBM SPSS v26 (Statistical Package for the Social Sciences) software. A two-tailed P < 0.05 was considered statistically significant. RESULTS: A total of 870 samples (290 of each of A, B, and O blood groups) were tested for IgG and IgM antibody titers. IgG anti-A and anti-B titers were significantly higher in blood group O as compared to blood groups B and A. Anti-B IgM titers in group A were significantly higher than anti-A IgM titer in B. The antibody titer showed decreasing trend with age. There was no correlation with gender. CONCLUSIONS: The study will help hospitals and blood centers in formulating policies regarding ABO antibody titer estimation in platelet donors and renal transplant recipients in the context of out-of ABO group platelet transfusions and ABO-incompatible renal transplants, respectively.

Longitudinal detection of prion infection in preclinical sheep blood samples compared using 3 assays

AbstractVariant Creutzfeldt-Jakob disease (vCJD) is a devastating disease caused by transmission of bovine spongiform encephalopathy to humans. Although vCJD cases are now rare, evidence from appendix surveys suggests that a small proportion of the United Kingdom population may be infected without showing signs of disease. These “silent” carriers could present a risk of iatrogenic vCJD transmission through medical procedures or blood/organ donation, and currently there are no validated tests to identify infected asymptomatic individuals using easily accessible samples. To address this issue, we evaluated the performance of 3 blood-based assays in a blinded study, using longitudinal sample series from a well-established large animal model of vCJD. The assays rely on amplification of misfolded prion protein (PrPSc; a marker of prion infection) and include real-time quaking-induced conversion (RT-QuIC), and 2 versions of protein misfolding cyclic amplification (PMCA). Although diagnostic sensitivity was higher for both PMCA assays (100%) than RT-QuIC (61%), all 3 assays detected prion infection in blood samples collected 26 months before the onset of clinical signs and gave no false-positive results. Parallel estimation of blood prion infectivity titers in a sensitive transgenic mouse line showed positive correlation of infectivity with PrPSc detection by the assays, suggesting that they are suitable for detection of asymptomatic vCJD infection in the human population. This study represents, to our knowledge, the largest comparison to date of preclinical prion detection in blood samples from a relevant animal model. The outcomes will guide efforts to improve early detection of prion disease and reduce infection risks in humans.

Diagnostic and prophylactic potential of a stabilized foot-and-mouth disease serotype Asia1 virus like particles designed through a structure guided approach

Intact capsids of foot-and-mouth disease virus (FMDV) play a vital role in eliciting a protective immune response. Any change in the physico-chemical environment of the capsids results in dissociation and poor immunogenicity. Structural bioinfomatics studies have been carried out to predict the amino acids at the interpentameric region that resulted in the identification of mutant virus-like particles(VLPs) of FMDV serotype Asia1/IND/63/1972. The insect cell expressed VLPs were evaluated for their stability by sandwich ELISA. Among 10 mutants, S93H showed maximum retention of antigenicity at different temperatures, indicating its higher thermal stability as revealed by the in-silico analysis and retained the antigenic sites of the virus demonstrated by Sandwich ELISA. The concordant results of the liquid phase blocking ELISA for estimation of antibody titre of known sera with stable mutant VLP as antigen in place of virus antigen demonstrate its diagnostic potential. The stable mutant VLP elicited a robust immune response with 85.6 % protection in guinea pigs against virus challenge. The stabilized VLP based antigen requires minimum biosafety and cold storage for production and transit besides, complying with differentiation of infected from vaccinated animals. It can effectively replace the conventional virus handling during antigen production for prophylactic and diagnostic use.

Titer and charge-based heterogeneity multiattribute monitoring of mAbs in cell culture harvest using 2D ProA CEX MS

Robust monitoring of heterogeneity in biopharmaceutical development is crucial for producing safe and efficacious biotherapeutic products. Multiattribute monitoring (MAM) has emerged as an efficient tool for monitoring of mAb heterogeneities like deamidation, sialylation, glycosylation, and oxidation. Conventional biopharma analysis during mAb development relies on use of one-dimensional methods for monitoring titer and charge-based heterogeneity using non-volatile solvents without direct coupling with mass spectrometry (MS). This approach requires analysis of mAb harvest by ProA for titer estimation followed by separate cation exchange chromatography (CEX) analysis of the purified sample for estimating charge-based heterogeneity. This can take up to 60–90 min due to the required fraction collection and buffer exchange steps. In this work, a native two-dimensional liquid chromatography (2DLC) mass spectrometry method has been developed with Protein A chromatography in the first dimension for titer estimation and cation exchange chromatography (CEX) in the second dimension for charge variant analysis. The method uses volatile salts for both dimensions and enables easy coupling to MS. The proposed 2DLC method exhibits a charge variant profile that is similar to that observed via the traditional methods and takes only 15 min for mass identification of each variant. A total of six charge variants were separated by the CEX analysis after titer estimation, including linearity assessment from 5 μg to 160 μg of injected mAb sample. The proposed method successfully estimated charge variants for the mAb innovator and 4 of its biosimilars, showcasing its applicability for biosimilarity exercises. Hence, the 2D ProA CEX MS method allows direct titer and charge variant estimation of mAbs in a single workflow.

Development and validation of streptavidin-biotin-based double antibody sandwich ELISA for ricin diagnosis

BackgroundRicin is a potential biowarfare agent. It is a phytotoxin isolated from castor seeds. At present there is no antidote available for ricin poisoning, patients only get supportive treatment based on their symptoms. This highlights the importance of early detection to avoid severity of accidents and reduce the risk factor. Considering this, our study aimed to develop a highly sensitive and specific sandwich ELISA for the detection of ricin. MethodsRicin was purified from castor seeds. Anti-ricin polyclonal and monoclonal antibodies were generated from rabbit antisera and hybridoma cell (1H6F1) supernatant using a protein A/G column. Antibody titer estimation was done using Indirect ELISA. A streptavidin–biotin-based sandwich ELISA was developed and the limit of detection (LOD), linear range, intra and inter-assay coefficient of variation (CV), and cross-reactivity with other similar toxins were determined. Interference of human plasma samples spiked with ricin was also checked. ResultsThe LOD of the ELISA was found to be 0.45 ng/ml, with a linear range of 0.90–62 ng/ml, intra and inter-assay CV ranged from 3.34 % to 5 % and 5.17 % to 10.80 % respectively. The assay was not cross-reactive with other similar ribosome-inactivating protein (RIP) toxins. Ricin was detected in spiked plasma samples. ConclusionThe developed assay is highly sensitive and specific for detecting ricin and is not cross-reactive with other similar types of toxins. The assay can detect ricin in spiked plasma samples, so it has the potential to be used for the analysis of clinical samples after ricin poisoning.

Automated Mass Photometry of Adeno-Associated Virus Vectors from Crude Cell Extracts.

Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5-10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer.

Estimation of ABO Anti-A and Anti-B Agglutinin Titers among Blood Donors at a Tertiary Care Referral Teaching Hospital Blood Centre in Southern India: A Cross-sectional Study

Introduction: Transfusion of blood Group O and its components with high ABO antibody titers to non O recipients has been shown to cause acute transfusion reactions, especially in platelet transfusions, as platelets contain significant amounts of ABO antigen on their surface as well as Anti-ABO alloisogglutinins in plasma. Aim: To estimate the ABO Anti-A and Anti-B agglutinin titers among blood donors at a tertiary care referral teaching hospital blood centre. Materials and Methods: This cross-sectional observational study was conducted at the Immunohaematology laboratory of the Department of Transfusion Medicine, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India, from March 2021 to June 2022. All blood donors presenting to the blood centre were screened for eligibility for blood donation. Donors who fulfilled the eligibility criteria as per the Drugs and Cosmetics Act, 1940, and Rules, 1945 were included. Anti-A and Anti-B titers were determined by the conventional tube method with dilutions of 1 in 2, 1 in 4, 1 in 8, 1 in 16, 1 in 32, 1 in 64, 1 in 128, 1 in 256, 1 in 512, and 1 in 1024. A titer of <64 was considered as a low titer, while a titer of >64 was considered as a high titer. Results: A total of 399 donors were included in the study, with 393 (98.5%) being males and 6 (1.5%) being females. The mean age of the study population was 28.49 years. Among blood group A, the percentage of individuals with an IgM anti-B titer of <64 was 85.44% (88) and >64 was 14.56% (15), whereas in blood group B, IgM anti-A titers of <64 were 65.5% (93) and >64 were 34.5% (49). In blood group O, the percentage of individuals with <64 and >64 titers of IgM anti-A were 52.60% (81) and 47.40% (73), respectively, whereas for anti-B, <64 and >64 titers were 77.92% (120) and 22.08% (34), respectively. Conclusion: It is recommended that a database be maintained in all institutes by estimating antibody titers for every A, B, O blood group donors. Whole blood or platelets from group O donors with IgM Anti-A and Anti-B antibody titers <64 can only be transfused across the ABO barrier. As the majority of group A donors had titers <64, group A platelets may be transfused across the ABO barrier in emergency situations.

Case series of spontaneous bilateral absorbed cataracts running in a family

There are only a handful of case reports and studies available regarding spontaneously absorbed cataracts and their management, including the surgical techniques and results following the management. In our study, on retrospective analysis, after a thorough search of the concerned literature, we encountered a series of cases of bilateral spontaneous absorbed cataracts running in a family with no such documentation. This retrospective study included 11 patients (aged between 3 and 65 years and of either sex) of the same family having bilateral absorbed cataracts. All patients underwent best-corrected visual acuity (BCVA); a thorough eye examination; rubella titer estimation; intraocular lens (IOL) power calculation; lens aspiration with or without posterior capsulotomy, anterior vitrectomy (AV), or IOL implantation; and visual rehabilitation. All patients had bilateral absorbed cataracts, which were acquired in an autosomal dominant pattern. Preoperative BCVA distance: 22 eyes had either counting fingers close to the face or counting fingers at 1–2 feet. The presence of high-frequency mixed bilateral nystagmus was noted in four patients. Anterior continuous curvilinear capsulorhexis was possible. Cortical aspiration was uneventful. In the capsular bag, implantation was possible in the majority of cases. No postoperative inflammation was noted, and the IOL was well-centered in all patients. To the best of our knowledge, this is the first study to report a case series of bilateral absorbed cataracts running in an Indian family with no syndromic association, no history of mental retardation, or infection. Anatomic disorganization of the absorbed lens makes surgery in such cases challenging. By using appropriate surgical methods and good visual rehabilitation, satisfactory surgical and visual outcomes can be achieved.

Comparative study of total antibody titer against SARS-CoV-2 spike protein between male and female COVID- 19 patients of different age groups in UAE

Abstract Introduction/Objective Currently, there is increasing evidence that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes men to experience more serious symptoms and have a higher mortality rate than women, but the associations between gender and age groups are still elusive. This study aimed to examine the associations with age, gender, and clinical parameters of total antibody responses against the spike protein of SARS- CoV-2 after infection in UAE population. Methods/Case Report In this study, spike protein antibody titer estimation for COVID-19 positive individuals (n = 1070) was determined. A cross-sectional study design was planned in which patients were divided into nine groups (based on their age) and further subdivided into two groups based on their genders (9 x 2). Serum was collected from the patients after 17 days of diagnosis as COVID-19 positive by RT-PCR method. Concentrations of SARS-CoV-2 Total antibody (COV2T) were measured by chemiluminescent immunoassay using ADVIA Centaur® XPT systems. We used a mixed effects model to investigate the potential associations of antibodies by bootstrapping t-tests of the putative effects of gender and age of the individuals. Results (if a Case Study enter NA) There were significant differences (P< 0.05) between males and females for SARS-CoV-2 total antibody reactivity. Total 645 (50.4%) male were found reactive compared to 425 (31.2%) female. Noteworthy, the group having middle-aged (41-50 years) participants depicted a higher antibody titer in both males and female against SARS-CoV-2. A significant decrease in titers also observed with different age groups. Highest number of reactive individuals were observed in 41-50 years age group (31%), followed by 31-40 years (27.34%), 51- 60 years (15.21%) and 21-30 years (13.04%). Same pattern of number of reactive individuals was found in male and female of different age group. Moreover, despite there being a weak significant positive association between age and total antibody titre in male patients, while there was no significant correlation between gender and age for the antibody response. Conclusion In a nutshell, total antibody (COV2T) reactivity was found significantly associated with male group and that was highest in 41-50 years age group, irrespective of the gender. These findings are crucial to treat and care for male and female patients infected with COVID-19 at hospitals.

Correlation between specific antibody response to wild-type BNT162b2 booster and the risk of breakthrough infection with omicron variants: Impact of household exposure in hospital healthcare workers

BackgroundThe emergence of omicron variants exhibiting antigenic changes has led to an increase in breakthrough infection among individuals with a wild-type SARS-CoV-2 vaccine booster. The correlation between post-booster spike-specific antibodies and omicron infection risk remains unclear. MethodsThis prospective cohort study included SARS-CoV-2-naive healthcare workers with three-dose BNT162b2. Post-booster spike-specific IgG and interferon-γ levels were measured. Breakthrough infection was documented during a 10-month omicron-predominant period. Household and healthcare contacts were followed to identify subsequent infections. The IgG titers were additionally measured at the end of follow-up, and the titers at exposure were estimated from the two-point titers. ResultsOf 333 participants, 89 developed infection, of whom 37 (41.6 %) were household contacts. Kaplan-Meier curves indicated that higher IgG titers were significantly correlated with lower cumulative infection incidence (p = 0.029), whereas the interferon-γ levels were not (p = 0.926). Multivariate Cox analysis showed that increasing IgG titers were associated with a reduced hazard ratio (HR) of 0.26 (95% CI, 0.12–0.55). Household exposure posed a greater infection risk than healthcare exposure (HRs, 11.24 [6.88–18.40] vs. 2.82 [1.37–5.44]). The difference in geometric mean IgG titers of infected and uninfected participants was significant among household contacts (20,244 AU/mL vs. 13,842 AU/mL, p = 0.031). Estimation of IgG titers at exposure showed a significantly higher infection incidence in those exposed with titers of <3,000 AU/mL than in those with higher titers (79.2 % vs. 32.3 %, p < 0.001). ConclusionsSpike-specific antibodies induced by a wild-type SARS-CoV-2 vaccine booster are suggested to be effective in protecting against omicron infection. Household exposure would be a significant source of infection for hospital healthcare workers.

A systematic examination of anti-drug antibody titer estimation: Applied recommendations

Biologic drugs (therapeutic proteins or peptides) have become one of the most important therapeutic modalities over the past few decades. Drug-induced immunogenicity is a significant concern as it may affect safety, tolerability, and efficacy. With more sensitive and drug-tolerant screening assays in use today, reliable estimation of anti-drug-antibody (ADA) titer has become more important for understanding clinically relevant ADA levels. Titer is commonly defined as the dilution factor resulting in an assay signal equal to a pre-specified cut point factor. Given its influence on the resulting titer precision, the choice of a titer cut point factor warrants careful consideration. In this paper, we discuss the theoretical dilution model, investigate how titer variability depends on the cut point factor and propose a standardized cut point factor to increase precision of titer estimates. Additionally, we demonstrate that non-linear regression-based titer estimation provides both improved precision and implementation efficiency relative to commonly used estimation approaches.

Quantitation and integrity evaluation of RNA genome in lentiviral vectors by direct reverse transcription-droplet digital PCR (direct RT-ddPCR)

Lentiviral vectors (LV) have proven to be powerful tools for stable gene delivery in both dividing and non-dividing cells. Approval of these LVs for use in clinical applications has been achieved by improvements in LV design. Critically important characteristics concerning quality control are LV titer quantification and the detection of impurities. However, increasing evidence concerning high variability in titration assays indicates poor harmonization of the methods undertaken to date. In this study, we developed a direct reverse transcription droplet digital PCR (Direct RT-ddPCR) approach without RNA extraction and purification for estimation of LV titer and RNA genome integrity. The RNA genome integrity was assessed by RT-ddPCR assays targeted to four distant regions of the LV genome. Results of the analyses showed that direct RT-ddPCR without RNA extraction and purification performs similarly to RT-ddPCR on purified RNA from 3 different LV samples, in terms of robustness and assay variance. Interestingly, these RNA titer results were comparable to physical titers by p24 antigen ELISA (enzyme-linked immunosorbent assay). Moreover, we confirmed the partial degradation or the incomplete RNA genomes in the prepared 3 LV samples. These results may partially explain the discrepancy of the LV particle titers to functional titers. This work not only demonstrates the feasibility of direct RT-ddPCR in determining LV titers, but also provides a method that can be easily adapted for RNA integrity assessment.

Yellow Leaf Disease Resistance and Melanaphis sacchari Preference in Commercial Sugarcane Cultivars.

Sugarcane yellow leaf disease (YLD) caused by sugarcane yellow leaf virus (ScYLV) is a major threat for the sugarcane industry worldwide, and the aphid Melanaphis sacchari is its main vector. Breeding programs in Brazil have provided cultivars with intermediate resistance to ScYLV, whereas the incidence of ScYLV has been underestimated partly due to the complexity of YLD symptom expression and identification. Here, we evaluated YLD symptoms in a field assay using eight sugarcane genotypes comprising six well-established commercial high-sucrose cultivars, one biomass yield cultivar, and a susceptible reference under greenhouse conditions, along with estimation of virus titer through RT-qPCR from leaf samples. Additionally, a free-choice bioassay was used to determine the number of aphids feeding on the SCYLV-infected cultivars. Most of the cultivars showed some degree of resistance to YLD, while also revealing positive RT-qPCR results for ScYLV and virus titers with non-significant correlation with YLD severity. The cultivars IACSP01-5503 and IACBIO-266 were similar in terms of aphid preference and ScYLV resistance traits, whereas the least preferred cultivar by M. sacchari, IACSP96-7569, showed intermediate symptoms but similar virus titer to the susceptible reference, SP71-6163. We conclude that current genetic resistance incorporated into sugarcane commercial cultivars does not effectively prevent the spread of ScYLV by its main aphid vector.

Serological Evaluation of Antibody Titers After Vaccination Against COVID-19 in 18-44-Year-Old Individuals at a Tertiary Care Center.

Background The evaluation of the effectiveness of the vaccines (ChAdOx1-nCOV; Covishield and BBV-152; Covaxin) against coronavirus disease 2019 (COVID-19) is necessary to assess their efficacy. Because most antibodies that neutralize the coronavirus are directed against the receptor binding domain within the spike protein of the virus, these antibodies serve as markers for viral neutralizers and, in turn, for vaccine response. The present study aimed to evaluate the anti-neutralizing antibody(receptor binding domain (RBD)) and immunoglobulin G2 (IgG2) titers following the completion of the vaccination schedule (both vaccines) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methodology In this longitudinal prospective study, conducted in a tertiary care center, 30 sequentially (two doses) vaccinated study participants between the ages of 18 and 44 years were sampled for estimation of anti-RBD antibody titer and IgG2. All statistical analysis was done using SPSS version 20 (IBM Corp., Armonk, NY, USA). P-values less than 0.05 were considered significant. Results There was a statistically significant increase in the neutralizing antibody titer after one month of the second dose (z = -4.597, p < 0.001), while a significant decrease was seen in the IgG2 levels (z = -3.075, p = 0.002). The results showed a significant neutralizing effect of the vaccines being used, with Covishield being more effective than Covaxin. The levels of neutralizing antibodies were independent of all demographic variables such as age, sex, and body mass index. Conclusions This study evaluating the efficacy of the two vaccines, namely, Covishield and Covaxin, is the first of its kind in the state of Chhattisgarh. The results of this study are similar to previous studies conducted in India and outside India, concluding that Covishield is a more effective vaccine.

Changes in anti-SARS-CoV-2 antibody titers of pooled plasma derived from donors in Japan: a potential tool for mass-immunity evaluation.

The anti-spike (S), anti-nucleocapsid (N), and neutralizing activities of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) of pooled plasma derived from donors in Japan from January 2021 to April 2022 were evaluated. Anti-S titers and neutralizing activities showed a wave-like trend affected by daily vaccinations and/or the number of reported cases of SARS-CoV-2 infections, whereas anti-N titers remained at negative levels. These results suggest that anti-S and neutralizing titers would fluctuate in pooled plasma in future. Pooled plasma may be potentially used for mass-immunity evaluation, and titer estimation in intravenous immunoglobulin, a derivative of pooled plasma.

Rapid analysis of titer, aggregate, and intact mass of antibody therapeutics using automated multi-dimensional liquid chromatography coupled with native mass spectroscopy.

Monoclonal antibodies are tetrameric complex proteins, primarily produced using mammalian cell culture. Attributes such as titer, aggregates, and intact mass analysis are monitored during process development/optimization. In the present study, a novel workflow such that the Protein-A affinity chromatography is performed in the first dimension for purification and titer estimation, whereas size exclusion chromatography is employed in the second dimension to characterize size variants using native mass spectrometry. The present workflow offers a significant advantage over the traditionally used standalone Protein-A affinity chromatography followed by size exclusion chromatography analysis in that it can monitor these four attributes in 8 min while requiring a minimal sample size (10-15μg) and not requiring any manual peak collection. In contrast, the traditional standalone approach requires manual collection of eluted peaks in Protein-A affinity chromatography followed by buffer exchange to a mass-compatible buffer, which can take up to 2-3h with considerable risk of sample loss, degradation, and induced modifications. As the biopharma industry moves to make analytical testing efficient, we believe that the approach proposed here would be of significant interest due to its ability to monitor multiple process and product quality attributes in a single workflow and via rapid analysis.

Rapid, simple, and cost-effective plaque assay for murine norovirus using microcrystalline cellulose

Murine norovirus (MNV) is used widely as a practical alternative to human norovirus (HuNoV). Plaque-forming assays for MNV are important for developing therapeutic agents against HuNoV infections. Although agarose-overlay MNV assays have been reported, recent improvements in cellulose derivatives suggest that they could be optimized further, particularly with respect to improving the overlay material. To determine which overlay material is optimal for the MNV plaque assay, we compared four typical cellulose derivatives [microcrystalline cellulose (MCC), hydroxyethyl cellulose (HEC), hydroxypropyl methylcellulose (HPMC), and carboxymethyl cellulose (CMC)] with conventional agarose. We found that 3.5% (w/v) MCC-containing medium provided clear round-shaped plaques on RAW 264.7 cells 1 day after inoculation; the visibility of plaques was comparable with that of the original agarose-overlay assay. Removing residual MCC powder from the MCC-overlay assay before fixing was important for obtaining distinct plaques that are clearly countable. Finally, after calculating the plaque diameter as a percentage of well diameter, we found that 12- and 24-well plates were better than other plates for accurate plaque counting. The MCC-based MNV plaque assay is cost-effective and rapid, and produces plaques that are easy to count. Accurate virus quantification using this optimized plaque assay will enable reliable estimation of norovirus titers.

A prospective observational study to evaluate effect of heat inactivation on ABO titers performed by column agglutination technology and conventional tube technique.

When determining ABO antibody titers, immunoglobulin G (IgG) antibodies can be masked by immunoglobulin M (IgM) antibodies. Hence, the measurement of actual concentration of IgG requires methods like heat inactivation (HI) of plasma. This study was aimed at determining the effects of HI on IgM and IgG titers performed by conventional tube technique (CTT) and column agglutination technique (CAT). This was a prospective, observational study conducted from October 2019 to March 2020. All consecutive A, B, and O group donors who gave consent for participation were included. All samples were consecutively tested by CTT and CAT, before and after HI (pCTT, pCAT). A total of 300 donors were included. IgG titers were found to be more than IgM titers. For group O, IgG titer results were higher for both anti-A and anti-B compared to group A and B. For group A, B, and O, pretreatment results were higher than posttreatment IgG titer results. Median anti-A titers were similar to median anti-B titers across all categories. Median IgM and IgG titers were higher for group O individuals than nongroup O individuals. There was reduction in IgG and IgM titers after HI of plasma. One log reduction in median titers was observed when ABO titers were performed by CAT and CTT. There is one log difference between median antibody titers estimated using heat inactivated and nonheat inactivated plasma. The use of HI for ABO isoagglutinin titer estimation can be considered in low resource settings.

Comparative Evaluation of Titer Estimation of ABO Isoagglutinins using Three Different Methods

Background and Aims: While automation has the advantage of objectivity, ease of use, and reproducibility with well-defined end points of agglutination reaction, standardization of these methods has still not been possible. The aim of the present study was to compare results obtained by manual conventional test tube technique (CTT) with semi-automated, column agglutination technique (CAT), and fully automated hemagglutination/solid-phase red cell adherence (HA/SPRCA) by calculating and comparing correlation and median immunoglobulin M (IgM) and immunoglobulin G (IgG) titer. Materials and Methods: This was a prospective, observational study conducted from October 2019 to March 2020. All consecutive A, B, and O group donors who consented to participate were included in the study. All samples were consecutively tested by CTT, CAT, and HA/SPRCA. Results: A total of 300 (100 each from A, B, and O blood groups) donors were included. IgG titers were higher than IgM titers in most group O individuals. This difference was more evident with the use of CAT. The correlation between CTT and CAT was found to be strong, whereas the correlation between CTT and HA/SPRCA was found to be variable for anti-A and anti-B IgG and IgM titers. When measured by CTT and CAT, median anti-A and anti-B titer results for group O individuals were similar. Anti-A and anti-B median titer results obtained by CTT and CAT for group A and B individuals were also similar. Conclusion: Semi-automated method (CAT) shows higher ability in detecting ABO isoagglutinins than the manual method (CTT) and automated method (HA/SPRCA). These methods cannot be used interchangeably.

Longitudinal neutralization activities on authentic Omicron variant provided by three doses of BBIBP-CorV vaccination during one year.

The majority of people in China have been immunized with the inactivated viral vaccine BBIBP-CorV. The emergence of the Omicron variant raised the concerns about protection efficacy of the inactivated viral vaccine in China. However, longitudinal neutralization data describing protection efficacy against Omicron variant is still lacking. Here we present one-year longitudinal neutralization data of BBIBP-CorV on authentic Omicron, Delta, and wild-type strains using 224 sera collected from 14 volunteers who have finished three doses BBIBP-CorV. The sera were also subjected for monitoring the SARS-CoV-2 specific IgG, IgA, and IgM responses on protein and peptide microarrays. The neutralization titers showed different protection efficacies against the three strains. By incorporating IgG and IgA signals of proteins and Spike protein derived peptide on microarray, panels as potential surrogate biomarkers for rapid estimation of neutralization titers were established. These data support the necessity of the 3rd dose of BBIBP-CorV vaccination. After further validation and assay development, the panels could be used for reliable, convenient and fast evaluation of the efficacy of vaccination.