Modelling the concentration of anti-SARS-CoV-2 immunoglobulin G in intravenous immunoglobulin product batches.

BACKGROUND: Plasma-derived intravenous immunoglobulin (IVIg) products contain a dynamic spectrum of immunoglobulin (Ig) G reactivities reflective of the donor population from which they are derived. We sought to model the concentration of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) IgG which could be expected in future plasma pool and final-product batches of CSL Behring's immunoglobulin product Privigen. STUDY DESIGN AND METHODS: Data was extracted from accessible databases, including the incidence of coronavirus disease 2019 and SARS-CoV-2 vaccination status, antibody titre in convalescent and vaccinated groups and antibody half-life. Together, these parameters were used to create an integrated mathematical model that could be used to predict anti-SARS-CoV-2 antibody levels in future IVIg preparations. RESULTS: We predict that anti-SARS-CoV-2 IgG concentration will peak in batches produced in mid-October 2021, containing levels in the vicinity of 190-fold that of the mean convalescent (unvaccinated) plasma concentration. An elevated concentration (approximately 35-fold convalescent plasma) is anticipated to be retained in batches produced well into 2022. Measurement of several Privigen batches using the Phadia™ EliA™ SARS-CoV-2-Sp1 IgG binding assay confirmed the early phase of this model. CONCLUSION: The work presented in this paper may have important implications for physicians and patients who use Privigen for indicated diseases.

Neutralising Antibodies against Enterovirus and Parechovirus in IVIG Reflect General Circulation: A Tool for Sero-Surveillance.

Non-polio enteroviruses (NPEV) and parechoviruses (PeV) are widespread pathogens that cause significant morbidity. Surveillance is based on culturing or genotyping of virus strains found in clinical samples. Sero-surveillance, by measuring neutralising antibodies (nAb) through virus neutralisation assays (VNA), could provide additional information as it offers a more comprehensive overview of exposure to circulating types in the general population. In our study we evaluated Intravenous immunoglobulins (IVIG) to generate sero-surveillance data. We performed VNA of nineteen NPEV and PeV with Dutch IVIG batches from two different time points (2010 and 2017) and an IVIG batch from Vietnam (2011). We compared our findings with geno- and sero-surveillance data and evaluated changes over time and between the two countries. Our findings show a good correlation with what is known from geno-surveillance data. The highest nAb titres were found against strains from Enterovirus B, while we did not observe nAb titres against strains belonging to Enterovirus C. In conclusion, we demonstrated that sero-surveillance by means of IVIG can be used to obtain insight into circulation of EV and PeV genotypes. This is of particular interest for public health, to evaluate changes over time and population susceptibility to emerging genotypes.

Characterization of antibodies in human immunoglobulin products from different regions worldwide.

AIM: The antibody levels against a broad spectrum of pathogens were assessed in commercial intravenous immunoglobulin (IVIG) manufactured from pooled plasma obtained from different global regions. METHODS: Twenty-four IVIG commercial lots from eight manufacturers corresponding to 12 brands were analyzed. The plasma was collected in 10 countries/regions. Depending on each pathogen, antibody levels were measured using specific commercial IgG-specific enzyme immunoassay kits or by cell culture neutralization test and guinea pig skin neutralization test. A principal component analysis was performed. RESULTS: For polio and diphtheria (reference markers of the US authorities), all IVIGs had relevant titers in accordance with reference levels. IVIGs from Canada, Australia, and the USA were positive for titers against globally distributed pathogens or those under vaccination programs in the developed world (parainfluenza, Epstein-Barr, varicella-zoster, influenza B, parvovirus B19, and measles viruses). IVIG from Taiwan and Hong Kong showed low antibody titers for these pathogens but high titers for Pseudomonas aeruginosa. IVIG from India had high titers for pathogens frequently found in developing countries (West Nile, dengue, chikungunya, and hepatitis E viruses and Streptococcus pneumoniae). IVIGs from Argentina, Spain, Israel, and Czechia showed intermediate antibody concentrations. CONCLUSION: The antibody profile in IVIG was greatly influenced by regional characteristics including climate, vaccination programs, and the prevalence of pathogens in the different countries and regions.

Machine learning and statistical analyses for extracting and characterizing "fingerprints" of antibody aggregation at container interfaces from flow microscopy images.

Therapeutic proteins are exposed to numerous stresses during their manufacture, shipping, storage and administration to patients, causing them to aggregate and form particles through a variety of different mechanisms. These varied mechanisms generate particle populations with characteristic morphologies, creating "fingerprints" that are reflected in images recorded using flow imaging microscopy. Particle population fingerprints in test samples can be extracted and compared against those of particles produced under baseline conditions using an algorithm that combines machine learning tools such as convolutional neural networks with statistical tools such as nonparametric density estimation and Rosenblatt transform-based goodness-of-fit hypothesis testing. This analysis provides a quantitative method with user-specified type 1 error rates to determine whether the mechanisms that produce particles in test samples differ from particle formation mechanisms operative under baseline conditions. As a demonstration, this algorithm was used to compare particles within intravenous immunoglobulin formulations that were exposed to freeze-thawing and shaking stresses within a variety of different containers. This analysis revealed that seemingly subtle differences in containers (e.g., glass vials from different manufacturers) generated distinguishable particle populations after the stresses were applied. This algorithm can be used to assess the impact of process and formulation changes on aggregation-related product instabilities.

Anti-Neuronal Antibodies Within the IVIg Preparations: Importance in Clinical Practice.

Our study objective was testing for anti-neuronal autoantibodies within commercially available intravenous immunoglobulin (IVIg) preparations. Sixteen samples from 5 different commercially available IVIg preparations were tested with cell-based assays (CBA) and enzyme-linked immunosorbent assay (ELISA) to detect and characterize common neuronal autoantibodies, and with immunohistochemistry on teased fibers from mouse sciatic nerve and on mouse brain sections to screen for nodal and not yet identified neuronal antigens. In 15/16 IVIg preparations, anti-GAD antibodies were detected in titers ranging from 40 to 1507 IU/mL, as typically seen in type 1 diabetes, but not in the range (> 2000 IU/mL) seen in GAD-positive neurological patients. None of the preparations was however positive with anti-GAD CBA. Antibodies to AQP4 were also detected by ELISA in 15/16 IVIg preparations with titers comparable to those seen in AQP4-seropositive NMO patients; with CBA, however, all IVIg samples were AQP4-negative. IVIg preparations contained IgG-anti-MAG antibodies by ELISA at statistically significant higher titers compared to controls. Two of the 16 IVIg samples were positive for human 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) antibodies. All IVIg preparations were negative for antibodies to MOG, NMDAR, anti-nodal, and other neuronal-specific proteins. IVIg preparations contain antibodies against GAD and AQP4 in titers comparable to those seen in autoimmune patients when tested by ELISA, but not by CBA or tissue immunohistochemistry, suggesting that the autoantibodies within the IVIg are against linear rather than structural epitopes, as part of the natural antibody immune repertoire. The information is clinically important for diagnosis when testing patients' sera after they have received therapy with IVIg to avoid false interpretation.

High level antibodies to TORCH in the IVIG preparation from Taiwanese.

BACKGROUND: Congenital TORCH (toxoplasmosis, other viruses [varicella-zoster virus, VZV, etc.], rubella, cytomegalovirus [CMV], Herpes simplex virus [HSV]) infections are major causes of prenatal, perinatal, and postnatal morbidity and mortality. Although treatment or prevention strategies are available for these pathogens, all drugs may not be safe during the pregnancy. The aim of this study is to measure the antibodies (Abs) concentration in the intravenous immunoglobulin (IVIG) preparation to evaluate the therapeutic potential for TORCH infection. METHODS: We tested the only one commercial IVIG preparation from Taiwanese for the presence of Abs against Toxoplasma gondii, VZV, Epstein-Barr virus (EBV), measles, mumps, rubella, CMV, HSV type 1 (HSV-1), and HSV type 2 (HSV-2) by using enzyme-linked immunosorbent assay or chemiluminescent microparticle immunoassay. RESULTS: In our study, the median level (range) of anti-CMV immunoglobulin G (IgG) is > 250 (All > 250) (arbitrary unit, AU)/mL, anti-EBV > 200 (All > 200) (relative unit, RU)/mL, anti-HSV > 200 (152.75 to >200) RU/mL, anti-VZV > 5000 (All > 5000) IU/L, anti-measles > 5000 (All > 5000) IU/L, anti-mumps > 200 (156.5 to > 200) RU/mL, anti-rubella 209.8 IU/mL (192.7 to 238.5), and anti-Toxoplasma is 14.05 (12.3 to 16) IU/mL. There was not any immunoglobulin M (IgM) against HSV, VZV, mumps, measles, rubella, CMV, EBV, and Toxoplasma in the "Taiwan Blood Services Foundation" IVIG preparations. CONCLUSION: There was high activity against T. gondii, VZV, EBV, measles, mumps, rubella, CMV, HSV-1, and HSV-2 in all IVIG batches. Further investigation is warranted to confirm the efficacy of IVIG from Taiwanese for congenital TORCH infections.

Measles virus neutralizing antibodies in immunoglobulin lots produced from plasma collected in Europe or the United States.

Vaccination against measles has reduced disease, although measles virus antibody (MVAb) levels are lower after vaccination than natural infection. Immunoglobulin (IG) preparations thus contain decreasing MVAb titers. US IG lot release requires a minimum titer of MVAb, yet equivalent information is not available for other geographies. Using a measles virus neutralization assay, IG fractionated from US or EU plasma is shown to contain similar levels of MVAb always above US regulatory requirements, supportive of equivalent protection against MV infection. Thus, the dosage for post-exposure prophylaxis in the EU could be aligned with the US FDA's treatment recommendations.

Rapid Quantification of Protein Particles in High-Concentration Antibody Formulations.

Current technologies for monitoring the subvisible particles that may be generated during fill-finish operations for protein formulations are cumbersome. Measurement times are generally too long for real-time analysis, and the high protein concentrations that are characteristic of many antibody products interfere with common optical techniques for particle analysis. To rapidly monitor protein particle levels in high-concentration protein solutions, we developed a fluorescence-based method that uses extrinsic fluorescent dyes such as 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid that are sensitive to the presence of aggregated protein. To test the method, antibody formulations containing various concentrations of protein particles were generated by application of various mechanical and freeze-thaw stresses. After addition of fluorescent dyes, fluorescence intensities were measured and compared to fluorescence intensities in particle-free formulations. The differences in fluorescence intensities were linearly proportional to protein particle levels, which for calibration purposes were measured offline by fluid imaging microscopy and protein assays. Protein particle levels could be measured without requiring sample dilution, even in high-concentration (e.g., 40 mg/mL) antibody formulations.

Analysis of sialic acid levels in Chinese intravenous immunoglobulins by high-performance liquid chromatography with fluorescence detection.

Intravenous immunoglobulin (IVIg) is increasingly used for the treatment of autoimmune and systemic inflammatory diseases with both licensed and off-label indications. Recent studies indicated that IVIg-mediated immunomodulation and anti-inflammation are closely associated with the IgG sialylation, especially with IgG crystallizable fragment (Fc) sialylation. The sialic acid levels of the IgG molecules and Fc fragments in 12 IVIg preparations from six Chinese manufacturers were evaluated. The Fc fragments were derived from the papain digestion of IVIg, followed by affinity and size exclusion chromatography. The sialic acid levels in Fc fragments and IVIg preparations were determined by high-performance liquid chromatography with fluorescence detection, after the sialic acid residues were released from the proteins. The results showed that the sialic acid levels in Chinese IVIg preparations ranged from 0.875 (mol/mol IgG) to 1.085 (mol/mol IgG), and the sialic acid levels in Fc fragments were from 0.321 (mol/mol Fc) to 0.361 (mol/mol Fc). Furthermore, the sialic acid levels of IVIg preparations and Fc fragments from different Chinese manufactures were significantly different. These findings will contribute to an increased understanding of Chinese IVIg preparations and the relationship between the sialic acid levels in IVIg preparations and their clinical efficacy in future clinical studies.

Intravenous immunoglobulin therapy among pediatric patients: labeled and off-labeled indications

ABSTRACT This study was designed to evaluate utilization patterns and clinical outcome of intravenous immunoglobulin (IVIG) therapy among pediatric patients in a tertiary hospital. Demographic data, IVIG prescribed, and clinical outcome were retrospectively reviewed from the pharmacy dispensing data and patient medical records between 2007 and 2014. One hundred and fifteen instances of IVIG administration to 108 pediatric patients were recorded. A total of 61 cases (53%) and 54 cases (47%) of the IVIG administered were for labeled and off-labeled indications, respectively. Age, weight, specialty, total IVIG usage, length of hospital stays, and mortality rate were found to be significantly associated with the indication being labeled or off-labeled (p<0.05). However, there was no significant difference in terms of adverse reactions between labeled and off-labeled indications (p>0.05). Guidelines should be developed and implemented for rational and evidence-based use of IVIG to avoid unnecessary wastage.

Antibodies against Hepatitis A and Hepatitis B Virus in Intravenous Immunoglobulin Products.

The worldwide seroprevalence of hepatitis A virus (HAV) and hepatitis B virus (HBV) has changed over the last two decades, indicating a declining incidence of HAV and HBV infections. Therefore, vaccinations against HAV and HBV are recommended for unimmunized people before traveling to an endemic area. Unfortunately, primary antibody deficiency (PAD) patients can only obtain humoral immunity through intravenous immunoglobulin G (IVIG) replacement and not from vaccination because of a defect in antibody production. However, few studies have analyzed the titers of antibodies against HAV or HBV in IVIG products. In this study, the titers of anti-HAV and anti-HBs antibodies were measured in nineteen lots of IVIG products from five manufacturers from three countries (A, B from Korea; C, D from Japan; and E from the USA), and trough titers in plasma were estimated. Concentrations of anti-HAV antibody ranged from 1,888-8,927 mIU/mL and estimated trough titers exceeded the minimal protective value in all evaluated IVIG products. Concentrations of anti-HBs antibody ranged from 438-965 mIU/mL in products A and B and were 157, 123, and 1,945 mIU/mL in products C, D, and E, respectively. Estimated trough titers in products A, B, and E exceeded the minimal protective value but those in products C and D did not reach this threshold. These data demonstrated that available IVIG products generally provide sufficient antibodies against HAV and HBV to protect patients with PAD, although the trough concentrations of anti-HBs antibody in two IVIG products did not reach the minimum protective value.

Eccema dishidrótico secundario a la infusión de inmunoglobulinas intravenosas: presentación de 2 casos / Dyshydrotic Eczema Secondary to Intravenous Immunoglobulin Infusion: A Report of 2 Cases

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Phenotyping and follow up of forty-seven Iranian patients with common variable immunodeficiency

BACKGROUND: Common variable immune deficiency (CVID) is a heterogeneous syndrome with a wide variety of signs and symptoms. This study describes the phenotyping and survival of the CVID patients in the allergy and clinical immunology department of Rasol-E-Akram Hospital of Iran University of Medical Sciences in Tehran. METHOD: We retrospectively reviewed hospital files of CVID patients in our department until January 2014. All patients were diagnosed with standard diagnostic criteria of CVID, treated and visited monthly, during the follow-up period. We divided the patients into four phenotypes; infection only, cytopenia, polyclonal lymphocytic infiltration and unexplained enteropathy. The immunologic, demographic and clinical findings in different phenotypes were analysed. RESULTS: The study included 47 CVID patients with mean age at onset of symptoms and diagnosis of 11.2 and 20.2 years, respectively. Phenotyping of our patients was: only infection (62%), cytopenia (26%) and PLI (19%) and 94% of cases had only one phenotype. We did not find a significant relation between the clinical phenotypes and immunologic or demographic data. Rate of parental consanguinity in our cases was 47%. Parental consanguinity was related to lower age at onset, lower age at diagnosis and higher baseline IgG levels. Patients with malignancy and autoimmunity had significantly higher age at onset. Our patients were followed-up for 6.9 years and the mortality rate during this time was 6%. CONCLUSIONS: Parental consanguinity and age at onset of CVID symptoms may have important roles in CVID manifestations

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Neutralization of Enterovirus D68 isolated from the 2014 US outbreak by commercial intravenous immune globulin products.

BACKGROUND: In 2014, an outbreak of Enterovirus D68 (EV-D68) was recorded as the largest in the US with cases confirmed in 49 states. Intravenous immune globulin (IVIG) has been used to treat enterovirus infections in neonates and is an accepted replacement therapy for immunodeficient patients. OBJECTIVES: This study aimed to detect the presence of neutralizing antibodies to EV-D68 viruses from the 2014 outbreak in commercially available IVIG products. STUDY DESIGN: Commercially available lots of IVIG preparations were obtained from five different manufacturers (2-10 preparations per manufacturer) and tested for neutralizing antibodies against the prototype EV-D68 virus and three EV-D68 isolates representing strains circulating during the 2014 outbreak. RESULTS: All lots of IVIG tested were positive for EV-D68 neutralizing antibodies, with high titers ranging from 9.5log2 to 17.5log2, and with comparable median titers to all four EV-D68 viruses. CONCLUSIONS AND DISCUSSION: Amino acid sequence differences in the regions of the predicted antigenic sites on the viral capsid may explain some of the differences in neutralization among the different strains. The neutralization titers suggests that the 2014 outbreak EV-D68 viruses share some antigenic sites with the prototype virus and also present some unique antigenic sites distinct from the prototype. However, the commercial IVIG lots tested all contained high levels of neutralizing antibodies against EV-D68.

Quantitative laser diffraction method for the assessment of protein subvisible particles.

Laser diffraction (LD) has been recognized as a method for estimating particle size distribution. Here, a recently developed quantitative LD (qLD) system, which is an LD method with extensive deconvolution analysis, was employed for the quantitative assessment of protein particles sizes, especially aimed at the quantification of 0.2-10 µm diameter subvisible particles (SVPs). The qLD accurately estimated concentration distributions for silica beads with diameters ranging from 0.2 to 10 µm that have refractive indices similar to that of protein particles. The linearity of concentration for micrometer-diameter silica beads was confirmed in the presence of a fixed concentration of submicrometer diameter beads. Similarly, submicrometer-diameter silica beads could be quantified in the presence of micrometer-diameter beads. Subsequently, stir- and heat-stressed intravenous immunoglobulins were evaluated by using the qLD, in which the refractive index of protein particles that was determined experimentally was used in the deconvolution analysis. The results showed that the concentration distributions of protein particles in SVP size range differ for the two stresses. The number concentration of the protein particles estimated using the qLD agreed well with that obtained using flow microscopy. This work demonstrates that qLD can be used for quantitative estimation of protein aggregates in SVP size range.

Anti-A and anti-B haemagglutinin levels in intravenous immunoglobulins: are they on the rise? A comparison of four different analysis methods and six products.

Recent reports of severe haemolytic reactions upon high dose treatment with new generation intravenous immunoglobulins (IVIGs) prompted us to examine the anti-A and anti-B haemagglutinin content of these therapeutics. We compared four different test methods, namely the indirect and direct haemagglutination test as described in the European Pharmacopoiea (Ph. Eur.) and two commercial gelcard systems with the aim to define the most reliable method for a large-scale comparison of different IVIG products. Absolute titres varied when the same samples were analyzed by the four methods, while the relative ranking of six different IVIG preparations representing different manufacturing classes was identical. New generation IVIGs showed 1-2 titre steps higher anti-A titres than the older products. Haemagglutinin titres of all 48 IVIG batches analyzed were within the current Ph. Eur. specification of ≤1:64 when tested by the official pharmacopoeial method. Based on efficiency, reliability and lower costs, the direct gelcard method could be a valid alternative to the official Ph. Eur. method to serve as a limit test. However, due to the highest intermediate precision, the official Ph. Eur. method seems to be most suitable to compare haemagglutinin titres of different IVIG products.

Flebogamma(®) DIF (intravenous immunoglobulin) purification process effectively eliminates procoagulant activities.

BACKGROUND: Studies have demonstrated that traces of activated factor XI (FXIa) present in specific brands of intravenous immunoglobulin (IVIG) concentrates may pose a thrombogenic risk. AIM: To characterize procoagulant activity during fractionation and the elimination capacity of the Flebogamma(®) DIF (Grifols' IVIG) manufacturing process. METHODS: Flebogamma(®) DIF fractionation steps included cryoprecipitate supernatant (Cryo/S), Fraction (Fr) I supernatant, and Fr II + III suspension. Purification steps included ultrafiltrate I, acid treatment, and pasteurization. Samples were assessed for total protein, IgG, and procoagulant activation markers. RESULTS: Cryo/S showed no procoagulant activity for prekallikrein activator (PKA), kallikrein-like, and non-activated partial thromboplastin time (NaPTT) with normal (-PPP) or FXI-deficient (-FXI) platelet poor plasma. Thrombin generation test (TGT)-PPP and TGT-FXI were <83-148 and <53-197 nM thrombin, respectively. Shortened NaPTTs (100-296 s), high PKA (51-119 IU/mL), kallikrein-like activities (0.043-0.075 ΔAU/min), positive TGTs (98-298 nM), and FXIa (9.5-14.0 ng/mL) were detected in Fr II + III. After pasteurization, no residual evidence of any procoagulant activity marker was observed, including the final IVIG concentrate at 5% or 10% protein. Results were similar in Fr II + III from different IVIG manufacturing facilities. CONCLUSIONS: The Flebogamma(®) DIF production process is capable of eliminating procoagulant activity because of its purification steps.

Dimeric IVIG contains natural anti-Siglec-9 autoantibodies and their anti-idiotypes.

BACKGROUND: Intravenous immunoglobulin (IVIG) preparations are increasingly used for the treatment of autoimmune and chronic inflammatory diseases. Naturally occurring autoantibodies against Siglec-9 and Fas are thought to contribute to the anti-inflammatory effects of IVIG via cell death regulation of leukocytes and tissue cells. Dimeric IVIG fractions are suspected to contain idiotypic (Id)-anti-idiotypic complexes of antibodies, which might also include anti-Siglec-9 and anti-Fas autoantibodies. METHODS: Dimeric IVIG fractions were separated from monomeric IVIG by size-exclusion chromatography and remonomerized by low pH treatment. Binding studies of total, monomeric, and dimeric IVIG were performed using surface plasmon resonance and flow cytometry on primary human neutrophils. RESULTS: Anti-Siglec-9 and anti-Fas autoantibodies were contained in both monomeric and dimeric IVIG fractions, but anti-Siglec-9 antibodies were highly enriched in dimeric IVIG. The propensity to engage in dimer formation was paratope dependent. IVIG binding to Siglec-9 was specific and sialylation independent. Interestingly, we detected anti-idiotypic antibodies (anti-Ids) against anti-Siglec-9 autoantibodies in dimeric, but not in monomeric fractions of IVIG. CONCLUSIONS: Our study supports the concept that idiotype-anti-idiotype (Id-anti-Id) interactions contribute to the dimer formation in IVIG preparations. To our knowledge, this is the first description of Id-anti-Id dimers of death receptor-specific antibodies in IVIG. Such Id-anti-Id interactions might determine the activity of immunomodulatory antibodies present both in IVIG and the patient.

Rheological and syringeability properties of highly concentrated human polyclonal immunoglobulin solutions.

This study of highly concentrated polyvalent immunoglobulin solutions, IgG, aimed at analyzing the relationships between protein concentration and aggregation on the one hand and viscosity on the other hand. Viscosity variations as a function of IgG concentration showed two well-defined behaviours: a Newtonian behaviour for low-concentrated solutions and a shear-thinning behaviour for highly concentrated ones. The viscosity data fitted very well with the Mooney model, suggesting the absence of intermolecular interactions in the IgG solutions that behaved like a non-interacting suspension of hard particles. The polyclonal nature of IgG seems to prevent intermolecular interaction. The shape factor, determined from Mooney fitting, revealed a non-spherical shape of the polyclonal IgG molecules. The rheological properties were also correlated with the injection force (F) through hypodermic needles by syringeability tests. Here, F was mainly affected by three parameters: the solution viscosity, the injection flow rate, and the needle characteristics. In fact, syringeability tests showed that F increased with IgG concentration and flow rate and decreased with the internal diameter of the needle. A zone for optimal injection conditions was then identified taking into account the different affecting parameters and mainly a maximum force for manual injection, which was fixed at 30N.

Measurement of anti-Abeta1-42 antibodies in intravenous immunoglobulin with indirect ELISA: the problem of nonspecific binding.

Improvement in cognitive scores in patients with Alzheimer's disease (AD) has been reported in two trials in which intravenous immunoglobulin (IvIg) preparations were administered. IvIg's benefits in AD patients have been suggested to be due to antibodies to amyloid-beta (Abeta). Our previous study using indirect enzyme-linked immunosorbent assay (ELISA) indicated that much of IvIg's apparent binding to Abeta1-42 is nonspecific; it is detectable even when IvIg is incubated on "specificity controls" (bovine serum albumin [BSA] and Abeta reverse sequence Abeta42-1) rather than Abeta1-42. The objective of this study was to evaluate procedures that might reduce this nonspecific binding. The IvIg preparation used was Gamunex (Talecris Biotherapeutics). Multiple blocking agents were evaluated, but even the most effective blocker only reduced nonspecific binding by 48%. Dissociating Gamunex's antibody-antigen complexes had no effect on specific binding when Abeta42-1 was used as the specificity control, although it increased this binding when BSA was the specificity control. Decreasing Gamunex's dilution from 1:1,500 to 1:500 resulted in a slight (7.4%) but significant (p=0.027) increase in specific binding. Using a sandwich ELISA to measure Gamunex's anti-Abeta antibodies resulted in even less specific binding to Abeta1-42 than with the indirect ELISA. Despite Gamunex's low percentage of specific binding to Abeta1-42, it inhibited Abeta oligomer formation. We conclude that, when anti-Abeta antibodies in IvIg are measured by indirect ELISA, extensive nonspecific binding occurs despite procedures taken to prevent it. This must be subtracted from total binding to accurately measure specific anti-Abeta antibody concentrations.