Total Antibody Binding Research Articles

SARS-CoV-2 IgG antibodies in COVID-19 convalescent plasma and conventional plasma units.

The Association for the Advancement of Blood and Biotherapies guidelines recommend the use of high-titer COVID-19 convalescent plasma (CCP) for patients with SARS-CoV-2 at high risk of disease progression, including those who are immunocompromised. We hypothesized that conventional plasma units have comparable neutralizing antibody levels to CCP. Conventional plasma and CCP units were obtained from blood suppliers. Quantitatively measured antibodies to SARS-CoV-2 were assessed using the MesoScale Discovery multiplex electrochemiluminescence immunoassay. Binding antibody distributions were compared with Wilcoxon rank-sum tests. SARS-CoV-2 neutralizing antibodies were analyzed using the GeneScript ELISA-based neutralization assay. The proportion of conventional and CCP units with a percent signal inhibition of ≥80% (as defined by the United States Food and Drug Administration for CCP in 2021) and exact binomial confidence intervals (CIs) were calculated. Among 218 conventional plasma units and 74 CCP units collected between September 2023 and July 2024, the distribution of total antibody binding levels largely overlapped between conventional plasma and CCP, though statistically significant differences in median nucleocapsid and spike Omicron variant concentrations were observed. Median percent signal neutralization was 97.5% (range 3.4%-98.6%) among conventional plasma units and 97.7% (range 95.4%-98.6%) among CCP units. For conventional plasma, 95.0% (95% CI = 91.2%-97.5%) met the neutralization antibody threshold for high-titer CCP. For CCP, 100% (95% CI = 95.1%-100.0%) met the neutralization threshold for high-titer CCP. Conventional plasma units demonstrate similar median antibody concentration to CCP units. In countries or regions where licensed CCP is unavailable and titers are unknown, transfusion of multiple conventional plasma units may be of clinical utility.

Characterization of human preformed antibodies to non-SLA xenoantigens on RBC from genetically-engineering pigs

Abstract BACKGROUND Genetic engineering pig models of pig-to-human xenotransplantation may overcome a growing shortage of available solid organs. Development of pigs deficient in galactose α1,3 galactose and N-glycolylneuraminic acid have significantly improved cell survival when challenged by human antibody and complement in vitro. The b4GalNT2 glycosyltransferase may also synthesize xenoantigens in pigs. In this study, we used RBC, as a cell model, to assess human antibody-mediated response to non-SLA xenoantigens of pigs deficient in these carbohydrate-modifying genes. MATHODS AND MATERIALS RBC were isolated from wild type pigs and pigs deficient in GGTA1/CMAH knockout (DKO) and GGTA1/CMAH/β4GalNT2 knockout (TKO). Human ABO sera were purchased from Valley Biomedical. Human ABO RBC were isolated from healthy people. Total antibody binding was assessed by hemagglutination assay; human IgG or IgM binding to RBC was characterized by flow cytometry; opsonized RBC phagocytosis by THP-1 macrophages was analyzed using confocal microscope. RESULTS Phenotype of TKO RBC was characterized by flow cytometry. Silencing the three genes reduced hemagglutination levels of TKO RBC compared with that of DKO RBC by all three human blood-type A, O, and AB sera but except blood-type B sera, and decreased both of IgG and IgM binding to TKO RBC compared with DKO RBC. Confocal image data showed that phagocytosis of opsonized TKO RBC was lower than that of opsonized DKO RBC by THP-1 cells. DISCUSSION Non-SLA xenoantigenicity of pig RBC can be disrupted using gene engineering tools, which greatly reduced the humoral barrier to xenotransplantation

The Editor takes a closer look at some of this month's articles

Clinical & Experimental AllergyVolume 45, Issue 10 p. 1477-1477 Editor's ChoiceFree Access The Editor takes a closer look at some of this month's articles First published: 15 September 2015 https://doi.org/10.1111/cea.12629AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Viral immunity: key differences between the gut and lung in asthma Rhinoviruses play an important part in asthma exacerbations, and viral antibody titres against these members of the enterovirus genus are higher in asthmatic children than healthy controls. Iwasaki et al. (pp. 1523–1530) have asked whether this was also the case for the gut tropic enteroviruses echovirus 30, which causes an acute febrile illness in children, and the Sabin 1 poliovirus. They found that IgG titres against echovirus, but not poliovirus, were lower in asthmatic children compared to matched healthy controls. Whether this was due to a protective effect of echovirus infection on the development of asthma or a diminished antibody immune response to echovirus in asthmatics is not clear. However, the study emphasizes the potential importance of differential, organ-specific, mucosal immune responses in the development of asthma. Jua Iwasaki Total (a) and species-specific (b) IgG1 antibody binding to echovirus 30 VP1 and human Sabin 1 poliovirus VP1 antigens in Emergency Department (ED) asthmatics and nonasthmatic children. [See Figure 1b in J. Iwasaki et al. (pp. 1523–1530)]. Predicting anaphylaxis with bee venom immunotherapy: a role for the basophil test? Measuring the sensitivity of basophil activation to in vitro allergen challenge, either by mediator release or changes in receptor expression, has been a test without an established clinical role for a long time. Korošec et al. (pp. 1579–1589) have investigated which factors including basophil sensitivity predict allergic reactions to ultra-rush immunotherapy for bee venom anaphylaxis. They studied a range of clinical and immunological measurements in 93 patients, 13 of whom had severe systemic reactions (5 had to have their IT stopped). Pre-treatment basophil sensitivity was the only independent predictor of both a severe systemic reaction and having to stop treatment. A useful test therefore for groups which undertake this approach to bee venom IT. Peter Korosec A bee. (Photo credit: James Petts, London, England; Wikimedia Commons). Prostaglandin E2: how can we harness its protective properties? It has been known for many years that prostaglandin E2 has bronchoprotective properties on both airway inflammatory cells and airway smooth muscle contraction, but so far this potent inhibitory pathway has not been exploited in drug development 1. In part, this is because the exact mechanisms involved remain unclear. Further clarity is brought to the subject by Torres et al. (pp. 1590–1600) who have investigated the effects of PGE2 in the asthma allergen challenge model using two strains of mice; BALB/c which is a mast cell-dependent model and C57BL/6 mice which is mast cell independent. They convincingly demonstrate that the inhibitory properties of PGE2 on inflammation and airway hyperresponsiveness are mast cell dependent and mediated through the mast cell E prostanoid 2 (EP2) receptor. There must be a therapeutic target somewhere in this pathway. Rosa Torres and Mariona Serra The molecular structure of Prostaglandin E2. (Photo credit: NEUROtiker; Wikimedia Commons). Reference 1Pavord ID, Tattersfield AE. Bronchoprotective role for endogenous prostaglandin E2. Lancet 1995; 345: 436– 8. Caption to cover illustration: Immune mechanisms of tolerance and sensitization to foods. [See figure 1 of M. C. Berin (pp. 1483–1496)]. This logo highlights the Editor's choice articles on the cover and the first page of each of the articles. Volume45, Issue10October 2015Pages 1477-1477 ReferencesRelatedInformation

ISCOMATRIX™ adjuvant promotes epitope spreading and antibody affinity maturation of influenza A H7N9 virus like particle vaccine that correlate with virus neutralization in humans

In a previously reported phase I clinical trial, subjects vaccinated with two doses of an unadjuvanted H7N9 virus like particle (VLP) vaccine responded poorly (15.6% seroconversion rates with 45μg hemagglutinin (HA) dose). In contrast, 80.6% of subjects receiving H7N9 VLP vaccine (5μg HA) with ISCOMATRIX™ adjuvant developed hemagglutination-inhibition (HI) responses. To better understand the role of adjuvant, complete antibody epitope repertoires of post-vaccination sera were investigated using Whole Genome Fragment Phage Display Library (GFPDL). In addition, antibody affinity maturation following vaccination was measured against HA1 and HA2 antigenic domains using real time Surface Plasmon Resonance (SPR) based kinetic assays. Unadjuvanted H7N9-VLP vaccine generated primarily antibodies targeting the C-terminus of the HA1 domain, predicted to be mostly buried on the native HA spikes, while adjuvanted VLP vaccine generated antibodies against large epitopes in the HA1 spanning the receptor binding domain (RBD). SPR analysis using a functional H7-HA1 domain demonstrated that sera from adjuvanted H7N9-VLP vaccine induced higher total binding antibodies and significantly higher antibody affinity maturation to HA1 compared to sera from unadjuvanted vaccine. Total antibody binding and affinity to the HA1 (but not HA2) domain correlated with HI and neutralization titers. This study demonstrates that ISCOMATRIX™ adjuvanted vaccine promotes higher quality antibody immune response against avian influenza in naïve humans.

Erythrocytes from GGTA1/CMAH knockout pigs: implications for xenotransfusion and testing in non-human primates.

Pig erythrocytes are potentially useful to solve the worldwide shortage of human blood for transfusion. Domestic pig erythrocytes, however, express antigens that are bound by human preformed antibodies. Advances in genetic engineering have made it possible to rapidly knock out the genes of multiple xenoantigens, namely galactose α1,3 galactose (aGal) and N-glycolylneuraminic acid (Neu5Gc). We have recently targeted the GGTA1 and CMAH genes with zinc finger endonucleases resulting in double knockout pigs that no longer express aGal or Neu5Gc and attract significantly fewer human antibodies. In this study, we characterized erythrocytes from domestic and genetically modified pigs, baboons, chimpanzees, and humans for binding of human and baboon natural antibody, and complement-mediated lysis. Distribution of anti-Neu5Gc IgG and IgM in pooled human AB serum was analyzed by ELISA. Erythrocytes from domestic pigs (Dom), aGal knockout pigs (GGTA1 KO), aGal and Neu5Gc double knockout pigs (GGTA1/CMAH KO), baboons, chimpanzees, and humans were analyzed by flow cytometry for aGal and Neu5Gc expression. In vitro comparative analysis of erythrocytes was conducted with pooled human AB serum and baboon serum. Total antibody binding was accessed by hemagglutination; complement-dependent lysis was measured by hemolytic assay; IgG or IgM binding to erythrocytes was characterized by flow cytometry. The pooled human AB serum contained 0.38 μg/ml anti-Neu5Gc IgG and 0.085 μg/ml anti-Neu5Gc IgM. Both Gal and Neu5Gc were not detectable on GGTA1/CMAH KO erythrocytes. Hemagglutination of GGTA1/CMAH KO erythrocytes with human serum was 3.5-fold lower compared with GGTA1 KO erythrocytes, but 1.6-fold greater when agglutinated with baboon serum. Hemolysis of GGTA1/CMAH KO erythrocytes by human serum (25%) was reduced 9-fold compared with GGTA1 KO erythrocytes, but increased 1.64-fold by baboon serum. Human IgG binding was reduced 27-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but markedly increased 3-fold by baboon serum IgG. Human IgM binding was decreased 227-fold on GGTA1/CMAH KO erythrocytes compared with GGTA1 KO erythrocytes, but enhanced 5-fold by baboon serum IgM. Removal of aGal and Neu5Gc antigens from pig erythrocytes significantly reduced human preformed antibody-mediated cytotoxicity but may have complicated future in vivo analysis by enhancing reactivity from baboons. The creation of the GGTA1/CMAH KO pig has provided the xenotransplantation researcher with organs and cells that attract fewer human antibodies than baboon and our closest primate relative, chimpanzee. These finding suggest that while GGTA1/CMAH KO erythrocytes may be useful for human transfusions, in vivo testing in the baboon may not provide a direct transplantation to the clinic.

Confirmation of the validity of the current characterization of immunochemical reactions by kinetic exclusion assay

Prior observations that questioned the validity of kinetic exclusion assays were based on the mistaken assumption that the assays quantified the fraction of those antibody molecules that had unoccupied binding sites. Instead, the standard KinExA assay quantifies the fraction of total antibody binding sites that are unoccupied, regardless of the number of unoccupied sites on each antibody molecule. Although the standard KinExA analysis assumes that there is only a small probability of antibody-site capture by the affinity matrix, the results of numerical simulations demonstrate the reliability of dissociation constants obtained by the standard KinExA analysis for capture probabilities as high as 30%. This finding further strengthens the potential of kinetic exclusion assays as the procedure of choice for the rapid and accurate characterization of immunochemical reactions that forms part of screening processes in the search for therapeutic antibodies.

A Bio-Material of Polymer Beads Based on Antibody Immobilization via Oxidized Glycochains Linkage for Flow Cytometric Suspension Array

A preparation of a bio-material of polymer beads used for immuno-assay is presented, in which the probe antibody is site-specific immobilized on the polystyrene microbead by the surface hydrazide activation and the oxidized glycochains linkage of antibody. Using adipic dihydrazide, free hydrazide-bearing matrix was acquired on carboxyl polystyrene microbead. An iron (III)-phenanthroline spectrophotometric method was developed to evaluate the activation efficiency. The dependence of probe antibody oxidization on the reaction time and the oxidant concentration was investigated. Also, an alternative strategy of antibody immobilization via traditional COOH/-NH2 linkage was illustrated for a comparison with this site-specific immobilization in the aspects of the total antibody binding amount and the site-specific binding amount. The results showed that the site-specific efficacy using glycochains linkage-based binding was three times better than that using traditional amino-based binding, which indicated a higher sensitivity for application in immuno-assay.

Development and Characterization of a Bead-Based, Multiplex Assay for Estimation of Recent HIV Type 1 Infection

Estimation of HIV-1 incidence is an important public health tool for understanding the status of the epidemic, identifying high-risk populations, and assessing various intervention strategies. Several laboratory-based methods have been developed for distinguishing recent from long-term HIV-1 infection; however, each exhibits some degree of misclassification, particularly among AIDS patients and those taking antiretroviral therapy (ART). To improve upon the limitations associated with measuring responses to a single analyte, we have developed a bead-based, multiplex assay for determination of HIV recent infection based on total antibody binding and antibody avidity to multiple analytes. An HIV-specific, multiplex panel was created by coupling the recombinant HIV-1 proteins p66, gp120, gp160, and gp41 to Bio-Plex COOH microspheres. Longitudinal plasma specimens from recent seroconverters were tested for reactivity to the coupled microspheres using the Bio-Plex 200 System. For each analyte, HIV-specific antibody binding and avidity increased for 1-2 years post-seroconversion, leading to a significant difference in reactivity between recent and long-term specimens. While the potential for misclassification of individuals diagnosed with AIDS or receiving ART appears to be minimal with avidity measures, the impact on total antibody binding was variable, depending on the individual analyte. This bead-based, HIV-specific multiplex assay measures several distinct immune responses in a single assay plate, allowing for sampling of multiple analytes in the determination of recent infection, which could aid in the development of improved statistical methods or algorithms that will more accurately estimate HIV incidence.

Effects of Monensin on Ethanol‐Induced Alterations in Function of Hepatocellular Asialoglycoprotein Receptor Subpopulations

Chronic ethanol consumption is associated with multiple impairments in receptor-mediated endocytosis (RME) in hepatocytes. RME mediated by the asialoglycoprotein receptor seems to be especially impaired by ethanol. In the present study, we determined susceptibility of RME to alterations in ethanol-fed and pair-fed control animals by the addition of a carboxylic ionophore, monensin. Monensin inhibits acidification of prelysosomal vesicular compartments, which results in a decrease in the rate of receptor-ligand dissociation within the cell. Low levels (25 microM) of monensin have been shown to preferentially affect receptor-ligand dissociation of one subset (state 2) of asialoglycoprotein receptor, whereas dissociation in a second subset (state 1 receptors) seems to be relatively unaffected. We examined whether ethanol treatment preferentially affected one or another of these receptor subpopulations. Male Wistar rats were fed a standard ethanol-containing (36% of calories) or control diet for 10 to 14 weeks, and hepatocytes were prepared from the animals. Similar to previous results from our laboratory, surface and total ligand and antibody binding were decreased by 30 to 45% (p < 0.01) in ethanol animals, compared with controls. An ethanol-induced impairment of receptor-ligand dissociation was also apparent in these cells, as shown by increased ratios of bound-to-free ligand during continuous endocytosis. After monensin treatment, surface receptors on both ethanol and control cells showed a similar pattern of redistribution to the cell interior and intracellular inactivation. When kinetics of intracellular receptor-ligand dissociation were examined upon addition of monensin, the bound-to-free ligand ratios in both control and ethanol cells increased dramatically and to an equal extent. These results indicate that, in the ethanol animals, the pattern of sensitivity to monensin is unchanged relative to controls. Thus, the relative proportion of state 1 and state 2 receptor populations do not seem to be affected after long-term feeding, and ethanol may be a perturbant that affects both state 1 and state 2 receptor function.

Automated classification of patients with chronic lymphocytic leukemia and immunocytoma from flow cytometric three-color immunophenotypes.

The goal of this study was the discrimination between chronic lymphocytic leukemia (B-CLL), clinically more aggressive lymphoplasmocytoid immunocytoma (LP-IC) and other low-grade non-Hodgkin's lymphomas (NHL) of the B-cell type by automated analysis of flow cytometric immunophenotypes CD45/14/20, CD4/8/3, kappa/CD19/5, lambda/CD19/5 and CD10/23/19 from peripheral blood and bone marrow aspirate leukocytes using the multiparameter classification program CLASSIF1. The immunophenotype list mode files were exhaustively evaluated by combined lymphocyte, monocyte, and granulocyte (LMG) analysis. The results were introduced into databases and automatically classified in a standardized way. The resulting triple matrix classifiers are laboratory and instrument independent, error tolerant, and robust in the classification of unknown test samples. Practically 100% correct individual patient classification was achievable, and most manually unclassifiable patients were unambiguously classified. It is of interest that the single lambda/CD19/5 antibody triplet provided practically the same information as the full set of the five antibody triplets. This demonstrates that standardized classification can be used to optimize immunophenotype panels. On-line classification of test samples is accessible on the Internet: http://www.biochem.mpg.de/valet/leukaem1.html Immunophenotype panels are usually devised for the detection of the frequency of abnormal cell populations. As shown by computer classification, most the highly discriminant information is, however, not contained in percentage frequency values of cell populations, but rather in total antibody binding, antibody binding ratios, and relative antibody surface density parameters of various lymphocyte, monocyte, and granulocyte cell populations.

Recombinant staphylokinase variants with altered immunoreactivity. III: Species variability of antibody binding patterns.

The "charged cluster-to-alanine" substitution variants SakSTAR(K35A,E38A,K74A,E75A,R77A) and SakSTAR(K74A,E75A,R77A,E80A,D82A), previously identified as SakSTAR.M38 and SakSTAR.M89, respectively, induce less antibody formation in patients than wild-type recombinant staphylokinase (SakSTAR), but their specific activities are reduced by 50%. Therefore, the effect of the reversal of one or more of these substituted amino acids on the ratio of activity to antigenicity was studied. Fourteen mutants with one to four "alanine-to-wild-type" reversals were expressed in Escherichia coli and highly purified (> 95%). In rabbits immunized with wild-type SakSTAR, the combined K35,E38,K74,E75,R77 or K74,E75,R77,E80,E82 epitope accounted for only 30% of antibody absorption from plasma, and no clear immunodominant residue could be identified. In baboons immunized with SakSTAR, the K35,E38 and K74,E75,R77 epitopes or the K74,E75,R77 and E80,D82 epitopes contributed equally to account for 50% of total antibody binding, but no immunodominant residues were apparent. In pooled plasma from patients with peripheral arterial occlusion treated with wild-type SakSTAR, about 40% of the antibodies depended on K74 of epitope K74,E75,R77 for binding, whereas epitopes K35,E38 and E80,D82 had a negligible contribution toward antibody recognition. The recognition pattern by SakSTAR variants of antibodies induced with wild-type SakSTAR differs markedly among species. This implies that a systematic evaluation of reduced antigen recognition and antibody induction in humans will require the development of human or humanized systems. Surprisingly, SakSTAR(K74), with a single substitution of Lys74 with Ala, had an intact specific activity but did not absorb 40% of the antibodies induced in patients by treatment with wild-type SakSTAR.

Measurement of antibody binding to intact bacteria using flow cytometric techniques

Laboratory based assays are used routinely to measure antibody binding to bacteria of their subunits and have become extremely important to the study of immune responses to these pathogens. We have developed a two-color fluorescence flow cytometric assay to measure the binding of bovine antibodies to intact Brucella abortus. Intact, irradiated B. abortus organisms, strain 19, were incubated with bovine plasma from B. abortus strain 19 vaccinated cattle. Bacteria with bound bovine immunoglobulin were labeled using an FITC conjugated anti-bovine immunoglobulin antiserum (green fluorescence), fixed to stabilize the antibody binding and to permeabilize the bacterial cell wall and then treated with propidium iodide (red fluorescence) to label the bacterial DNA. Dual labeled bacteria were analyzed with a flow cytometer using red fluorescence to identify bacteria from sample debris and green fluorescence to measure antibody binding. The advantages of such an assay are that intact bacteria can be used, labeling procedures are simple and readily automated and data represents the measurement of antibodies bound to a large number of individual bacteria which are used to determine the total antibody binding estimate. This assay was used to measure specific antibody levels using a single dilution of test plasma and was subject to less intertest variation than an enzyme linked immunosorbent assay.

Safety and immunogenicity of immunotherapy with accelerated dosage schedules of polymerized grass and ragweed in patients with dual inhalant sensitivity

Immunotherapy with individually polymerized grasses (IPG) and immunotherapy with polymerized ragweed (PRW) have been demonstrated to be immunogenic and safe and to result in lowering of symptom-medication scores compared to placebo. We conducted this study to evaluate the immunogenicity and safety of immunotherapy with concomitantly administered accelerated dosage schedules of IPG and PRW in 12 patients with dual inhalant sensitivities. Patients were treated in nine weekly visits with IPG, comprising 71,950 PNU; they were treated in 11 weekly visits with PRW comprising 2955 allergy units. Eleven additional patients who had been previously treated with IPG received only PRW. There were no systemic reactions and no clinically significant changes in routine laboratory parameters, including hepatic and renal functions, with injections. There were significant rises in IgG titers by ELISA to each grass-pollen allergen administered, orchard, timothy, and Bermuda, and in total antibody binding of antigen E. Changes in IgE against orchard, timothy, Bermuda, and antigen E were minor. Thus, IPG and PRW administered concomitantly in accelerated dosage schedules are safe and immunogenic in patients with dual inhalant sensitivities.

Pulmonary and immunologic evaluation of foundry workers exposed to methylene diphenyldiisocyanate (MDI)

A cross-sectional evaluation was performed of workers in a steel foundry in which methylene diphenyldiisocyanate (MDI) was used as a component of a binder system used to make cores and molds. Preshift and postshift spirometry and clinical evaluations were performed on 26 currently exposed (group I), on six formerly exposed (group II), and on 14 nonexposed workers to MDI (group III). Serum samples were assayed for total antibody binding, specific IgG by ELISA, and specific IgE by the RAST method to MDI-human serum albumin (HSA). Symptoms compatible with occupational asthma were elicited from seven (27%) of 26 group I workers and from three of six group II workers. No symptoms were reported by group III workers. Intrashift change in FEV 1 (a mean decrease of 0.049 L) in group I workers was significantly decreased compared to that in unexposed group III workers (a mean increase of 65 ml; p = 0.043). Specific IgG and total antibody responses to MDI-HSA were detected only in workers with current or former exposure to MDI. Only one worker was identified with IgE-mediated occupational asthma exhibiting a positive prick test and elevated RAST to MDI-HSA of 25.5% bound. In this occupational setting, polyclonal immune responses to MDI-HSA and clinical sensitization to MDI were demonstrated to occur.

Neuronal acetylcholine receptors: fate of surface and internal pools in cell culture

Chick ciliary ganglion neurons have nicotinic ACh receptors that mediate synaptic input to the cells. Ultrastructural studies with a monoclonal antibody that recognizes the neuronal ACh receptor have previously shown that, in addition to a predominantly synaptic location for the receptors on the neuron surface in vivo, substantial amounts of intracellular receptor are present as well. Here we report that intracellular receptor and smaller receptor-related components make up at least two-thirds of the total antibody binding sites associated with the ciliary ganglion neurons in cell culture. The intracellular sites for the most part represent integral membrane components that bind to concanavalin A when solubilized, indicating that the components are glycosylated. Sucrose gradient analysis shows that the intracellular material includes a 10 S component, likely to represent assembled receptor, along with species sedimenting in the 5-9 S range. Blocking the surface sites with unlabeled antibody and measuring the appearance of the new receptor on the cell surface with radiolabeled antibody indicates that the receptors are inserted into the plasma membrane at a rate equivalent to about 4% of the total surface receptor per hour. The transit time for newly synthesized receptor to reach the cell surface appears to be 2-3 hr. These observations suggest that about 5% of the intracellular receptors are transported to the cell surface in culture. The half-life of ACh receptors in the plasma membrane was estimated by 2 different approaches to be about 22 hr. Surface and internal sites respond in a qualitatively similar way to external agents that specifically modulate cholinergic receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

THE CALCULATION OF THE INTRINSIC AFFINITY CONSTANT K FOR ANTIBODY BINDING TO PRP OLIGOSACCHARIDES FROM HAEMOPHILUS INFLUENZAE TYPE B

We postulate that the affinity of antibody against polyribosylribitol phosphate (PRP) may be as important as concentration in host defense against Haemophilus influenzae b (Hib), and may differ for T-independent and T-dependent antigens. Using oligosaccharides (OS) from Hib we measured the intrinsic affinity (K) of antigen antibody binding rather than the relative avidity of antibody for multivalent antigen. PRP-OS were produced by acid hydrolysis and gel filtration; sized according to the number of repeating units (T/R); and radiolabelled. Affinities of purified bacterial polysaccharide hyperimmune globulin (BPIG) and of sera from vaccinated adult donors were measured by a modified Farr assay, and the total antibody binding sites (Abt) calculated. BPIG bound to OS of T/R=4, 3 and 2 with K=3.5, 2.3 and 1.5×105 M−1 respectively, while Abt was constant. Serum from ten adults vaccinated with one dose of PRP-diphtheria toxoid conjugate (PRP-D) had an average K=5.1±4.1×105M−1, compared to nine PRP vaccinated subjects with an average K=6.5±4.1×105 M−1 (p>0.1). We conclude that anti-PRP IgG binds PRP-OS with low affinity that decreases with the size of OS. Since total antibody binding sites did not vary by OS size, these data suggest that there were no subpopulations of antibody with binding restricted by OS size. Finally, the intrinsic affinity of the primary antibody response is similar regardless of vaccination with PRP or PRP-D, though further studies may show different affinities with secondary antibody responses.

Safety and immunogenicity of immunotherapy with polymerized tree, grass, and ragweed in patients with multiple inhalant sensitivities

Immunotherapy with polymerized ragweed (PRW) and individually polymerized grasses (IPG) previously has been demonstrated to be safe and effective. We conducted this trial in order to assess safety and immunogenicity of individually polymerized trees (IPT) and to assess safety of concomitantly administering IPT, IPG, and PRW in patients with multiple inhalant sensitivities. Sixteen patients with typical tree pollinosis were recruited; all but one had grass pollinosis, and all had ragweed pollinosis. Patients were treated in 12 weekly visits with allergens to which they were sensitive, 48,850 PNU of IPT; 2668 AU (50,000 PNU) of PRW; and 48,850 PNU of IPG. There were no systemic reactions and no changes in laboratory parameters, including renal and hepatic functions with injections. Blood was drawn for immunologic studies before and after the injection series. There were significant rises in IgG titers by ELISA to each tree allergen administered, oak, elm, and box elder. There were significant rises in total antibody binding of ragweed AgE and rye grass group I in patients treated with PRW and IPG, respectively. Changes in IgE to oak, elm, box elder, AgE, and rye grass group I were minimal. In summary, IPT is safe, immunogenic, and can be administered concomitantly with PRW and IPG in patients with multiple inhalant sensitivities.

IgE against ethylene oxide-altered human serum albumin in patients with anaphylactic reactions to dialysis

We have measured total antibody and IgE directed against ethylene oxide-altered human serum albumin (ETO-HSA) in the sera of 24 patients who have experienced anaphylaxis during hemodialysis and of 41 patients who have not had such episodes during hemodialysis. ETO is used to sterilize dialyzers and other medical equipment. The geometric mean level of IgE to ETO-HSA in patients with reactions (0.9 ng ETO-HSA bound to IgE per milliliter of serum) is significantly higher than in nonreacting patients (0.1 ng/ml, p < 0.0001). Sixteen of 24 patients with reactions had detectable levels of IgE to ETO-HSA, whereas only three of 41 nonreacting patients had detectable levels, ( p < 0.0001 chi-square). The geometric mean level of total antibody to ETO-HSA is also significantly higher in patients with reactions (270 ng ETO-HSA bound per milliliter) than in nonreacting patients (31 ng/ml, p < 0.0001). Fourteen of 24 patients with reactions but only four of 39 nonreacting patients had total antibody binding of ETO-HSA ( p < 0.0001 chi-square). These data extend our previous observations on a small group of 13 patients receiving hemodialysis (seven patients with reactions, and six nonreacting patients) and clearly demonstrate an association between the presence of IgE or total antibody to ETO-HSA and immediate anaphylactic reactions in this group of 65 patients receiving hemodialysis.

IgE and IgG antibody against human (recombinant DNA) insulin in patients with systemic insulin allergy.

We demonstrated IgE and IgG antibodies to human (rDNA) insulin as well as to bovine and porcine insulin in the serum of two patients with systemic insulin allergy by an enzyme-linked immunosorbent assay. We also demonstrated IgE and total antibody binding to bovine insulin with the use of radioimmunoassays. Both patients had cutaneous reactivity to all three insulins. When the serum of one patient was preincubated with human, porcine, or bovine insulin, there was inhibition of binding of the patient's IgE and IgG antibodies to human insulin. The other patient had very low levels of IgE antibodies to insulin and thus only IgG inhibition was possible. Preincubation with human insulin inhibited binding of each patient's antibodies to bovine or porcine insulin. We conclude that, for these two patients, human insulin has all the antigenic determinants that bovine and porcine insulin have. Therefore, human insulin for these two patients will not eliminate insulin allergy in all patients with systemic allergy to animal insulin, because there are patients whose antibodies recognize determinants common to commercial human, bovine, and porcine insulin.

5 Respiratory Allergy to Synthetic Resins

Human hypersensitivity responses to acid anhydride compounds have been recognized with increased frequency. The most extensive use of acid anhydride compounds is in the production of synthetic resins. Clinical sensitization to phthalic anhydride (PA), hexahydrophthalic anhydride (HHPA), tetrachlorophthalic anhydride (TCPA) and himic anhydride (HA) usually presents as an asthma/rhinitis syndrome. Workers sensitized to trimellitic anhydride (TMA) may also develop IgE-mediated asthma/rhinitis, but in addition they may exhibit late respiratory symptoms (LRSS) and a unique syndrome characterized by pulmonary infiltrates and anaemia (PDA). Non-immunological irritant effects may also occur after overexposure to any of these compounds. Clinical diagnosis is established by a careful occupational history, skin tests, serial pulmonary function tests and confirmed by bronchial challenge conducted under controlled laboratory conditions. Phthalic anhydride IgE-mediated reaction skin tests can be performed with either unconjugated PA or PA carrier protein reagents. Skin tests with TCPA- or TMA-protein conjugates have been used successfully for in vivo diagnosis of workers developing the asthma/rhinitis syndrome after sensitization to these substances. Several in vitro immunological tests are also useful for diagnosis and serial surveillance studies in the workplace. Total IgE, specific IgE, total antibody binding and specific IgG antibody responses can be measured accurately for these purposes. Total IgE and specific IgE reactions are associated with IgE-mediated asthma while total antibody and specific IgG levels are more apt to be higher in patients with LRSS. Specific IgG antibodies may be protective in some workers who develop high levels of specific IgE without having clinical sensitivity. Antibody responses to these materials probably involve specificities directed toward haptens and new antigenic determinants within the carrier proteins. Patients who develop severe respiratory symptoms to any of these acid anhydride compounds should be removed from further contact or exposure with these materials.