Antibody-bound Antigen Research Articles

Immune Complex Vaccine Strategies to Combat HIV-1 and Other Infectious Diseases.

Immune complexes (ICs) made of antibody-bound antigens exhibit immunomodulatory activities exploitable in a vaccination strategy to optimize vaccine efficacy. The modulatory effects of ICs are typically attributed to the Fc fragments of the antibody components, which engage Fc receptors, complement and complement receptors on various immune cells. These Fc-mediated functions facilitate the critical interplay between innate and adaptive immune systems to impact the quality and quantity of the elicited adaptive responses. In addition to the Fc contribution, the Fab fragment also plays an immunoregulation role. The antigen-binding domains of the Fab fragment can bind their specific epitopes at high affinity to sterically occlude these antigenic sites from recognition by other antibodies. Moreover, the Fab-mediated binding has been demonstrated to induce allosteric alterations at nearby or distant antigenic sites. In this review article, we survey published studies to illuminate how the immunomodulatory functions of ICs have been investigated or utilized in a vaccination strategy to fight against an array of infectious pathogens, culminating with IC vaccine designs aimed at preventing HIV-1 infection. In particular, we highlight IC vaccine candidates that exploit Fab-mediated steric and allosteric effects to direct antibody responses away or toward the V1V2 domain, the V3 loop, and other antigenic sites on the HIV-1 envelope gp120 glycoprotein. Like other HIV-1 vaccine approaches, the path for IC-based vaccines to reach the clinic faces major hurdles yet to be overcome; however, investigations into this vaccine strategy have provided insights into the multifaceted activities of antibodies beyond their conventional roles in the host defense against HIV-1 and other microbial pathogens.

A proteome-wide immuno-mass spectrometric identification of serum autoantibodies

BackgroundAutoantibodies are produced when tolerance to self-antigens is broken and they can be mediators of tissue injury and systemic inflammation. They are excellent biomarkers because they are minimally invasive to screen and are highly abundant in serum due to limited proteolysis and slow clearance. Conventionally used methods of identifying autoantibodies in patient sera include indirect immunofluorescence, enzyme-linked immunoabsorbent assays (ELISAs) and protein microarrays. Here we present a novel proteome-wide immuno-mass spectrometric method to identify serum autoantibody targets.MethodsSerum samples from patients with inflammatory bowel disease (IBD) were analyzed by ELISA for the presence of autoantibodies to CUB and zona pellucida-like domain-containing protein 1 (CUZD1). Protein was extracted from the human pancreas as well as 16 other human tissues to make a complex tissue lysate protein mixture. Antibodies in patient sera were immobilized and purified on protein G magnetic beads and subsequently incubated with pancreatic lysate containing CUZD1 or the aforementioned complex tissue lysate. After extensive washing, antibody-bound protein antigens were trypsin-digested and identified using shotgun mass spectrometry.ResultsThe protocol was optimized for the immunoaffinity purification of autoantibody targets from tissue lysate, using CUZD1 from pancreatic lysate and anti-CUZD1 autoantibodies present in IBD patient serum as a proof-of-concept. Pancreatic secretory granule membrane major glycoprotein 2, whose autoantibodies are a known biomarker of Crohn’s disease, was also immunoprecipitated from IBD patient serum, as an additional internal positive control.ConclusionsThis study demonstrates the effectiveness of a proteomic approach to identify serum autoantibody targets, using immunoaffinity purification followed by tandem mass spectrometry. Our methodology is applicable for proteome-wide analysis of autoantibody targets in a wide variety of clinical settings.

A generalized quantitative antibody homeostasis model: maintenance of global antibody equilibrium by effector functions.

The homeostasis of antibodies can be characterized as a balanced production, target-binding and receptor-mediated elimination regulated by an interaction network, which controls B-cell development and selection. Recently, we proposed a quantitative model to describe how the concentration and affinity of interacting partners generates a network. Here we argue that this physical, quantitative approach can be extended for the interpretation of effector functions of antibodies. We define global antibody equilibrium as the zone of molar equivalence of free antibody, free antigen and immune complex concentrations and of dissociation constant of apparent affinity: [Ab]=[Ag]=[AbAg]=KD. This zone corresponds to the biologically relevant KD range of reversible interactions. We show that thermodynamic and kinetic properties of antibody–antigen interactions correlate with immunological functions. The formation of stable, long-lived immune complexes correspond to a decrease of entropy and is a prerequisite for the generation of higher-order complexes. As the energy of formation of complexes increases, we observe a gradual shift from silent clearance to inflammatory reactions. These rules can also be applied to complement activation-related immune effector processes, linking the physicochemical principles of innate and adaptive humoral responses. Affinity of the receptors mediating effector functions shows a wide range of affinities, allowing the continuous sampling of antibody-bound antigen over the complete range of concentrations. The generation of multivalent, multicomponent complexes triggers effector functions by crosslinking these receptors on effector cells with increasing enzymatic degradation potential. Thus, antibody homeostasis is a thermodynamic system with complex network properties, nested into the host organism by proper immunoregulatory and effector pathways. Maintenance of global antibody equilibrium is achieved by innate qualitative signals modulating a quantitative adaptive immune system, which regulates molecular integrity of the host by tuning the degradation and recycling of molecules from silent removal to inflammatory elimination.

Discrepancy Between Tumor Antigen Distribution and Radiolabeled Antibody Binding in a Nude Mouse Xenograft Model of Human Melanoma.

Biodistribution of antibodies is vital to successful immunoscintigraphy/immunotherapy, and it is assumed to be similar to antigen distribution. We measured and compared the binding pattern of radiolabeled antibody to tissue antigen distribution in a nude mouse xenograft model of human melanoma. We transplanted 107 FEM-XII human melanoma cells into the right flank of five nude mice. For the control, we transplanted 5 × 106 LS174T human colon cancer cells into the left flank. Two weeks later, 10 μCi of 131I-labeled melanoma-associated 96.5 monoclonal antibody (targeting p97 antigen) was intravenously injected. Three days later, we sacrificed the mice and evaluated 96.5 antibody binding and concentration in the tumors by ex vivo quantitative autoradiography (QAR). Two months later, we incubated adjacent tumor tissue slices in various concentrations of 125I-labeled 96.5 MoAb and evaluated the distribution/concentration of p97 antigen by in vitro QAR. p97 antigen distribution was homogeneous in the tumors (total antigen concentration [Bmax] = 17.36-38.36 pmol/g). In contrast, radiolabeled 96.5 antibody binding was heterogenous between location within the tumor (estimated bound antigen concentration = 0.7-6.6 pmol/g). No quantifiable parameters were found to be related with radiolabeled antibody binding and tumor antigen distribution. Antibody-bound tumor antigen to total antigen ratios ranged between 2% and 38%. Heterogeneous features of target antibody binding were observed in contrast to relatively homogenous feature of tumor antigen. We did not identify any correlations between p97 antigen distribution and 96.5 antibody binding in melanoma tissue. Radiolabeled 96.5 antibody binding patterns within melanoma cannot be predicted based on p97 antigen distribution in the tumor, which needs to be further studied with several other methods and more subjects in the future.

Antigen-Specific Antibody Glycosylation Is Regulated via Vaccination.

Antibody effector functions, such as antibody-dependent cellular cytotoxicity, complement deposition, and antibody-dependent phagocytosis, play a critical role in immunity against multiple pathogens, particularly in the absence of neutralizing activity. Two modifications to the IgG constant domain (Fc domain) regulate antibody functionality: changes in antibody subclass and changes in a single N-linked glycan located in the CH2 domain of the IgG Fc. Together, these modifications provide a specific set of instructions to the innate immune system to direct the elimination of antibody-bound antigens. While it is clear that subclass selection is actively regulated during the course of natural infection, it is unclear whether antibody glycosylation can be tuned, in a signal-specific or pathogen-specific manner. Here, we show that antibody glycosylation is determined in an antigen- and pathogen-specific manner during HIV infection. Moreover, while dramatic differences exist in bulk IgG glycosylation among individuals in distinct geographical locations, immunization is able to overcome these differences and elicit antigen-specific antibodies with similar antibody glycosylation patterns. Additionally, distinct vaccine regimens induced different antigen-specific IgG glycosylation profiles, suggesting that antibody glycosylation is not only programmable but can be manipulated via the delivery of distinct inflammatory signals during B cell priming. These data strongly suggest that the immune system naturally drives antibody glycosylation in an antigen-specific manner and highlights a promising means by which next-generation therapeutics and vaccines can harness the antiviral activity of the innate immune system via directed alterations in antibody glycosylation in vivo.

Positive-unlabeled learning for the prediction of conformational B-cell epitopes.

BackgroundThe incomplete ground truth of training data of B-cell epitopes is a demanding issue in computational epitope prediction. The challenge is that only a small fraction of the surface residues of an antigen are confirmed as antigenic residues (positive training data); the remaining residues are unlabeled. As some of these uncertain residues can possibly be grouped to form novel but currently unknown epitopes, it is misguided to unanimously classify all the unlabeled residues as negative training data following the traditional supervised learning scheme.ResultsWe propose a positive-unlabeled learning algorithm to address this problem. The key idea is to distinguish between epitope-likely residues and reliable negative residues in unlabeled data. The method has two steps: (1) identify reliable negative residues using a weighted SVM with a high recall; and (2) construct a classification model on the positive residues and the reliable negative residues. Complex-based 10-fold cross-validation was conducted to show that this method outperforms those commonly used predictors DiscoTope 2.0, ElliPro and SEPPA 2.0 in every aspect. We conducted four case studies, in which the approach was tested on antigens of West Nile virus, dihydrofolate reductase, beta-lactamase, and two Ebola antigens whose epitopes are currently unknown. All the results were assessed on a newly-established data set of antigen structures not bound by antibodies, instead of on antibody-bound antigen structures. These bound structures may contain unfair binding information such as bound-state B-factors and protrusion index which could exaggerate the epitope prediction performance. Source codes are available on request.

Abstract 2893: Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo

Abstract Harnessing the power of adaptive immunity to combat cancer has been a long-term goal of translational immunotherapy. Tumor-specific immunity, where present, is typically at low frequency and affinity with compromised effector function. By contrast, immunity against persistent herpesviruses in man is characterised by high affinity cytotoxic T-lymphocytes (CTL) at high frequency with potent effector function. Furthermore, the immunosubversive mechanisms employed by herpesviruses show striking parallels to tumors, yet the associated anti-viral immunity limits these to life-long asymptomatic infections. We reasoned that the delivery of immunodominant viral peptide epitopes to the tumor surface might facilitate passive-loading of peptides into empty MHC class-I molecules, effectively mimicking viral infection, rendering tumors susceptible to lysis by anti-viral immunity. To address this we developed a new class of targeting antibodies: APEC (Antibody Peptide Epitope Complexes) that are able to deliver an antigenic payload at the cell surface through proteolytic release of covalently-coupled peptide antigens. As a proof-of-concept we used clinically-validated antibodies cetuximab (anti-EGFR) and rituximab (anti-CD20) to develop APECs that are able to target human tumors. We screened 15 HLA-A*0201+ EGFR-expressing NCI-60 cell lines, CD20+ lymphoma cell lines, 20 primary CD20+ CLL tumor samples and four healthy B-cells against a library of 190 cetuximab-APECs (cAPEC) or rituximab (rAPEC) incorporating the immunodominant cytomegalovirus (CMV) pp65495-503 epitope and candidate protease cleavage sites following co-incubation with CMV-specific CTL (CMV-CTL). The most effective cAPEC and rAPEC were those incorporating MMP2, MMP9, Cathepsin B and Cathepsin D protease recognition domains. Very few (2/190) rAPEC were able to redirect CMV-CTL against healthy cells. Heterogeneity was observed for primary CLL tumors but a limited number of rAPEC were effective in all cases (5/190). Mechanistic studies demonstrated that: (i) peptide loading occurred at the cell surface, (ii) required the expression of target antigens at the cell surface and (iii) T-cell recognition could be inhibited by unconjugated antibody (92%) or by incubation with protease inhibitors (83%). T-cell specificity was examined using rAPEC treated tumor targets co-incubated with various HLA-matched effector T-cell populations. No activation of CD4+ was observed including CD4+CD25hi regulatory T-cell populations. Incubation with CD8+ T-cells revealed that only pp65495-503-specific CD8+ T-cells engaged with APEC-treated tumor cells. Lastly, xenograft studies using EGFR+ and CD20+ tumor cell lines demonstrated efficacy of both cAPEC and rAPEC to eliminate tumors in vivo by redirecting anti-viral CTL. These data indicate that APECs represent a powerful new approach to combat cancer. Citation Format: David G. Millar, Laura Morton, Manuela Carvalho Gaspar, Punam Mistry, Hugo De La Peña, Ricky Joseph, Sarah Penny, Oliver C. Goodyear, Margaret Goodall, Guy E. Pratt, Mark Cobbold. Engineered release and presentation of antibody-bound viral antigens: A highly specific and novel immunotherapeutic approach to target cancer in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2893. doi:10.1158/1538-7445.AM2014-2893

Correlation of Fc Gamma RIIA Polymorphisms with EBV-Positivity and LMP1 Expression in Patients with Low Grade B-Cell Lymphomas

Abstract 1600 Background:It is well known that Fc gamma receptors expressed on monocytes and macrophages enhance the presentation of antibody-bound antigens. Human Fc gamma receptors are coded by genes located on chromosome 1q23. Fc gamma RIIA (CD32) is a transmembrane protein expressed on neutrophils. Efficacy of phagocytosis by polymorphonuclear neutrophils is known to vary between allotypes of Fc gamma RIIa. Polymorphisms of Fc gamma RIIA have been linked to susceptibility to infections by encapsulated bacteria (N. meningitides, H. influenzae) and severe sepsis by several authors. Moreover, susceptibility to perinatal HIV-1 infection has been attributed to polymorphisms of Fc gamma RIIA, and this is the only association of FC gamma RIIA with viral diseases in the literature. FcγRIIA gene has two condominantly expressed alleles the 131-Arg (R131) and the 131-His (H131) allele. R131 binds IgG2 much less avidly than H131. Aim of the study:To propose Fc gamma RIIA polymorphisms as a genetic risk factor for acquisition and latency of EBV infection in patients with lymphoproliferative diseases. Patients and Methods:Forty Greek patients with EBV-unrelated low grade B-cell leukemic lymphomas (chronic lymphocytic leukemia: 23, marginal zone lymphoma: 11, mantle cell lymphoma: 3, hairy cell leukemia: 2, follicular lymphoma: 1), were included in the study. Patients' sera were tested with ELISA for the presence of EBV-VCA IgG antibodies. DNA from peripheral blood was studied by quantitative real time - PCR for the EBV-R gene, while RNA from peripheral blood was studied by RT-PCR for the EBV-LMP1 oncoprotein. Genomic DNA from peripheral blood was tested for Fc gamma RIIA 131Histidin(H)/arginine(R) SNP, by PCR, followed by restriction fragment length polymprphim (RFLP) using the appropriate enzyme (BstUI, Fermentas, Vilnius, Lithuania). We used Pearson Chi-square for the statistical analysis of the results. Results:All but 2 patients were positive for EBV-VCA IgG antibodies. Nineteen patients (47.5%) were EBV-positive. LMP1 was expressed in 13/19 (68.4%) EBV-positive patients. The vast majority (16/19, 84.2%) of EBV positive patients carried the R131 allele (13 were heterozygous and 3 homozygous), while only 3 were homozygous for the H131 allele. Among 21 EBV-negative patients, only 6 (28.5%) carried the R131 allele (4 heterozygous and 2 homozygous) (2-sided p=0001). R131 allele was present in 11/13 (84.6%) LMP1-positive patients in comparison to 6/21 (28.0%) EBV-negative patients (2-sided p=0.002). Discussion:The high prevalence of FC gamma RIIA polymorphisms among EBV-positive patients indicates a possible pathogenetic role of the FC gamma RIIA in acquisition and chronicity of EBV infection. LMP1 is the major oncoprotein of EBV. The correlation of this polymorphism to LMP1 expression is an indication that it may play a major role in the expression of latency phase proteins, a fact that may further be implicated in the pathogenesis of lymphoproliferative diseases in these patients. We continue the study in our centre, increasing the number of patients enrolled. Disclosures:No relevant conflicts of interest to declare.

Effect of antigen turnover rate and expression level on antibody penetration into tumor spheroids

Poor tissue penetration is a significant obstacle to the development of successful antibody drugs for immunotherapy of solid tumors, and diverse alterations to the properties of antibody drugs have been made to improve penetration and homogeneity of exposure. However, in addition to properties of the antibody drug, mathematical models of antibody transport predict that the antigen expression level and turnover rate significantly influence penetration. As intrinsic antigen properties are likely to be difficult to modify, they may set inherent limits to penetration. Accordingly, in this study, we assess their contribution by evaluating the distance to which antibodies penetrate spheroids when these antigen properties are systematically varied. Additionally, the penetration profiles of antibodies against carcinoembryonic antigen and A33, two targets of clinical interest, are compared. The results agree well with the quantitative predictions of the model and show that localizing antibody to distal regions of tumors is best achieved by selecting slowly internalized targets that are not expressed above the level necessary for recruiting a toxic dose of therapeutic. Each antibody-bound antigen molecule that is turned over or present in excess incurs a real cost in terms of penetration depth-a limiting factor in the development of effective therapies for treating solid tumors.

Improved immunomatrix methods to detect protein:protein interactions

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein–protein interactions. These methods utilize immobilized Protein A or Protein G to isolate antibody-bound target antigens. The main disadvantage of traditional IP and co-IP is that the conditions used to elute the precipitated antigen also release the antibody thus contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized Protein A or Protein G, or by coupling it directly to the resin (see Scheme 1). Antibody cross-linking can be done in 1 h while antibody coupling requires 4 h. IP or co-IP is accomplished by incubating the antibody resin with the protein sample. Washes and elutions are carried out in a spin column to reduce resin loss and decrease assay time. Target proteins are eluted with 0.1 M glycine (pH 2.8) and the resin-bound antibody is re-equilibrated in phosphate-buffered saline (PBS) for reuse. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yield IP and co-IP results similar to traditional protocols but eliminate the antibody heavy and light chain contamination.

Involvement of the C-terminal end of the prostrate-specific antigen in a conformational epitope: characterization by proteolytic degradation of monoclonal antibody-bound antigen and mass spectrometry.

Prostate-specific antigen (PSA), a 237-amino acid glycoprotein, encoded by the hKLK3 gene, is widely used as a serum marker for the diagnosis and management of prostate cancer. We report here the localization of a conformational epitope recognized by the anti-total PSA monoclonal antibody (mAb) 11E5C6, by proteolytic degradation of mAb-bound antigen followed by mass spectrometric analyses of the peptides generated. These two technologies, combined with molecular display, allowed the identification of amino acid residues contained within three different peptides distant on the PSA sequence, but close in the PSA three-dimensional structure, that may be part of the mAb 11E5C6 epitope. The last four C-terminal amino acid residues are included in this epitope, as well as certain other C-terminal residues between Y225 and T232. The involvement of the PSA C-terminal end in the mAb 11E5C6 epitope was confirmed by western blotting experiments with the recombinant protein proPSA-RP1, resulting from the cloning of an alternative transcript of the hKLK3 gene, in which the PSA C-terminal end was deleted and replaced by another sequence. Although the anti-total PSA mAb 5D5A5 used as a control bound proPSA-RP1, mAb 11E5C6 did not. The requirement of the C-terminal end for the recognition by mAb 11E5C6 may be useful for the discrimination of PSA-related forms.

Use of Immunomatrix Methods to Improve Protein-Protein Interaction Detection.

Immunoprecipitation (IP) and coimmunoprecipitation (co-IP) are key techniques for studying protein-protein interactions. These methods utilize immobilized protein A or protein G to isolate antibody-bound target antigens. The main disadvantage of traditional immunoprecipitation and coimmunoprecipitation is that the conditions used to elute the precipitated antigen also release the antibody, contaminating the antigen and destroying the antibody support. To overcome these problems, we describe two methods to generate a reusable antibody support by cross-linking the antibody to immobilized protein A or protein G, or by coupling it directly to the resin. Our studies have demonstrated that the immobilization efficiency for the antibody coupling method was similar for several species of antibody. Furthermore, we illustrate that using both methods of antibody immobilization yields IP and co-IP results similar to traditional protocols but eliminates the antibody heavy and light chains contamination.

Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping.

Lyme borreliosis is a multisystem disorder caused by the spirochete Borrelia burgdorferi that is transmitted to humans by the tick Ixodes dammini. The immune response against the 31 kDa OspA, which is one of the most abundant B. burgdorferi proteins, appears to be critical in preventing infection and tissue inflammation. Detailed knowledge of the immunological and molecular characteristics of the OspA protein is important for the development of reliable diagnostic assays. In this study, we characterized a new conformational epitope present within the middle part of B. burgdorferi OspA. Our approach used enzymatic proteolyses of the immune complex followed by mass spectrometric identification of the peptides bound to the antibody. It appears to be one of the first reports on the characterization of a discontinuous epitope using mass spectrometry.

DNA vaccination with glutamic acid decarboxylase (GAD) generates a strong humoral immune response in BALB/c, C57BL/6, and in diabetes-prone NOD mice.

The technique of DNA-based vaccination was used to generate a T-cell-dependent antibody response to glutamic acid decarboxylase (GAD) in BALB/c, C57BL/6, and non-obese diabetic (NOD) mice. Plasmids were constructed in which the expression of the rat GAD65 (rGAD65) or the rat GAD67 (rGAD67) gene was driven by the immediate early region promoter of the human cytomegalovirus (pCMV). This "naked" plasmid DNA was then injected into the regenerating muscles of the studied mice. In the vaccinated animals, antibody responses to GAD65 or to GAD67 were induced. Epitope recognition of GAD was studied by protein footprinting, a technique which makes use of a limited proteolysis of antibody-bound antigen. Different epitope recognition patterns were found, corresponding to strain-specific patterns. Mild trypsin treatment generated 50 kD, 46 kD, 40 kD, 30 kD, and 21 kD proteolytic fragments. In NOD mice, 50, 46 and 40 kD bands were the most prominent signals. In non-diabetes prone BALB/c mice, a faint 40 kD band appeared suggesting a rather weak protection of GAD from tryptic lysis. The pattern observed in C57BL/6 mice was more comparable to the NOD mice pattern with prominent 40 kD and 30 kD signals and a faint 21 kD fragment. Diabetes incidence was unchanged in NOD mice, and no diabetes was observed in C57BL/6 and BALB/c mice, respectively. The data demonstrate that genetic immunization is a suitable novel tool to stimulate and to manipulate an immune response against the diabetes-associated protein glutamic acid decarboxylase. Interestingly, our results indicate that, by genetic vaccination, distinct B-cell epitopes were generated in the various studied mouse strains.

Molecular characterization of a conformational epitope of hen egg white lysozyme by differential chemical modification of immune complexes and mass spectrometric peptide mapping.

A new approach for the characterization of conformationally dependent epitope structures in protein antigens is described using differential chemical modification of immune complexes in combination with mass spectrometric peptide mapping analysis. Well-established methods for epitope characterization are frequently not applicable to conformationally dependent epitopes, and direct methods of structure analysis such as X-ray crystallography of immune complexes have been successful only in a few cases. Our approach combines tertiary structure-selective chemical modification of immune complexes with the molecular characterization of reaction products by mass spectrometric peptide mapping. The comparison of the modification pattern of free and antibody-bound antigen provides the identification of residues protected from modification by the antibody. These residues hence are characterized as part of the epitope structure. The well-characterized hen egg white lysozyme and a corresponding monoclonal IgM-type antibody were investigated as a model system. Specific modification reactions for arginine, lysine, and tyrosine residues were performed, and the modification sites in free and antibody-bound antigen were determined by mass spectrometric peptide mapping. The R14 residue and residues K13 and K96 in the antibody-bound lysozyme were found to be protected from modification, comprising a surface of spatially adjacent residues by folding of the native protein. In contrast, other K and R residues as well as Y20 and Y23 showed no significant shielding from modification in the immune complex. These results provided an estimation of the molecular epitope surface area of native lysozyme.

A sensitive radioimmunoassay optimized for reproducible measurement of rat plasma insulin

There has been a notable lack of consistency in plasma insulin values reported in the literature. In an attempt to find a more reproducible and sensitive insulin radioimmunoassay (RIA) method for the measurement of plasma insulin, the charcoal adsorption method for separation of free from antibody-bound antigen was investigated. The applicability of this method towards the measurement of plasma insulin, however, has been known to be severely limited because of interference by plasma proteins with charcoal binding. Indeed, the assay resulted in negative fasting insulin values and unacceptable interassay variability, which was not improved by adding charcoal-extracted plasma (CEP) to the standard curve. In this paper, we demonstrate a process by which relatively small volumes (25 μL) of plasma from control, diabetic, and fasted rats can be assayed reproducibly with charcoal in the final separation step. It was observed that the assay of insulin-free plasma, in the form of CEP, could represent a distinct “zero insulin” level specific for plasma samples, in place of the zero insulin standard (insulin-free standard buffer) routinely used. Normalizing plasma samples against CEP resulted in lower intra-assay and interassay variability (coefficient of variation <10%) and enhanced sensitivity to 7 μU/mL. Recovery was tested by adding 25 μL of known insulin standards to plasma samples in which insulin levels had been determined previously. Whereas the recovery of total insulin in the presence of plasma was low (38%–89%), recovery of total insulin from two standards was >95%. Following correction, recovery of total insulin in 25-μL plasma was improved to >90%. Hence, this approach provides a simplified way of correcting for plasma effects and greatly enhances the feasibility of using the charcoal-separation technique for the measurement of plasma insulin.

Mass spectrometric mapping of protein epitope structures of myocardial infarct markers myoglobin and troponin T.

Monoclonal antibodies are widely used analytical tools in biochemical research. The knowledge of their corresponding epitopes is of major interest. One possible approach for epitope characterization is the application of protein antigen proteolysis in combination with mass spectrometric peptide mapping analysis. Two complementary analytical strategies were applied: (a) limited proteolysis of antibody-bound antigen followed by removal of nonbound peptides and detachment of the antigenic peptides (epitope excision) and (b) enzymatic digest of the antigen followed by extraction of the antigenic peptides with the antibody and detachment of antigenic peptides after removal of nonbinding fragments (epitope extraction). In the few examples published so far, immobilized antibodies were used for these studies. In this study we present a method for characterization of the epitope sequences without prior immobilization of the monoclonal antibody. The separation of nonepitope peptides from antibody-bound peptides was carried out by ultrafiltration. The epitope and nonepitope fractions were analyzed by MALDI-MS without further purification, and the epitope sequences were identified. The method was developed using a model system consisting of the synthetic C-terminal cyanogen bromide fragment CB3 of myoglobin and the commercial monoclonal anti-myoglobin MG1. In further investigations the epitope sequence of a synthetic 32 amino acid peptide derived from heart muscle protein troponin T toward a monoclonal antibody MAb-M7, which was raised against the intact protein, was characterized. With this approach the epitope binding site of this antibody was determined, and selective shielding of potential cleavage sites in the immune complex could be observed. Furthermore, statements about the three-dimensional structure of the bound antigen were made.

Characterization of monoclonal antibodies specific for human sperm : effect of CRL-10 on acrosome reaction

Monoclonal antibodies to human sperm were obtained from hyperimmunized BALB/c mouse spleen cells fused with myeloma NS-1 cells. Each antibody recognized definite regions in fresh unfixed sperm: equatorial region, acrosome, postacrosome, midpiece, tail. All the antibodies were specific for sperm. We selected CRL-10 monoclonal antibody, specific for acrosome, for a detailed study. The expression of the CRL-10 antibody-bound antigen was detected in other mammalian species. When CRL-10 antibody was added prior to sperm incubation in a capacitating medium, promotion of the acrosome reaction was observed.

Determination of prostaglandin metabolites in biological samples by competitive time-resolved fluoroimmunoassay

We describe a time-resolved fluoroimmunoassay method for the determination of 13,14-dihydro-15-ketoprostaglandin E 2 (PGEM) and 13,14-dihydro-15-ketoprostaglandin F 2α (PGFM) in femtomolar ranges. Polyclonal antibodies were raised in rabbits and the antigen was labelled with europium by the avidin-biotin technique. A second antibody, directed to rabbit IgG, was coated onto the solid phase. The IgG-PG-europium complex was bound by the second antibody, giving a rapid and complete separation of antibody-bound and free antigen. The detection limit was 0.2 pg/assay for PGFM and 1.2 pg/assay for PGEM. The intra-assay CV ranged from 4.6% to 9.3% and the interassay CV from 6.7% to 11.4%. A good correlation was obtained between the results from TR-FIA and RIA when the method was applied to the investigation of tissues from breast cancer. We conclude that the TR-FIA is more sensitive and much faster than the RIA and avoids the use of radioactive material.

Dual-label time-resolved fluoroimmunoassay for simultaneous detection of myoglobin and carbonic anhydrase III in serum

We developed a dual-labeled time-resolved fluoroimmunoassay for simultaneous quantification of myoglobin (Mb) and carbonic anhydrase III (CA III) in serum involving polyclonal antibodies and the fluorescent lanthanides europium (Eu3+) and samarium (Sm3+). This solid-phase immunoassay is based on competition between Eu(3+)- or Sm(3+)-labeled antigen and the sample antigen for polyclonal rabbit antibodies. Standards and patients' samples containing antigen inhibit binding of the lanthanide-labeled antigen to the antibody. A second antibody directed against rabbit IgG is coated on a solid phase and binds the IgG-antigen-lanthanide complex, giving rapid and complete separation of antibody-bound and free antigen. The assay requires only one incubation step. An enhancement solution dissociates Eu3+ and Sm3+ ions from the labeled CA III and Mb, respectively, into a solution where they form highly fluorescent chelates. Spectra of the fluorescent chelates in the microtitration-strip wells were run on a time-resolved fluorometer equipped with filters for Eu3+ (613 nm) and Sm3+ (643 nm), the fluorescence from each sample being inversely proportional to the concentration of antigens. The measurement range for both analytes is from 5 to 1500 micrograms/L. The mean within- and between-assay precisions (CV) were 4.6% and 6.2% for CA III and 5.9% and 7.3% for Mb, respectively. Good correlations were obtained with the results of CA III RIA and a commercial myoglobin RIA kit.