Background-free Detection of Mouse Antibodies on Mouse Tissue by Anti-isotype Secondary Antibodies

Immunodetection on mouse routinely processed tissue via antibodies raised in mice faces cross-reactivity of the secondary anti-mouse reagents with endogenous immunoglobulins, which permeate the tissue. Various solutions to this problem have been devised and include endogenous Ig block with anti-mouse Fab fragments or directly conjugated primary antibodies. Mouse isotype-specific antibodies, differently from reagents directed against both heavy and light chains, fail to detect endogenous Ig after fixation and embedding, while providing a clean and specific detection system for mouse antibodies on mouse routinely processed tissue.     

Autoantigen arrays for multiplex analysis of antibody isotypes

We describe here a microarray-based method for multiplexed, antigen-specific assessment of immunoglobulin (Ig) subclasses. We used 1152-feature arrays composed of 140 antigens or antigen fragments to detect isotype-specific mAb, to quantitatively monitor changes in isotype mAb concentration, and to profile antigen-specific antibody isotype production in a murine model of autoimmunity. This platform can be easily adapted to a variety of applications, and has the potential to elucidate mechanisms that govern development and evolution of antibody responses in in vivo and in vitro systems.     

Measurement of immunoglobulin synthesis using the ELISPOT assay

We describe a method for the detection of specific antibody-producing cells from either in vitro or in vivo immunization. These techniques are especially useful for detecting antibodies from developing hybridomas. We have successfully used the system to detect isotype-specific antibodies to a variety of bacterial antigens which were produced by heterohybridomas.     

Characterization of antibodies to idiotypic determinants of monoclonal anti-sperm antibodies

Rabbit polyclonal antibodies to the idiotype of murine monoclonal anti-sperm antibodies were developed and characterized. M29.6 and M42.15 are monoclonal antibodies (mAbs) that inhibit fertilization in vivo and in vitro. Sera from rabbits inoculated with purified mAbs (Ab1) were absorbed with normal mouse and isotype-specific immunoglobulin (Ig); the anti-idiotype Ig fraction (Ab2) was isolated by protein A-chromatography or by chromatography on the corresponding idiotype column. Binding specificity of Ab2 was confirmed by measuring the reactivity of Ab2 with homologous and heterologous mAbs. Ab2 competitively inhibited 125I-labeled Ab1 binding to mouse sperm, suggesting that the Ab2 preparation possessed subpopulations directed against idiotopes similar or adjacent to the antigen-binding site of the mAb. Anti-idiotype antibodies reactive with the antigen-combining site of the anti-sperm mAb may contain subpopulations that mimic the mouse sperm epitope recognized by Ab1. Immunization with Ab2 induced anti-(anti-idiotype) antibodies (Ab3), which competitively inhibited binding of 125I-labeled Ab1 to immobilized Ab2. These results are consistent with the hypothesis that immunization of mice with antibodies to the idiotype of sperm-specific mAbs can induce antibodies that share structural similarities with the anti-sperm mAb used for their induction. Immunization with anti-idiotype antibodies that mimic sperm antigen structure represents a possible strategy for induction of immunity to sperm.     

The use of antibodies against DNA modified by trans-diamminedichloroplatinum for identification of specific DNA sequences

DNA modified by trans-diamminedichloroplatinum (trans-DDP) is suggested as an effective probe for non-isotopic hybridisation. Highly specific anti trans-DDP-DNA antibodies and peroxidase-conjugated antibodies to antirabbit Ig and protein A system have been used for the hybrids visualisation. This method allows one to detect 2 pg/mm2 DNA.     

抗鹅免疫球蛋白轻链单克隆抗体的鉴定及其应用

免疫球蛋白是机体固有免疫系统的组成部分,是机体防御的第一道防线。本研究对抗鹅免疫球蛋白轻链单克隆抗体进行了特征分析并将其应用到不同免疫试验中用以检测鹅免疫球蛋白。用此单克隆抗体制备的免疫亲和层析柱用以分离血清中的鹅免疫球蛋白;偶联辣根过氧化物酶 (Horseradish peroxidase,HRP) 后的单克隆抗体用作第二抗体来检测鹅特异性抗体。此外,该单克隆抗体可以识别和定位外周血淋巴细胞中的SIg+淋巴细胞。研究表明,该单克隆抗体可在多种条件下检测或分离鹅免疫球蛋白并作为研究鹅体液免疫的有力工具。     

A role for isotype-specific binding factors in the regulation of IgA- and IgG-specific responses by the anti/contrasuppressor T cell circuit

Previous studies have shown that the isotype of an antibody response is selected, in part, by the inhibition of isotype-specific suppression. The antisuppressor model predicts that isotype selection is initiated through an interaction between Ag, Ig, and a T cell-derived factor within 6 h of immunization. This report characterizes some of these molecules and their contribution to isotype regulation. Cultures of murine spleen cells stimulated with the T cell-dependent Ag SRBC led to Ag-specific IgG and IgA responses that could be suppressed and then antisuppressed by a molecular complex produced by mixing purified serum Ig with the supernatant of Ag-pulsed macrophages co-cultured with T cells. The supernatants from separate cultures of Ag-pulsed macrophages and rIL-1 alpha stimulated CD4+ T cells, could be pooled and mixed with Ig to produce functional antisuppressive complexes thereby allowing the factors from the different cell types to be studied separately. Adsorption of the co-culture or the rIL-1 alpha stimulated T cell supernatants against monoclonal IgG or IgA, removed IgG and IgA binding factors, respectively, and abrogated the ability to enhance the corresponding isotype. The adherent material could be recovered and used to reconstitute enhancement by the supernatants depleted of the binding factors. When affinity purified IgG or IgA was used as the source of Ig within the antisuppressive complexes, the enhancement of the antibody response was limited to the isotype of the regulatory Ig used to form the complex. Thus, manipulation of the antisuppressive molecules has a predictable effect on isotype selection. Release of isotype-specific binding factors by CD4+ cells by rIL-1 alpha supports the hypothesis that T cell circuits play a role in initiating isotype regulation.     

Use of a monoclonal rat anti-mouse Ig light chain (RAMOL-1) antibody reduces background binding in immunohistochemical and fluorescent antibody analysis

Rat monoclonal antibodies (MAb) directed to mouse Ig heavy and light chain determinants were produced. A rat anti-mouse light chain MAb (RAMOL-1) which bound to all (24/24) mouse Ig of the kappa light chain type and with varying strength to 4/4 lambda light chain-bearing Ig was evaluated as a general secondary reagent, together with two MAb that bound to the heavy chain of mouse IgG. They were conjugated with biotin or FITC and used in immunohistochemical and immunofluorescence assays to detect mouse monoclonal antibodies binding to antigens expressed in rat and human tissues and cells. As compared to commercially available polyclonal reagents, RAMOL-1 gave higher staining contrast by showing lower background staining and equal or higher staining of the primary MAb tested. This was a result of two main effects. First, crossreactivity with endogenous Ig and tissue type-specific determinants was eliminated. With polyclonal anti-mouse Ig reagents, binding to endogenous Ig was noted in vascular spaces and on Ig-bearing cells, and to rat gastric mucosa and epithelial tumor tissue in frozen tissue sections, even when diluted in high concentrations of serum homologous to the tissue. Second, binding of the secondary reagent was reduced to cells and tissues prone to have high nonspecific binding capability, such as monocytes/macrophages and formalin-fixed, paraffin-embedded tissue. Owing to unlimited and reproducible access to this homogeneous reagent, RAMOL-1 is used as second antibody to standardize the procedure used for immunohistochemical grading of human malignant tumors by determination of blood group antigen expression detected with mouse MAb.     

Isotype-like suppression of T cell-mediated immunity in vivo. II. Suppression of the early component of contact sensitivity by a Ly-2+ T cell-derived suppressor factor that binds to contact sensitivity-initiating, antigen-specific, Ly-1+ T cell-derived factors that are of different antigen specificities

Recognition that delayed-type hypersensitivity (DTH) reactions, such as contact sensitivity (CS) in mice, are initiated by Ly-1+ T cell-derived, antigen-specific factors has led to identification of a new kind of suppressor T cell that regulates this initiation phase of CS. Regulation by these suppressor T cells is T cell isotype-like in that initiation of DTH of various antigenic specificities is suppressed, whereas, Ly-1+ T cells mediating the antigen/major histocompatibility complex-restricted, classic delayed phase of CS responses are not affected, nor are other T cell activities. This study shows that these isotype-specific suppressor T cells probably act by release of soluble, isotype-specific, suppressor factors. These isotype-specific T cell factors bind to and can be eluted from columns linked with antigen-specific Ly-1+ T cell factors that initiate CS, and are of different antigen specificities. These T cell regulating, anti-isotypic suppressor factors are derived from Lyt-2+ I-J- T cells and suppress CS-initiating T cells, but do not affect the delayed-acting T cells of CS. This is in contrast with antigen-specific T cell suppressor factors that affect the late-acting and not the early-acting T cells of CS. It is suggested that the antigen-binding, CS-initiating, T cell factors, and their regulatory, anti-isotypic T cell factors are, respectively, T cell analogues of immunoglobulin(Ig)E antibody, and IgE-binding factors, that regulate IgE antibody production by IgE+ B cells.     

II. Use of antibodies to DNA modified by trans-diaminodichloroplatinum, for identification of specific DNA sequences

DNA modified by trans diaminedichlorplatinum-II (transDDP) has been suggested as an effective probe for non-isotopic hybridization and high-specific anti-DNA-transDDP antibodies with horse radish peroxidase or alkaline phosphotase conjugated antibodies to rabbit Ig and protein A-peroxidase - for hybrids visualization. This method allows to detect 2 pg/mm DNA.     

Analysis of FcγRIII and IgG Fc Polymorphism Reveals Functional and Evolutionary Implications of Protein–Protein Interaction

Fcγ receptor III (FcγRIII), a low-affinity receptor for the Fc portion of immunoglobulin G (IgG Fc), targets antigen-antibody complexes in a variety of effector cells of the immune system. We have investigated FcγRIII and IgG Fc polymorphism and made comparative analysis of the functional and evolutionary implications of the interaction between these two molecules. Sequence analysis and comparison of the three-dimensional structure suggest that the C-terminal Ig domain of FcγRIII is associated with the binding of IgG. The polymorphic residues of FcγRIII are mainly located in the region of the C-terminal Ig domain that might be involved in IgG binding. Therefore, polymorphism and functional binding affinity seems to be related to each other as has been increasingly implicated in clinical observations. IgG Fcs, the natural ligand of FcγRs, also exhibit significant polymorphism. Three regions have been identified where polymorphism frequently occurs: the putative FcR binding site, the linker region, and the intermolecular domain-domain interface of the second Ig domain. The putative FcγR binding sites where polymorphic, and isotype-specific residues cluster are consistent with the regions that have been identified by mutagenesis and molecular modeling studies. The polymorphic residues of IgG Fc were mainly located in the molecular surface, which could be used in the recognition of other binding molecules. These observations suggest that polymorphic and isotype-specific residues in IgG Fc are closely related to their function and protein-protein interaction. Therefore, the colocalization of the polymorphic residues of FcγRIII and IgG Fcs at their docking sites implies that the polymorphic residues would affect the IgG-FcγRIII binding interactions to optimize their signaling through evolution. Received: 9 December 1999 / Accepted: 15 February 2001     

The use of specific helper T cell clones to study the regulation of isotype expression by antigen-stimulated B cell clones

In order to determine the mechanism by which helper T cells regulate the production of the various immunoglobulin (Ig) classes, a number of helper T cell clones specific for keyhole limpet hemocyanin (KLH) were generated. These helper T cell clones then were used in a modified splenic fragment system whereby cloned helper T cells and a source of B cells were limit-diluted into naive, lethally irradiated recipients. The B cell clones that were subsequently stimulated in such an assay system by the addition of the antigen 2,4-dinitrophenol (DNP)-KLH then were tested for the various isotypes produced. The results of these studies indicate that the use of a single helper T cell clone could result in the production of all known Ig isotypes including IgE. Moreover, the use of a single helper T cell clone could result in multiple isotype production by a single B cell clone. However, a comparison of the isotypes secreted by a number of different B cell clones that were stimulated with the same helper T cell clone indicated that a variety of isotypic patterns could be obtained. In addition, it was found that the majority of B cell clones produced in the presence of T cell clones secrete fewer numbers of different isotypes compared with B cell clones generated with a heterogeneous population of T cells. Finally, no evidence could be found for isotype-specific helper T cell clones, although a few of the T cell clones appeared to induce a somewhat restricted isotype pattern in which only two or three different isotypes were observed.     

T cells with FC receptors in myeloma; suppression of growth and secretion of MOPC-315 by T alpha cells

We have previously demonstrated that 1) BALB/c mice and patients with IgA myeloma developed a marked expansion of T cells with surface IgA-Fc receptors (T alpha cells), 2) the FcR alpha were induced by direct interaction of soluble myeloma IgA with T cells, and 3) the T alpha cells induced in IgA myeloma were Lyt-1-2+, IgA isotype-specific suppressor cells in normal immune responses. These findings established that the host with IgA myeloma responds to the large amounts of Ig produced by the tumor by activating an otherwise normal IgA isotype-specific suppressor circuit. In the present studies, we extend our previous observations and show that T alpha cells can suppress both the growth and secretion of MOPC-315 myeloma tumor cells. Thus, the isotype-specific immunoregulatory circuit activated in myeloma is capable of suppressing tumor cells as well as normal cells.     

Two isoforms of human membrane-bound alpha Ig resulting from alternative mRNA splicing in the membrane segment

Antibodies specific for membrane-bound Ig (mIg) but not for their secreted forms would be useful not only for studying the function of mIg but also for modulating B cell activities in vivo. We have proposed that the extracellular portions of the membrane anchor peptides of mIg can be used as antigenic sites for isotype-specific targeting of B cells. Clones containing the genes of human Ig alpha 1 or alpha 2 subclasses were isolated from a genomic DNA library. The gene segments encoding the membrane peptides and their flanking regions were amplified by polymerase chain reaction, subcloned into plasmid pUC19, and the DNA sequences were determined. Human alpha 1 and alpha 2 genes, like murine alpha gene, each has only one membrane exon. The sequences of the human alpha 1 and alpha 2 genes are almost identical in the membrane peptide-coding region. The mRNA from a human mIgA-expressing B cell line, DAKIKI, was isolated, its cDNA prepared, and the segments spanning the membrane peptide-coding region and a part of the constant domain 3 amplified by polymerase chain reaction. DNA sequences revealed that there are two isoforms of alpha 1-chain, resulting from the alternative splicing of the third constant domain of H chain to two acceptor sites in the membrane exon. One isoform has a segment of 32 and the other 26 amino acid residues in the extracellular portion of the membrane peptide. These segments may serve as isotype-specific antigenic epitopes for antibody targeting of mIgA-bearing B cells.     

Detection of endotoxins of Gram-negative bacteria on the basis of electromagnetic radiation frequency spectrum

Method of Gram-negative bacteria endotoxins detection on the basis of their own spectrum of electromagnetic radiation frequency was developed. Frequency spectrum typical for chemotype Re glycolipid, which is a part of lypopolysaccharides in the majority of Gram-negative bacteria, was used. Two devices--"Mini- Expert-DT" (manufactured by IMEDIS, Moscow) and "Bicom" (manufactured by Regumed, Germany)--were used as generators of electromagnetic radiation. Detection of endotoxin using these devices was performed by electropuncture vegetative resonance test. Immunoenzyme reaction with antibodies to chemotype Re glycolipid was used during analysis of preparations for assessment of resonance-frequency method specificity. The study showed that resonance-frequency method can detect lypopolysaccharides of different enterobacteria in quantities up to 0.1 pg as well as bacteria which contain lypopolysaccharides. At the same time, this method does not detect such bacteria as Staphylococcus aureus, Bifidobacterium spp., Lactobacillus spp., and Candida albicans. The method does not require preliminary processing of blood samples and can be used for diagnostics of endotoxinemia, and detection of endotoxins in blood samples or injection solutions.     

Rapid isolation of antigen-specific antibody-secreting cells using a chip-based immunospot array

Here we report a new method for isolating antigen-specific antibody-secreting cells (ASCs) using a microwell array chip, which offers a rapid, efficient and high-throughput (up to 234,000 individual cells) system for the detection and retrieval of cells that secrete antibodies of interest on a single-cell basis. We arrayed a large population of lymphoid cells containing ASCs from human peripheral blood on microwell array chips and detected spots with secreted antibodies. This protocol can be completed in less than 7 h, including 3 h of cell culture. The method presented here not only has high sensitivity and specificity comparable with enzyme-linked immunospot (ELISPOT) but it also overcomes the limitations of ELISPOT in recovering ASCs that can be used to produce antigen-specific human monoclonal antibodies. This method can also be used to detect cells secreting molecules other than antibodies, such as cytokines, and it provides a tool for cell analysis and clinical diagnosis.     

An immunoenzyme system for determining antibodies to Streptococcus pneumoniae polysaccharides in biological fluids

The enzyme immunoassay (EIA) system for the determination of antibodies to capsular polysaccharides of pneumococci, serotypes 1, 3, 6B, 8, 9N, 15F, 23F, and C-polysaccharide has been developed on the basis of poly-L-lysin-modified antigens. The use of isotype-specific conjugates in this system permits the detection of IgG and IgA antibodies in different biological fluids: blood serum, pleural fluid, saliva, milk. Samples obtained from children with pneumococcal infection and from nursing mothers have been studied. As shown in this study, the EIA system can be used for the evaluation of the dynamics of pneumococcal infection in children.     

Primary response to GAT in F344 rats: anti-GAT antibodies, nonspecific immunoglobulins, and expression of the GAT-13 idiotype

It has been reported that antigen induces differentiation of two populations of Ig-containing cells: the first one to appear, IgCC, synthesizes nonspecific Ig and the second, AbCC, synthesizes antibodies. Along with other arguments, the observation that nonspecific Ig bear idiotypic determinants, which cross-react with those of antibodies, had led to the hypothesis that IgCC are precursors of AbCC. However, the synthesis of such idiotype-positive nonspecific Ig before the appearance of the antibodies has not yet been proven. This problem was investigated by analyzing the primary response to poly(Glu60-Ala30-Tyr10) (GAT) in F344 rats. Kinetics studies of cells synthesizing Ig expressing a major idiotype (GAT-13), and of cells synthesizing Ig not expressing GAT-13 idiotype, revealed that these two cell populations were undetectable before the appearance of the anti-GAT antibodies. This demonstrates that IgCC differentiation is not a necessary condition for the development of all antibody responses.     

Regulation of an idiotype+ B cell lymphoma. Effects of antigen and anti-idiotopic antibodies on proliferation and Ig secretion

The B cell surface Ig molecule plays an important regulatory role in delivering inductive/tolerogenic signals to the cell. In this paper, the effect of Ag and anti-idiotopic antibodies on the in vitro proliferation and Ig secretion of a B cell tumor was studied. The tumor (BCL1), which had been transfected with the TEPC-15 VH and VL Ig genes, expresses surface Ig and secretes antibody that binds the hapten phosphorylcholine. We found that Ag (C polysaccharide and phosphorylcholine carrier Ag) and two different anti-idiotopic antibodies, in the absence of T cells, all inhibited the proliferation of the T15+ transfectant cell line. The anti-idiotopic antibodies, but not Ag, also inhibited the secretion of T15 Ig by this cell line, suggesting different functional roles for Ag vs anti-Id in the regulation of B cell inactivation. The inhibition of secretion and proliferation appears to be cell cycle phase related. In addition, mouse rIL-4 could override the inhibition of proliferation induced in these studies. These phenomena, demonstrating that binding of surface Ig can result in the transduction of negative growth signals to a B cell tumor, can be viewed as a manifestation of immunologic tolerance. These findings collectively demonstrate that Ag and anti-Id mediate different signals to B cells via interaction with the surface Ig. Because of the monoclonal nature of the T15 transfectant and the anti-idiotypic antibodies, this system can be used to investigate the underlying molecular reactions involved in the B cell response and induction of tolerance.     

A new approach to studies of cell-antibody interactions using fluorescent lipid probes

The interaction of poly- and monoclonal antibodies against the L-chain of human Ig with Burkitt lymphoma EB-3 cells was studied using a fluorescent lipid probe, anthrylvinyl-labelled sphingomyelin, incorporated into the cell plasma membrane. Binding of the antibodies to Ig receptors on the surface was shown to induce changes in the fluorescence polarization of the probe. The high sensitivity of the method allows one to detect less than 100 antibody molecules per cell. The possibility of using cells or liposomes carrying antigens and fluorescent lipids for the determination of antibodies in solution is discussed.