Antibodies In Normal Sera Research Articles

Many chronic lymphocytic leukemia antibodies bind apoptotic cells with exposed non-muscle myosin heavy chain IIA in a similar manner as natural antibodies (101.13)

Abstract Chronic lymphocytic leukemia (CLL) mAbs containing an IGHV1-69, IGHD3-16 and IGHJ3 rearrangement with a nearly identical or stereotyped sequence (subset 6) recognize non-muscle myosin heavy chain IIA (MYHIIA). Immunohistochemistry showed that MYHIIA becomes exposed on the surface of apoptotic cells. Flow cytometry with 7-amino-actinomycin D and Annexin V-Phycoerytherin showed that MYHIIA exposure (and concomitant subset 6 CLL mAb binding) occurs in a subgroup of both early and late apoptotic cells. Because other CLL mAbs bind apoptotic cells, the binding of MYHIIA exposed apoptotic cells (MEACs) to a panel of CLL mAbs, only two from subset 6, was measured by flow cytometry. Most CLL mAbs (16/26) bound MEACs well. The MEAC binding level inversely correlated with the degree of IGHV mutation. CLL patients with unmutated IGHV (≤ 2%) tend to have shorter survival times. Similarly, CLL mAbs with high binding to MEACs correlated with shorter patient survival. Natural antibodies are characterized by unmutated IGHV and binding to apoptotic cells. Thus, binding of normal serum antibodies to MEACs was tested. Serum samples had reactivities comparable to CLL mAbs with high binding to MEACs, whereas an antigen-selected human antibody specific for CD20 had little MEAC reactivity. Thus, many CLL leukemic clones may derive from natural antibody-expressing B cells, such as B-1 cells, and stimulation by MEACs could lead to development and/or expansion of CLL leading to poor patient outcome.

Mechanisms of action of immune globulin

Mechanisms of action of immune globulin

Reactivity of human monoclonal IgM with nerve glycosphingolipids

We examined the reactivity of monoclonal IgM of sera from patients with neuropathy and monoclonal IgM, with or without antibody activity to myelin-associated glycoprotein (MAG), as well as sera from non-neurologic patients with Waldenström's macroglobulinaemia, with various nerve glycolipids extracts or with purified gangliosides. As expected from previous studies, all (five cases) anti-MAG IgM stained two glycolipids, the chemical characteristics of which corresponded to sulphated glucuronyl-paragloboside (SGPG) and sulphated glucuronyl-lactosaminyl-paragloboside (SGLPG). Five of 12 sera from patients with neuropathy whose IgM was devoid of anti-MAG reactivity stained nerve extracts greatly enriched (98%) with SGPG and SGLPG. Three of these five sera reacted with additional glycolipids and/or gangliosides. Two of 16 sera from patients with macroglobulinaemia without neuropathy reacted strongly with both SGPG and SGLPG. The latter finding as well as the detection of low titre of anti-sphingolipid antibodies in normal sera may cast a doubt on the pathogenetic significance of this antibody activity.

Serological identification and bioinformatics analysis of immunogenic antigens in osteosarcoma

In order to improve disease-free survival and potentially a cure, it is necessary to identify more potent osteosarcoma antigen. Here, we defined the osteosarcoma-associated antigen (OSAA) recognized by the humoral immune system for the first time. We have applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on osteosarcoma, followed by DNA sequencing and analyzing of positive clones. Then, the reactivity of normal and other bone tumor sera with positive clones were performed. Seven distinct novel antigens reactive with IgG antibodies were identified by SEREX analysis on osteosarcoma patients and were characterized according to cDNA sequence and the reactivity with allogeneic sera. Five of the seven antigens identified in this study were recognized by IgG antibodies in normal sera, and the remaining two were recognized exclusively by sera from allogeneic osteosarcoma patients but not by normal donor sera, suggested that the immune response to the two antigens are osteosarcoma related. Crude lysate ELISA methodology indicated that the optical density value of OSAA-3 and OSAA-5 were significantly higher in OS patients than in healthy donors. The two immunogenic antigens detected by immune responses might facilitate the identification of epitopes recognized by antigen-specific cytotoxic T lymphocytes (CTL). In summary, the antigens identified in this study may be potential candidates for diagnosis and targets for immunotherapy in osteosarcoma (OS).

Serological identification of immunogenic antigens in acute monocytic leukemia

In order to improve disease-free survival and potentially a cure, it is necessary to identify more potent leukemia antigen. Here, we defined the acute monocytic leukemia-associated antigen (LAA) recognized by the humoral immune system for the first time. We have applied the method of serologic analysis of recombinant cDNA expression library (SEREX) on acute monocytic leukemia (FAB M5), followed by DNA sequencing and analyzing of positive clones. Then, the reactivity of normal and other leukemia sera with positive clones were performed. Thirty-five distinct novel antigens reactive with autologous IgG were identified by SEREX analysis on an acute monocytic leukemia patient and were characterized according to cDNA sequence and the reactivity with allogeneic sera. Twenty of the 35 antigens identified in this study were recognized by IgG antibodies in normal sera, and the remaining 15 were recognized exclusively by sera from allogeneic leukemia patients but not by normal donor sera, suggested that the immune response to these 15 antigens are leukemia related. The 15 immunogenic antigens detected by immune responses in the autologous host facilitate the identification of epitopes recognized by antigen-specific cytotoxic T lymphocytes (CTL) and are potential candidates for diagnosis and immunotherapy in acute myeloid leukemia (AML).

The neutralization of pseudorabies virus by anti-α-galactocyl natural antibody in normal serum

Pseudorabies virus (PrV), a member of herpesviridae alphaherpes subfamily, can infect human cells in vitro. However, the transmission to Old World primates including humans is strongly restricted. In this study, we report the neutralizing activity of normal human serum against PrV grown in CPK cells derived from pig. PrV grown in all Old World primates-derived cells, which was tested in this study, were not neutralized by normal human serum. The virion of PrV grown in CPK cells harbored Galα1–3Galβ1–4GlcNAc-R (α-gal epitope) on its surface, while PrV grown in Vero cells did not. Depletion of antibodies reacting to surface antigens of CPK cells negated the neutralization activity of human serum. Blockade of anti-α-gal antibodies by adding soluble Galα1–3Gal to normal human serum also prevent the inactivation of PrV grown in CPK cells. Although normal swine serum did not neutralize PrV grown in CPK cells, swine serum supplemented with exogenous anti-α-gal antibodies did. These results indicate that anti-α-gal antibodies in normal human serum contribute to the neutralization of PrV. Anti-α-gal antibodies in normal human serum may prevent transmission of PrV into humans.

Specific recognition of protein carboxy-terminal sequences by natural IgM antibodies in normal serum.

Our previous study indicated that normal serum contains complement-fixing natural IgM antibodies reacting with a large variety of randomly generated protein carboxy-termini. Here we show that the "carboxy-terminal" IgM (C-IgM) antibodies specifically react with short peptide sequences located immediately at the protein carboxy-terminus. The specificity of C-IgM-peptide interactions is tentatively defined by three to four amino acid residues. All carboxy-terminal peptides in a large peptide library apparently react with C-IgM antibodies. Immobilized synthetic peptides also react with C-IgM antibodies. No interaction of C-IgM antibodies with internal peptide sequences has been observed. C-IgM antibodies are present in germ-free and in athymic adult rats and are absent in newborn rats. The natural ubiquity of protein carboxy-termini in biological structures suggests that C-IgM could play an important role in antigen clearance and presentation to the immune system. From a practical viewpoint, the recognition of carboxy-terminal peptides by complement-fixing C-IgM antibodies has profound implications for the use of peptide- and protein-derivatized delivery vehicles and artificial materials.

Serum resistance in Haemophilus ducreyi requires outer membrane protein DsrA.

Haemophilus ducreyi is resistant to killing by normal serum antibody and complement. We discovered an H. ducreyi outer membrane protein required for expression of serum resistance and termed it DsrA (for "ducreyi serum resistance A"). The dsrA locus was cloned, sequenced, and mutagenized. An isogenic mutant (FX517) of parent strain 35000 was constructed and characterized, and it was found to no longer express dsrA. FX517 was at least 10-fold more serum susceptible than 35000. DsrA was expressed by all strains of H. ducreyi tested, except three naturally occurring, avirulent, serum-sensitive strains. FX517 and the three naturally occurring dsrA-nonexpressing strains were complemented in trans with a plasmid expressing dsrA. All four strains were converted to a serum-resistant phenotype, including two that contained truncated lipooligosaccharide (LOS). Therefore, serum resistance in H. ducreyi does not require expression of full-length LOS but does require expression of dsrA. The dsrA locus from eight additional H. ducreyi strains was sequenced, and the deduced amino acid sequences were more than 85% identical. The major difference between the DsrA proteins was due to the presence of one, two, or three copies of the heptameric amino acid repeat NTHNINK. These repeats account for the variability in apparent molecular mass of the monomeric form of DsrA (28 to 35 kDa) observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Since DsrA is present in virulent strains, is highly conserved, and is required for serum resistance, we speculate that it may be a virulence factor and a potential vaccine candidate.

Antibodies to different isoforms of the heavy neurofilament protein (NF-H) in normal aging and Alzheimer's disease.

Sera of normal controls and of patients with neurological diseases contain antineurofilament antibodies. Recent studies suggest that biochemically and immunologically distinct subclasses of neurofilaments occur in different types of neurons. Alzheimer's disease (AD), the major cause of dementia, is associated with a marked degeneration of brain cholinergic neurons. In the present work we characterized the repertoire and age dependence of antineurofilament antibodies in normal sera and examined whether the degeneration of cholinergic neurons in AD is associated with serum antibodies directed specifically against the neurofilaments of mammalian cholinergic neurons. This was performed by immunoblot assays utilizing neurofilaments from the purely cholinergic bovine ventral root neurons and from the chemically heterogeneous bovine dorsal root neurons. Antibodies to the heavy neurofilament protein NF-H were detected in normal control sera. Their levels were significantly higher in older (aged 70-79) than in younger (aged 40-59) subjects. These antibodies bound similarly to bovine ventral root and dorsal root NF-H and their NF-H specificity was unchanged during aging. In contrast, the levels of IgG in AD sera that are directed against ventral root cholinergic NF-H were higher than those directed against the chemically heterogeneous dorsal root NF-H. Immunoblot experiments utilizing dephosphorylated ventral root and dorsal root NF-H and chymotryptic fragments of these molecules revealed that AD sera contain a repertoire of antimamalian NF-H IgG. A subpopulation of these antibodies binds to phosphorylated epitopes that are specifically enriched in ventral root cholinergic NF-H and that are located on the carboxy terminal domain of this molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

326 IGG SUBCLASSES AND ANTIBODIES TO STREPTOCOCCUS PNEUMONIAE IN IMMUNODEFICIENT CHILDREN WITH CHRONIC LUNG SUPPURATIONS

We studied 17 immunodeficient children 4-14 years old (9 with hypogammaglobulinemia, 8 with selective IgA deficiency) all having chronic lung suppurations. The highest rate of S.pneumoniae colonization was in children with IgA deficiency (up to 50%) with noncapsular H.influenzae found in 43%. Antibody levels to S.pneumoniae capsular polysaccharides, C-polysaccharide, protein antigens were studied in serum, saliva and bronchoalveolar lavage fluid by ELISA; IgG subclasses in serum - by radial immunodiffusion. All patients had absent or very low levels of pneumococcal IgA-antibody in secretions. In spite of the presence of serum IgG-antibody (even their high levels in selective IgA deficiency) all children had severe suppurations indicating the major role of local immune response in bacterial clearance from the respiratory tract. It also suggests that serum IgG antibodies do not pass in sufficient quantity into bronchial secretion due to a peribronchial fibrosis. Low serum IgG-antibody levels were found in children with hypogammaglobulinemia, however two patients with combined IgA - IgG subclass deficiency (IgA plus IgG1, IgG3, IgG4 and IgA plus IgG4) had almost normal serum antibody level probably due to the relation of pneumococcal antibody to IgG2. Clinical data indicate that different rate of hypogammaglobulinemia expression may explain the diversity in manifestations of primary humoral immunodeficiency.

Global analysis of antibody repertoires. II. Evidence for specificity, self‐selection and the immunological “homunculus” of antibodies in normal serum

The serum IgM repertoires of C57BL/6, DBA/2 and BALB/c mouse strains were analyzed using a recently developed global and quantitative assay that measures antibody reactivities to a very large number of antigens. A characteristic repertoire could be assigned to each strain. The different repertoires could be successfully classified with multivariate statistics. Many common reactivities were also observed among the different strains, which allows the definition of a mouse-specific repertoire. Analysis of human sera support this notion. To investigate the impact of minor genetic differences on the serum IgM repertoire, the congenic strains B10.D2/oSn and B10.D2/nSn, which differ in the expression of the C5 component of complement, were analyzed. The two strains could be separated based on the reactivity profiles obtained. The analysis of the results reveals that many antigenic proteins are not recognized at all by natural antibodies, while others are disproportionately reactive, the resulting patterns giving rise to what could be the definition of an "immunological homunculus". The relevance of this type of analysis for clinical applications is discussed.

SHIN, a low frequency red cell antigen, found in two Japanese blood donors.

A low frequency red cell antigen, SHIN, was detected in two Japanese blood donors. Red cells of the first propositus reacted, in pretransfusion tests, with the serum of a previously untransfused male patient. The second propositus was disclosed when anti-SHIN was used to screen red cells from 3,000 random blood donors in Tokyo: a frequency of 0.03% in this population. Family studies showed SHIN to be inherited as an autosomal dominant character and not to be part of the MNS, FY, JK or P1 blood group systems. Anti-SHIN is not a common antibody in normal sera but was found in about 10% of multispecific sera used in investigation of low frequency antigens.

Actions of Complement on Junin Virus

Fresh sera from normal rhesus monkeys, guinea pigs, and rabbits inactivated 90%-99% of the infectivity of Vero cell-passaged, attenuated strains of Junin virus (JV) within 60 minutes. Selective depletion studies showed that inactivation occurred by the classical complement pathway. Complement had little effect on virulent JV strains. Adsorption of the fresh sera with JV-infected Vero cells showed that inactivation was not mediated by low levels of antibodies in normal sera. The cells used for propagation of the virus affected inactivation: virus passaged in Vero cells (a continuous African green monkey kidney line) was more susceptible than virus passaged in FRhL-2 cells (a diploid strain derived from fetal rhesus monkey lung). Complement was important for in vitro neutralization of virulent JV strains by immune sera but was unnecessary for neutralization of attenuated strains. Thus, complement may be important in host resistance to Argentine hemorrhagic fever in two ways: first, complement activation may contribute to the attenuated phenotype of some strains; and second, complement may be necessary for efficient neutralization of virulent strains.

Heart-reactive antibodies in rabbit anti-Streptococcus mutans sera fail to cross-react with Streptococcus mutans.

Immunization of rabbits with Streptococcus mutans antigens results in the production of serum antibodies that bind in vitro to human, rabbit, and monkey cardiac muscle. Antibodies to heart, however, have also been reported to occur at lower titers in the sera of unimmunized rabbits. In this study, the specificities of heart-reactive antibodies (HRA) in sera of unimmunized and S. mutans-immunized rabbits were compared using indirect immunofluorescence, Western blot, and Bio-Dot immunoassays. Both groups of sera gave striational indirect immunofluorescence-staining patterns on thin sections of native human and monkey cardiac muscle. Western blot analyses revealed that antibodies in normal sera bound 9 to 20 components of human, rabbit, and monkey heart. The major bands had Mr of 205,000, 160,000, 135,000, and 70,000. Several of the normal sera did not have antibody activity to S. mutans antigens, indicating that these HRA do not cross-react with these bacteria. Although immunization of rabbits with S. mutans caused increased titers of HRA (two to three doubling dilutions), Western blot assays using anti-S. mutans sera showed banding patterns qualitatively similar to those of normal sera on heart extracts. Antibodies to skeletal muscle myosin were detected in both serum groups. Of eighteen normal rabbit sera sixteen had antimyosin titers of 10 to 40, whereas all eighteen anti-S. mutans sera had titers of 10 to 160. Affinity-purified antimyosin antibodies isolated from anti-S. mutans serum did not bind to S. mutans components. Conversely, affinity-purified antibodies to S. mutans antigens did not bind to myosin or to other cardiac muscle components. Among these were antibodies to the 185-kDa cell wall protein (also known as B, I/II, IF, Spa A, and P1) previously believed to possess antigenic mimicry. HRA were removed from anti-S. mutans sera by absorption with S. mutans but this effect was not specific, because a non-cross-reactive internal standard antibody was also absorbed to the same extent. Because previous evidence for antigenic mimicry between S. mutans and cardiac muscle was based on serum cross-absorption experiments, this immunologic relationship is not substantiated. These results indicated that naturally occurring antibodies to cardiac muscle components are present in the sera of unimmunized rabbits and that immunization with S. mutans does not stimulate production of new heart-reactive antibody, but rather serves to boost antibody production by preexisting clones of self-reactive B-lymphocytes.

Spontaneous expression of antibodies to DNA of various species origin in sera of normal subjects and patients with systemic lupus erythematosus.

To investigate mechanisms for the induction of anti-DNA antibodies in systemic lupus erythematosus (SLE), the specificity of anti-DNA antibodies was determined in sera from SLE patients and normal control subjects. As a marker of these responses, the reactivity to single-stranded DNA of various mammalian and bacterial species origin was tested by enzyme-linked immunosorbent assay. Patients with SLE demonstrated serum antibodies to all six types of DNA tested, whereas normal control subjects showed appreciable antibody responses only to DNA obtained from Micrococcus lysodeikticus (MC) and Staphylococcus epidermidus (SE). Anti-DNA antibodies in normal sera appeared to recognize unique sites on the DNA because MC DNA failed to inhibit antibody binding to SE DNA, and vice versa; in contrast, SLE antibody binding to MC DNA could be inhibited by SE as well as other DNA, suggesting recognition of a more widely shared epitope. The expression in normal sera of antibodies specific for certain bacterial DNA is consistent with their induction by structural determinants on these DNA molecules that are immunogenic. DNA may therefore represent another bacterial macromolecule capable of inducing cross-reactive antibodies in human autoimmune disease.

Human anti-estrogen receptor antibodies: assay, characterization, and age- and sex-related differences.

A binding assay was developed to measure the estrogen receptor (ER)-binding activity in serum. The method employed immunoglobulin G (IgG) adsorption with protein A-bearing Staphylococcus aureus cells before incubation with tritium-labeled ER. Specificity studies employing chromatographic and electrophoretic analyses suggested that an IgG antibody (Anti-ER) was responsible for the serum activity. Anti-ER was found in human, rat, and mouse serum and exhibited species cross-reactivity. The antibody recognized both the 8S and 5S forms of the ER. Anti-ER was measured in 262 individuals ranging in age from 1-85 yr. The antibody was detected in all serum samples examined, suggesting its natural occurrence. When the study group was divided into three arbitrary age groups (young, 1-13 yr; middle age, 13-51 yr; older, greater than 51 yr), significant differences in levels of antibody were found, with highest levels in the young, followed by the elderly, and lowest levels in the middle years. An examination of sex- as well as age-related differences in the population revealed a striking sex difference. Thus, the young male population had a lower level of antibody than the corresponding female population, and these levels in males declined throughout life to reach their lowest point in old age, whereas the high levels in young females declined in middle years and then increased significantly in the elderly. We postulate that the antibody is a subgroup of IgG of multifactorial etiology. The idiotypic network theory offers an explanation for the occurrence of these antibodies in normal serum, whereas an autoimmune mechanism could account for the secondary rise of anti-ER in the aging female population.

The fine specificity of IgG antiguanosine antibodies in systemic lupus erythematosus

The antigen specificity, isotype, and subclass of antinuclear antibodies may be related to their pathogenicity in systemic lupus erythematosus (SLE). Our laboratory found that IgG antibodies that bound the nucleoside, guanosine, occurred frequently in SLE patients. In contrast, sera from healthy subjects contained IgM but not IgG antiguanosine antibodies. The present studies were designed to characterize the fine specificity of IgG antiguanosine antibodies in SLE and compare them with IgM antiguanosine antibodies in normal sera. Serum antinuclear antibodies from six healthy subjects and six SLE patients were isolated by affinity binding to guanosine and measured by an enzyme-linked immunosorbent assay (ELISA). IgM in normal sera, and both IgM and IgG in SLE sera bound guanosine. IgM antiguanosine antibodies in normal sera were polyspecific and bound other nucleosides and 1-methylguanosine but not denatured DNA (ssDNA). In contrast, IgG antiguanosine antibodies from the SLE patients bound guanosine and ssDNA but not other nucleosides or 1-methylguanosine. SLE IgM antiguanosine antibodies had the same fine specificity and bound guanosine and ssDNA but not any of the other nucleosides. These results suggest that SLE IgG and IgM antiguanosine antibodies have fine specificity in contrast to the polyspecific IgM antibodies in normal sera. In addition, subclass analysis indicated that all SLE patients had either IgG 1 or IgG 3 subclass of antiguanosine antibodies that bind complement. Characterizing the isotype, subclass, and fine antigen specificity of antiguanosine antibodies should assist in evaluating their potential pathogenicity in SLE.

Enhanced detection of anti-tissue antibody by anti-immunoglobulin bound glucose oxidase.

An immunohistochemical procedure, using glucose oxidase (GO) linked to anti-immunoglobulin serum, has been adapted to detect anti-tissue antibody to smooth muscle, parietal cells, and mitochondria. With this procedure antibody to smooth muscle was detected in a high percentage of normal sera. Anti-tissue antibodies were frequently detected in sera of patients clinically suspected of having an autoimmune disease when an immunofluorescence (IF) test to detect the antibodies was negative. The glucose oxidase immunohistochemical procedure to detect anti-tissue antibodies is feasible and highly sensitive, and produces permanent results. The clinical usefulness of this procedure will have to be carefully evaluated. The detection of anti-tissue antibody in normal sera may indicate that such antibody is normally present in low concentration and that it may possibly have physiologic functions.

Characterization of the antibodies in normal rabbit serum responsible for the lysis of NK-sensitive target cells

Normal rabbit serum has previously been reported to lyse a spectrum of murine lymphoid cell lines characterized by being susceptible to natural killer (NK) cells. Although we have failed to confirm that a simple correlation exists between the susceptibility of target cells to NK cells and to normal rabbit serum, we were able to characterize a number of naturally occurring anti-carbohydrate antibodies which selectively lyse NK-susceptible lymphoma cells. The antibodies present in normal rabbit serum responsible for lysing an NK susceptible clone derived from the DBA/2 lymphoma L5178Y (27v−IC2) were found to be directed against the terminal β- N-acetylgalactosamine sugar present on the neutral glycolipid gangliotriosylceramide. Interestingly, normal rabbit serum did not lyse an NK-insusceptible variant, derived from the same lymphoma, which lacked gangliotriosylceramide. YAC-1 target cells, the prototype target for murine NK cells, were insusceptible to normal rabbit serum. However, following neuraminidase treatment, such cells were lysed by an antibody in normal serum directed against terminal β-galactose residues. The anti-β-galactose reactivity was directed against the carbohydrate structure of gangliotetraosylceramide. These findings are discussed in light of the hypothesis that NK cells interact with susceptible target cells through cell surface glycoconjugates.

Humoral bactericidal systems: nonspecific and specific mechanisms

Bactericidal systems in normal serum from month-old rabbits and guinea pigs were characterized in terms of activity against rough- and smooth-phase species of gram-negative bacteria. Killing of the rough strains in the absence of natural antibody required complement and at least one additional nonspecific component exhibiting lability to freezer storage and to heating at 52 degrees C. Bactericidal action against the smooth-phase organisms, on the other hand, appeared to require natural antibody in addition to complement and the labile component. Both nonspecific and specific bactericidal systems were also functional in normal serum from adult animals. In immune systems, the labile nonspecific component was not essential for bactericidal activity. Whereas immune systems were subject to inhibition by homologous endotoxins, the normal serum bactericidal systems were not, indicating that the natural antibodies in normal serum were specific for surface antigens other than the O-somatic polysaccharides. The significance of these observations is discussed in relation to probable complement pathways that mediate killing in each of the systems described.