Immunoglobulin classes of anti-Sendai virus antibody detected by ELISA in infected nude mouse serum

Sendai virus-infected nude mouse sera obtained on the seventh day after infection or later, in which anti-Sendai virus antibodies were undetectable by hemagglutination-inhibition and neutralization tests, were found to be reactive with the virus antigen by ELISA using horseradish peroxidase-conjugated anti-mouse IgG rabbit IgG. The reactivity was blocked by rabbit anti-Sendai virus antiserum and was not observed against influenza virus which served as a control antigen. Anti-Sendai virus antibody activity of fractions from Sephadex G-200 gel filtration was detected in the IgM fraction when anti-mouse mu chain-specific antiserum was used and in both IgG and IgM fractions when heavy and light chain-specific anti-mouse IgG serum was employed in ELISA. ELISA of the fractions from protein A-Sepharose affinity chromatography of Sendai virus-infected nude mouse sera showed that the eluates at pH 6.0 and pH 3.5 contained IgG1 and IgG2b anti-Sendai virus anti-bodies, respectively, and that the eluate at pH 4.5 contained both IgG2a and IgG3 antibodies.     

Photodynamic Action on Native and Denatured Transforming Deoxyribonucleic Acid from Haemophilus influenzae

The photodynamic inactivation of native or denatured transforming deoxyribonucleic acid (DNA) from Haemophilus influenzae is described. The inactivation at the same pH was higher for denatured than native DNA. At acidic pH, the inactivation both for native and denatured DNA was faster than at alkaline pH. The guanine content of photoinactivated native DNA at neutral pH was less than untreated DNA. The inactivation of biological activity was more extensive than the alteration of guanine. The absorption spectrum of photoinactivated native or denatured DNA was only slightly different than the control DNA at the different experimental conditions.     

Influence of duodenal secretions and its components on release and activities of human brush-border enzymes

The in vitro effects of human duodenal secretions and various combinations of its components on activity and release of enzymes from the human brush border were examined. Sucrase retained activity for 90 min in duodenal secretions, and maltase was almost as stable; lactase lost activity rapidly and alkaline phosphatase was of intermediate stability. Inactivation of lactase could only be partly (50%) attributed to luminal proteases, bile salts and phospholipids played no role. Rate of release of an enzyme from the brush border bore no relationship to its rate of inactivation. When individual proteases were studied, elastase was the most potent for releasing disaccharidases from the brush border; trypsin was ineffective alone but augmented the effect of elastase. Sucrase and maltase were activated by proteolytic release, but activation was abolished by simultaneous exposure of brush borders to bile salts. Lactase was released and rapidly inactivated by proteinases, while alkaline phosphatase appeared to be inactivated without significant release. These results show that there are significant interactions between luminal factors which have been inapparent when studying them in isolation. Loss of functionally useful enzyme does not follow release of sucrase or maltase from the brush border into the lumen but does follow release of lactase. Study of the susceptibility of lactase to inactivation by luminal factors in the various forms of lactose intolerance is warranted.     

Pneumocystis carinii: immunoblotting and immunofluorescent analyses of serum antibodies during experimental rat infection and recovery

Serum antibodies to Pneumocystis carinii were measured in rats by the indirect fluorescent antibody and immunoblotting techniques. Serum IgG and IgM antibodies developed with environmental exposure to P. carinii, were low or absent during immunosuppression to induce P. carinii pneumonia, and rose when immunosuppression was withdrawn. The IgG and IgM antibodies formed at the same time, but the titers of each antibody varied in individual rats. Serum IgG antibodies by immunoblotting recognized bands of 45, 50, and 116 kDa as the major reactive moieties of P. carinii. The bands were detected with sera from all rat groups in a temporal pattern which closely paralleled antibody formation by indirect immunofluorescence. The pattern of immunoblotting reactivity varied among individual rats, particularly with immunosuppression. Additional bands were detected with prolonged exposure to P. carinii. Thus, the rat makes both IgG and IgM antibodies to P. carinii, and specific P. carinii antigens identified in this immune response might be targeted for future serologic studies.     

Natural and immune human antibodies reactive with antigens of virulent Neisseria gonorrhoeae: immunoglobulins G, M, And A

Natural and immune human antibodies reactive with heat-labile and heat-stable antigens of virulent Neisseria gonorrhoeae were studied by use of an indirect fluorescent-antibody (IFA) procedure. The immunoglobulin class of the reactive antibodies was identified by using fluorescein-conjugated antisera specific for human IgG, IgA, or IgM in the IFA procedure. The effects of heat and mercaptoethanol on IFA reactivities were also studied. It appeared that antibodies of the IgG, IgM, and IgA classes present in the sera of both infected persons (immune antibodies) and normal persons with no history of gonococcal infection (natural antibodies) react with heat-stable somatic antigens. Immune IgG antibodies, however, were distinguishable from natural IgG antibodies by their ability to recognize heat-labile surface antigens. The distinction between natural and immune IgM antibodies was less obvious. IgM antibodies from both infected and normal persons appeared to react with heat-labile antigens. Some, but not all, infected persons had immune IgA antibodies to heat-labile as well as to heat-stable antigens. Treatment of sera with mercaptoethanol had no effect on IgG antibodies. The IFA activity of IgM antibodies was decreased, but not abolished. The effects of mercaptoethanol on IgA antibodies were variable. Some sera showed a decrease in IgA titer, and others showed an increase in IgA activity to certain antigens. Immune IgG antibodies were more resistant to heating than were natural IgG antibodies. Natural and immune IgM antibodies appeared equally sensitive to heating. IgA activity, on the other hand, was increased by heating sera at 60 C, but was decreased at higher temperatures. Thus, it appears that natural and immune human IgG antibodies to N. gonorrhoeae may be distinguished by their interactions with heat-labile antigens and by their resistance to heating.     

Rabbits immunized with mixtures of staphylococcal protein A and autologous IgG produce anti-human IgG antibodies

The results of this study provide evidence that protein A may render IgG immunogenic in the autologous host. Antibodies to human but not rabbit IgG were detected in sera of rabbits immunized with a mixture of autologous serum and protein A. Anti-human IgG antibodies appeared within 2 weeks at which time the antibodies were of the IgM class. Upon further immunization, both IgM and IgG antibodies were produced with the IgG class predominating. The antibodies elicited by a mixture of protein A with autologous IgG resembled those which arise in response to autologous IgG that has been denatured by physicochemical means, in that they react mainly with foreign species IgG and weakly, if at all, with IgG of rabbit origin.     

INACTIVATION OF CRYSTALLINE TRYPSIN

1. The rate of inactivation of crystalline trypsin solutions and the nature of the products formed during the inactivation at various pH at temperatures below 37°C. have been studied. 2. The inactivation may be reversible or irreversible. Reversible inactivation is accompanied by the formation of reversibly denatured protein. This denatured protein exists in equilibrium with the native active protein and the equilibrium is shifted towards the denatured form by raising the temperature or by increasing the alkalinity. The decrease in the fraction of active enzyme present (due to the formation of this reversibly denatured protein) as the pH is increased from 8.0 to 12.0 accounts for the decrease in the rate of digestion of proteins by trypsin in this range of pH. 3. The loss of activity at high temperatures or in alkaline solutions, just described, is very rapid and is completely reversible for a short time only. If the solutions are allowed to stand the loss in activity becomes gradually irreversible and is accompanied by the appearance of various reaction products the nature of which depends upon the temperature and pH of the solution. 4. On the acid side of pH 2.0 the trypsin protein is changed to an inactive form which is irreversibly denatured by heat. The course of the reaction in this range is monomolecular and its velocity increases as the acidity increases. 5. From pH 2.0 to 9.0 trypsin protein is slowly hydrolyzed. The course of the inactivation in this range of pH is bimolecular and its velocity increases as the alkalinity increases to pH 10.0 and then decreases. As a result of these two reactions there is a point of maximum stability at about pH 2.3. 6. On the alkaline side of pH 13.0 the reaction is similar to that in strong acid solution and consists in the formation of inactive protein. The course of the reaction is monomolecular and the velocity increases with increasing alkalinity. From pH 9.0 to 12.0 some hydrolysis takes place and some inactive protein is formed and the course of the reaction is represented by the sum of a bi- and monomolecular reaction. The rate of hydrolysis decreases as the solution becomes more alkaline than pH 10.0 while the rate of formation of inactive protein increases so that there is a second point at about pH 13.0 at which the rate of inactivation is a minimum. In general the decrease in activity under all these conditions is proportional to the decrease in the concentration of the trypsin protein. Equations have been derived which agree quantitatively with the various inactivation experiments.     

Tumor cell reactivity mediated by IgM antibodies in sera from melanoma patients vaccinated with GM2 ganglioside covalently linked to KLH is increased by IgG antibodies

 Natural IgM antibodies against the melanoma cell-surface ganglioside GM2, and IgM antibodies induced by vaccination with GM2 adherent to bacillus Calmette-Guerin, have been correlated with increased disease-free and overall survival in melanoma patients in previous phase I and II clinical trials. A vaccine containing GM2 covalently attached to keyhole limpet hemocyanin (KLH) plus the immunological adjuvant QS-21 now induces higher-titer, longer-lasting IgM antibodies against GM2 and has recently entered phase III clinical trials. For the first time this new vaccine also induces IgG antibodies against GM2 in the majority of immunized patients. With regard to immunity against bacteria, IgM antibodies have been described to be 1000-fold more effective than IgG antibodies at opsonification, complement-mediated cytotoxicity and protection from bacterial challenge. Though IgG antibodies have the theoretical advantage of being able to mediate antibody-directed cell-mediated cytotoxicity (ADCC), they may inhibit complement mediated IgM effector mechanisms against melanoma cells. Our goal was to confirm the functional characteristics of the anti-GM2 IgM and IgG antibodies induced by vaccination and to determine the impact that IgG antibodies might have on IgM antibody reactivity with GM2-positive tumor cells. Post-immunization sera from seven immunized patients were separated by size-exclusion chromatography into IgM and IgG fractions and a variety of serological assays were performed with the individual fractions and their combinations. Assays identifying specific IgM or IgG reactivity demonstrated partial inhibition by the opposite fraction. However, when the endpoint was complement-mediated lysis or overall antibody binding, which may more faithfully predict in vivo complement-mediated opsonification and lysis, the combinations of IgM and IgG fractions consistently demonstrated higher reactivity than either fraction alone. In addition, ADCC was induced in all seven patients. The results were the same whether the sera were obtained after 2 months or 2 years of immunizations. These findings suggest that IgG antibodies induced by the GM2-KLH plus QS-21 vaccine will not inhibit and should further augment the clinical impact of induced IgM antibodies. Received: 25 April 1996 / Accepted: 21 October 1996     

Ethylenediamine-NN''-tetra-acetate-dependent amino acid-stimulated inactivation of mouse ovarian alkaline phosphatase activity.

1. Preincubation of partially purified preparations of mouse ovarian alkaline phosphatase in the presence of both EDTA and glycine at alkaline pH resulted in a pronounced inactivation of alkaline phosphatase activity. Inactivation did not occur on preincubation with EDTA or glycine alone. 2. The rate of inactivation was first-order with respect to the concentration of enzyme, and was independent of EDTA concentration above a threshold value. 3. The process was pH-dependent with a pK at 9.85, and inactivation was not dependent on the stereochemistry of the amino acid. A free alpha-amino group and a free carboxyl group at a specific spatial separation were essential for inactivation. 4. Inactivation involved the formation of an enzyme--metal ion--amino acid complex, the amount formed being dependent on both the nature and concentration of the amino acid. This complex then decayed to a derivative that was then acted on by EDTA, yielding an inactive form of the enzyme.     

Reaction of poliovirus RNAs with antibodies to double-stranded RNA demonstrated by an immunochemical binding assay.

An immunochemical binding assay was used to investigate the reactivity of radioactively labeled viral RNAs from poliovirus-infected cells with antibodies to the synthetic double-stranded RNA, poly(I)-poly(C). A RNase-free antibody-containing serum fraction was employed. Poliovirus replicative form reacted with the antibodies to poly(I)-poly(C) as well as or better than poly(I)-poly(C). Poliovirus replicative intermediate reacted with the antibodies to a greater extent than poliovirus single-stranded RNA, but both were less reactive than replicative form. The use of the immunochemical binding assay with sucrose-gradient fractions demonstrated that for both poliovirus single-stranded RNA and replicative form the peak of reactivity with the antibodies was coincident with the peak of radioactive material precipitated by trichloroacetic acid. The proportion of replicative intermediate that reacted with the antibody increased in sucrose-gradient fractions containing the more slowly sedimenting RI RNA.     

Serum antibody and cellular immune response in mice to dextran B512.

Serum antibodies to dextran started to appear 3 days after immunization of C57BL/6 mice. Synthesis of IgM antibodies was followed by IgG3 and IgGA. Other immunoglobulin classes (IgG1, IgG2b, and IgG2a) were very low or absent. The immune response to dextran was also thymus independent with regard to IgG3 and IgA synthesis as demonstrated by the use of nu/nu mice. CBA and C57BL/6 mice were high responders to dextran with regard to IgM synthesis. C57BL/6 mice produced high levels of IgG3 and IgA antibodies, whereas CBA, A/J, and A.TL only synthesized IgM antibodies. A/J and A.TL strains were most frequently low responders with regard to IgM synthesis and CBA/N mice were completely nonresponders with regard to all immunoglobulin classes. The ability to produce anti-dextran antibodies increased with age in high responder strains. This was most pronounced for IgG3 and IgA antibodies, which reached adult levels 3 months after birth. The affinity of anti-dextran antibodies was high and homogeneous in antisera from C57BL/6 mice. Preimmune matural antibodies and antibodies from immunized low responder strains had a low and variable affinity for dextran.     

Potent neutralization by monoclonal human IgM against SARS‐CoV‐2 is impaired by class switch

To investigate the class‐dependent properties of anti‐viral IgM antibodies, we use membrane antigen capture activated cell sorting to isolate spike‐protein‐specific B cells from donors recently infected with SARS‐CoV‐2, allowing production of recombinant antibodies. We isolate 20, spike‐protein‐specific antibodies of classes IgM, IgG, and IgA, none of which shows any antigen‐independent binding to human cells. Two antibodies of class IgM mediate virus neutralization at picomolar concentrations, but this potency is lost following artificial switch to IgG. Although, as expected, the IgG versions of the antibodies appear to have lower avidity than their IgM parents, this is not sufficient to explain the loss of potency.     

Enhanced binding of antibodies to neutralization epitopes following thermal and chemical inactivation of human immunodeficiency virus type 1

Inactivation of viral particles is the basis for several vaccines currently in use. Initial attempts to use simian immunodeficiency virus to model a killed human immunodeficiency virus type 1 (HIV-1) vaccine were unsuccessful, and limited subsequent effort has been directed toward a systematic study of the requirements for a protective killed HIV-1 vaccine. Recent insights into HIV-1 virion and glycoprotein structure and neutralization epitopes led us to revisit whether inactivated HIV-1 particles could serve as the basis for an HIV-1 vaccine. Our results indicate that relatively simple processes involving thermal and chemical inactivation can inactivate HIV-1 by at least 7 logs. For some HIV-1 strains, significant amounts of envelope glycoproteins are retained in high-molecular-weight fractions. Importantly, we demonstrate retention of each of three conformation-dependent neutralization epitopes. Moreover, reactivity of monoclonal antibodies directed toward these epitopes is increased following treatment, suggesting greater exposure of the epitopes. In contrast, treatment of free envelope under the same conditions leads only to decreased antibody recognition. These inactivated virions can also be presented by human dendritic cells to direct a cell-mediated immune response in vitro. These data indicate that a systematic study of HIV-1 inactivation, gp120 retention, and epitope reactivity with conformation-specific neutralizing antibodies can provide important insights for the development of an effective killed HIV-1 vaccine.     

Fractionation of immunoglobulins by liquid-liquid partition chromatography in aqueous two-phase systems

In this paper we show that although immunoglobulins are easily precipitated in solutions containing polyethylene glycol (PEG), especially at pH's where the conformation of the proteins should be close to native, human and rabbit IgG can be solubilized in aqueous dextran/PEG two-phase systems containing glycine and sodium chloride at pH 7.0 and that human IgA and IgM can be solubilized in such systems if the pH is increased to 9.0. Liquid-liquid partition chromatography (LLPC) on Li-ParGel was used to separate immunoglobulins into subfractions. Human IgG, IgM, and IgA all gave three peaks in the system used. These results indicate the possibility of separating different classes of immunoglobulins with this method. Specific IgG antibodies isolated from a rabbit antiserum against human serum proteins gave only two peaks in the LLPC system while the total IgG population gave three, as did human IgG. Thus, partitioning of immunoglobulins seems to be related to antibody activity.     

Monoclonal anti-human tumor antibodies of six isotypes in cytotoxic reactions with human and murine effector cells

Eighty-seven murine monoclonal antibodies (MAb) produced against human tumors of various origins and representing six different immunoglobulin classes were tested for antitumor reactivity in antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) assays. Mouse splenocytes, thioglycolate-elicited mouse peritoneal macrophages, freshly obtained nonadherent human peripheral blood lymphocytes, and human monocytes were used as effector cells, and human or rabbit serum as the source of complement. Of all four effector cell types tested, mouse macrophages showed the highest cytotoxic activity, based on net cytotoxicity, minimum requirement for Mab concentration, and effector cell number. Different immunoglobulin classes were associated with characteristic patterns of reactivity with the various effector cells or complement, independent of the target cell type used. MAb able to mediate ADCC were found among all IgG subclasses, with IgG2a and IgG3 MAb inducing lysis with all effector cell types. IgM and IgA MAb were nonreactive in the various ADCC assays, but IgM MAb were highly cytotoxic with complement.     

Immune response to deoxyribonucleic acid (DNA) of African trypanosomes

Kushimo J. B. and Akinrimisi E. O. Immune response to deoxyribonucleic acid (DNA) of African trypanosomes. International Journal for Parasitology12: 537–540. Rabbits immunized with methylated bovine serum albumin (MBSA) complexes of nuclear DNA from either T. brucei or T. vivax which have low content of Adenine + Thymine (A + T) produced only IgM antibodies to denatured DNA. On the other hand rabbits immunized with MBSA complexes of denatured kinetoplast DNA (K-DNA) from either T. brucei or T. vivax which are rich in A + T content elicit both IgM and IgG antibodies. However the amount of IgG antibodies was higher than IgM antibodies. There seems to be a correlation between % AT content of immunogen and average ratio of maximum precipitable IgG to IgM (IgG)/(IgM).     

Western Blot analysis of the antigens of Toxoplasma gondii recognized by human IgM and IgG antibodies

Western Blot analysis revealed that both IgM and IgG antibodies present in the sera of humans infected with Toxoplasma gondii recognize three major antigens with apparent m.w. of 32,000, 22,000, and 6000, respectively. In addition, IgG antibodies recognized at least 17 other antigenic components. After subcellular fractionation, enrichment of the three major antigens recognized by IgM and IgG antibodies by the membrane fraction was observed. Solubilization of membrane-enriched preparations with a mixture of sodium dodecyl sulfate and sodium deoxycholate did not reveal any new antigenic structures that reacted with IgM or IgG antibodies. Treatment of Toxoplasma lysate preparations and various fractions obtained after differential centrifugation with NaIO4 diminished the reactivity of the antigens with both IgM and IgG antibodies. Lipase treatment had no effect on the number or nature of antigens recognized by IgM antibody. Treatment with pronase and trypsin eliminated the 32,000 and 22,000 m.w. antigenic components detected by IgM antibodies, whereas such treatment had no effect on the 6000 m.w. component. Periodic acid-Schiff staining of polyacrylamide gels of Toxoplasma sonicates revealed the presence of three components corresponding to m.w. of 62,000, 45,000, and 6000, respectively. At least 15 components, including the 6000 m.w. component, directly bound concanavalin A.     

Protonation-dependent inactivation of Na,K-ATPase by hydrostatic pressure developed at high-speed centrifugation

Irreversible inactivation of membranous Na,K-ATPase by high-speed centrifugation in dilute aqueous solutions depends markedly on the protonation state of the protein. Pig kidney Na,K-ATPase is irreversibly inactivated at pH 5 but is fully protected at pH 7 and above. Shark rectal gland Na,K-ATPase is irreversibly inactivated at neutral or acidic pH and partially protected at an alkaline pH. The overall Na,K-ATPase activity and the K-dependent pNPPase activity were denatured in parallel. Cryoprotectants such as glycerol or sucrose at concentrations of 25-30% fully protect both enzymes against inactivation. The specific ligands NaCl and KCl protect the Na,K-ATPase activity partially and the pNPPase activity fully at concentrations of 0.2-0.3 M. Electron microscope analysis of the centrifuged Na,K-ATPase membranes revealed that the ultrastructure of the native membranes is preserved upon inactivation. It was also observed that the sarcoplasmic reticulum Ca-ATPase and hog gastric H, K-ATPase are susceptible to inactivation by high-speed centrifugation in a pH-dependent fashion. H,K-ATPase is protected at alkaline pH, whereas Ca-ATPase is protected only in the neutral pH range.     

IgG autoantibody activity in normal mouse serum is controlled by IgM

In the serum of normal BALB/c mice, IgG antibody reactivity to mouse actin and tubulin, DNA, and TNP groups was very low compared to that of the IgM. This activity was considerably increased when IgG was separated, by affinity chromatography on protein A-Sepharose, whereas no difference in the IgM activity was observed. Addition of IgM to IgG isolated from the same serum resulted in the inhibition of IgG binding to these Ag. Isolation of IgG antibodies on actin, TNP, and tubulin immunoadsorbents has indicated that at least part of the IgG antibodies is polyreactive. In order to understand this inhibition better, experiments with F(ab')2 fragments of IgG were performed. IgM inhibited the binding of F(ab')2 to the antigens in a dose-dependent manner and reacted with immobilized F(ab')2. IgM isolated on F(ab')2 immunoadsorbent, as compared to the initial IgM preparation, were less active toward the Ag but more inhibitory for IgG binding to the Ag. In some pathologic situations, IgM failed to inhibit some IgG antibody activities. The anti-DNA IgG activity from (NZB x NZW)F1 mice was not affected by autologous IgM. Similarly the anti-tubulin IgG from mice infected with Trypanosoma cruzi were less inhibited by IgM from autologous serum than antitubulin IgG from normal mice. These results are compatible with the existence in normal mice of an idiotypic-like network, regulating via an IgM population in the serum, the binding of IgG autoantibodies to self Ag. Modifications of this idiotype-anti-idiotype system might lead to the expression and/or expansion of autoreactive IgG-producing clones.     

Ordering of antibodies, that react with cardiac muscle fiber and interstitial connective tissue antigens, in different immunoglobulin classes

Sera of patients suffering from rheumatic diseases and myocarditis were examined on the sections of human and bovine myocardial tissue by indirect immunofluorescence with the use of pure IgG antibodies or monospecific sera against IgG, IgA and IgM. It was shown that antibodies reacting with different myofibers and interstitial connective tissue of the heart belong to the main immunoglobulin classes (IgG, IgA and IgM). There was a significant predominance of IgG antibodies as shown by the frequency of their detection and by the titer height. The predominance of antibodies to certain classes of immunoglobulins did not correlate with a specific disease entity. The frequency of detecting antibodies to a certain immunoglobulin class was in good agreement with the time of the disease onset. Moreover, the frequency of positive reactions due to IgG, IgA, and IgM antibodies correlated with the level of the appropriate immunoglobulins in the test sera.