Comparative functional characterization of canine IgG subclasses

To date, very little is known about the functional characteristics of the four published canine IgG subclasses. It is not clear how each subclass engages the immune system via complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC), or how long each antibody may last in serum. Such information is critical for understanding canine immunology and for the discovery of canine therapeutic monoclonal antibodies. Through both in vitro and ex vivo experiments to evaluate canine Fc's for effector function, complement binding, FcRn binding, and ADCC, we are now able to categorize canine subclasses by function. The subclasses share functional properties with the four human IgG subclasses and are reported herein with their function-based human analog. Canine Fc fusions, canine chimeras, and caninized antibodies were characterized. Canine subclasses A and D appear effector-function negative while subclasses B and C bind canine Fc gamma receptors and are positive for ADCC. All canine subclasses bind the neonatal Fc receptor except subclass C. By understanding canine IgGs in this way, we can apply what is known of human immunology toward translational and veterinary medicine. Thus, this body of work lays the foundation for evaluating canine IgG subclasses for therapeutic antibody development and builds upon the fundamental scholarship of canine immunology.     

Immunoglobulin G subclass distribution in canine leishmaniosis: a review and analysis of pitfalls in interpretation

Infection with Leishmania may have different outcomes in genetically distinct individuals and the course of infection is determined by the nature of the host innate and adaptive immune response. Thus in experimentally infected mice, and in naturally infected dogs or humans, the protective (self-healing or asymptomatic) phenotype is associated with the induction of Th1-regulated cell-mediated immunity. By contrast, a Th2-regulated humoral immune response is associated with severe symptomatic disease. In the murine model system there is strong correlation between clinicopathological phenotype and the nature of the antigen-specific humoral immune response. Symptomatic infection and Th2-regulation is associated with elevation in antigen-specific IgG1 and IgE, whereas asymptomatic infection with Th1-regulation is linked with IgG2a production. IgG subclass restriction is less clear in human disease with only some clinical forms being correlated to a specific serological profile. Although numerous studies have questioned whether infected dogs develop skewed IgG subclass usage, the results of these have been conflicting-suggesting bias towards IgG1 or IgG2 or neither subclass in different investigations. This confusion could relate to the specificity of the commercially available polyclonal antisera used to detect the canine IgG1 and IgG2 subclasses. More meaningful results might be obtained by the use of the panel of monoclonal antibodies with well-validated specificity for all four canine IgG subclasses.     

Production and characterization of monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2

Monoclonal antibodies specific for sheep IgG subclasses IgG1 or IgG2 were produced using conventional cell fusion techniques. Monoclonal antibodies detected by preliminary screening assays were further characterized and their specificity verified by titration of ascites in radioimmunoassay or passive haemagglutination using pure sheep IgG1 or IgG2. Further evidence for the subclass specificity of the antibodies was obtained from immunoelectrophoretograms of sheep serum or purified proteins developed with monoclonal antibodies. Reaction of monoclonal antibodies with various IgG fragments showed that the determinants recognised were located on the pFc' portion of the heavy chain.     

Detection of specific antibody responses to vaccination in variable flying foxes (Pteropus hypomelanus)

Megachiropteran bats are biologically important both as endangered species and reservoirs for emerging human pathogens. Reliable detection of antibodies to specific pathogens in bats is thus epidemiologically critical. Eight variable flying foxes (Pteropus hypomelanus) were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each bat received monthly inoculations for 2 months. Affinity-purified IgG was used for production of polyclonal and monoclonal anti-variable flying fox IgG antibodies. ELISA and western blot analysis were used to monitor immune responses and for assessment of polyclonal and monoclonal antibody species cross-reactivity. Protein G, polyclonal antibodies, and monoclonal antibodies detected specific anti-DNP antibody responses in immunized variable flying foxes, with protein G being the most sensitive, followed by monoclonal antibodies and then polyclonal antibodies. While the polyclonal antibody was found to cross-react well against IgG of all bat species tested, some non-specific background was observed. The monoclonal antibody was found to cross-react well against IgG of six other species in the genus Pteropus and to cross-react less strongly against IgG from Eidolon helvum or Phyllostomus hastatus. Protein G distinguished best between vaccinated and unvaccinated bats, and these results validate the use of protein G for detection of bat IgG. Monoclonal antibodies developed in this study recognized immunoglobulins from other members of the genus Pteropus well, and may be useful in applications where specific detection of Pteropus IgG is needed.     

Evaluation of enzyme-linked immunosorbent assay for quantitation by subclass for bovine antibodies.

The enzyme-linked immunosorbent assay (ELISA) was evaluated for use in the quantitative measurement of bovine immunoglobulin IgG1 and IgG2 antibodies. A method for standardization was devised in which IgG1 or IgG2 was directly adsorbed to polystyrene tubes and the actual degree of binding was calculated by using different input amounts of 125I-labeled IgG1 or IgG2. Values for quantity of IgG1 antibodies to human serum albumin were only slightly higher when measured by the ELISA than when measured by quantitative precipitation although the value measured by the ELISA for IgG2 antibodies was twice that determined by quantitative precipitation. This discrepancy could result from conjugate cross reactivity, differences in affinity between antibodies of the 2 subclasses, or the occurrence of IgG2 nonprecipitating antibodies. The danger of overlooking subclass anti-globulin cross reactivity because of the failure to detect it by immunoprecipitation, also is illustrated. In addition, only enzyme-antibody conjugates prepared with specifically purified antibodies were effective, and reproducibility of individual data points required that 4 replicate determinations be performed. Advantages, pitfalls, and limitations of the ELISA are discussed.     

Use of an ELISA for detection of antibody responses in Argentine boa constrictors (Boa constrictor occidentalis)

OBJECTIVE: To develop mouse monoclonal and rabbit polyclonal antibodies against immunoglobulin of Argentine boa constrictors and to demonstrate the ability of these reagents to detect antibody responses in boa constrictors by use of an ELISA and western blot analysis. ANIMALS: Two 3-year-old Argentine boa constrictors. Procedure-Boa constrictors were immunized with 2,4-dinitrophenylated bovine serum albumin (DNP-BSA). Each snake received biweekly inoculations of 250 microg of DNP-BSA (half SC, half IP) for a total of 6 inoculations followed by monthly inoculations for 3 months. Preimmune blood samples were collected. Subsequently, blood was collected immediately prior to each booster inoculation. Anti-DNP antibodies were isolated from immune plasma samples by affinity chromatography. Affinity-purified boa anti-DNP immunoglobulin was used for production of polyclonal and monoclonal antibodies. An ELISA and western blot analysis were used to monitor immune responses, for purification of boa anti-DNP immunoglobulin, and for assessment of polyclonal and monoclonal antibody specificity. RESULTS: A 6-fold increase in optical density (OD405) of immune boa plasma, compared with preimmune plasma, was detected by the polyclonal antibody, and a 12- and 15-fold increase was detected by monoclonal antibodies HL1787 and HL1785, respectively, between weeks 4 and 8. Results of western blot analysis confirmed anti-DNP antibody activity in immunized boa plasma and in affinity column eluates. Polyclonal and monoclonal antibodies detected specific anti-DNP antibody responses in immunized boas. CONCLUSIONS AND CLINICAL RELEVANCE: Polyclonal and monoclonal antibodies recognized boa constrictor immunoglobulin. These antibodies may be useful in serologic tests to determine exposure of snakes to pathogens.     

Bovine herpesvirus-1 infection of cattle: kinetics of antibody formation after intranasal exposure and abortion induced by the virus

The kinetics of antibody formation in Holstein heifers after primary and secondary intranasal inoculation of bovine herpesvirus-1 (BHV-1) and after BHV-1-induced abortion was determined. Sera were fractionated by gel filtration and ion-exchange chromatography. The antibody activity within serum immunoglobulin (Ig) isotypes was assessed, using a plaque-reduction neutralization assay. The primary immune response to BHV-1 infection was characterized by the appearance of IgM and IgG antibodies in serum by postinoculation day (PID) 7. Maximal IgG antibody activity occurred at PID 35 in nonpregnant heifers and at PID 14 in pregnant heifers. Thereafter, IgG antibody activity declined slowly in both groups of heifers. Maximal IgM antibody activity occurred at PID 14 in both groups of heifers and declined rapidly thereafter. The IgG antibody activity during primary immune responses was restricted to the IgG1 subclass. Secondary responses were characterized by anamnestic IgG antibody responses. Antibody activity was present within the IgG1 and IgG2 subclasses during secondary immune responses, but the increase in antibody activity during this period was primarily in the IgG2 subclass. Secondary IgM antibody formation was elicited by abortion induced by the intra-amniotic inoculation of BHV-1, but not by reexposure by the intranasal route. Abortion occurred in 1 heifer 28 days after intranasal BHV-1 inoculation. Abortion in this heifer was not associated with a secondary antibody response. The nature of BHV-1 antigenic exposure in the bovine determined the relative distribution of anti-BHV-1 antibody activity in serum IgM, IgG1, and IgG2. The formation of IgM antibody, with the exception of secondary intranasal exposure, indicated recent BHV-1 antigenic exposure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of IgG subclass expression during antibody responses in sheep

Experiments were undertaken to investigate IgG subclass expression during epitope-specific antibody responses in sheep. Animals were immunised with conjugates of bovine serum albumin-DNP (BSA-DNP), or killed Staphylococcus aureus-DNP (Sa-DNP) alone or with muramyl dipeptide (MDP), dextran sulphate (DXS), or staphylococcal exotoxins (toxin). Sheep received two injections of the same preparation intracutaneously at six weeks interval. Total and IgG subclass-specific anti-DNP, and anti-carrier (BSA or S aureus) antibody levels were measured by ELISA in blood taken at weekly intervals before and after immunisation. Anti-DNP antibody levels in animals given Sa-DNP alone were considerably greater than in those immunised with BSA-DNP alone. Toxin suppressed antibody responses to DNP and both carriers; MDP suppressed anti-hapten antibody responses below the levels obtained with antigen alone. Neither toxin nor MDP significantly altered the IgG subclass profile of antibody to DNP bound to either carrier. DXS did not significantly change total levels of anti-DNP antibody measured in sheep given BSA-DNP or S aureus-DNP. However, DXS promoted IgG1 and suppressed IgG2 anti-DNP antibody responses during the secondary response to Sa-DNP but not to BSA-DNP.     

H3N8亚型马流感病毒单克隆抗体的制备及鉴定

以灭活马流感病毒(EIV)A/Equine/Jilin/1/1989(H3N8)为免疫原,免疫Balb/c小鼠,经常规细胞融合后,用血凝抑制试验(H1)和间接ELISA方法筛选获得3株(3C2、5G10和5A10)能稳定分泌H3N8亚型马流感病毒单克隆抗体(mAb)的杂交瘤细胞株.其中3C2和5G10为IgG2α,5A...     

Comparison of monoclonal and polyclonal antibodies for the detection of canine IgG1 and IgG2, and associations with infection outcome in Leishmania infantum naturally infected dogs

In murine models of leishmaniasis, IgG subclass expression is a proxy measure for Th1/Th2 cellular immune response bias. However, in dogs, the reservoir of zoonotic visceral leishmaniasis, no consistent association has been described between IgG subclass ratios and disease resistance. Inconsistent results may reflect lack of specificity of commonly used commercial antibodies. Our aim was to measure IgG1 and IgG2 responses to crude Leishmania antigen using commercial polyclonal antibodies for comparison with a panel of commercially unavailable monoclonal antibodies, in a cohort of 60 naturally infected dogs, and to compare associations between subclass responses and clinical or parasitological outcomes. IgG1 and IgG2, measured by both antibodies, were higher in clinically symptomatic than in asymptomatic dogs (P ≤ 0.03), reflecting general upregulation of IgG in infected dogs. Unlike the murine model, canine IgG2:IgG1 ratios were not predictive of clinical or parasitological outcomes of infection. Associations between subclass levels and positivity by bone marrow culture and PCR were not consistent when measured with different antibodies. Further research is needed to re-evaluate the specificity of commercially available IgG subclass antibodies.     

Mononuclear phagocyte receptors in rhesus monkeys (Macaca mulatta) and their role in hemolytic disease of the newborn

Hemolytic disease of the newborn does not develop in rhesus monkeys because placentally-transferred maternal antibodies do not induce immune clearance of the newborn's erythrocytes. In an in vitro RBC adherence assay, rhesus peripheral blood monocytes did not bind newborn's RBC which had been coated in utero or in vitro with maternal antibodies. Nevertheless, rhesus phagocytes possess receptors that are specific for the Fc portion of IgC and for the C3b. Using purified human IgG subclasses as inhibitors of RBC adherence, rhesus Fc receptors preferentially bind IgG1 and IgG3. Thus, it may be that maternal antibodies are non-opsonic because they belong to IgG subclasses that do not bind effectively to rhesus Fc receptors. Also, RBC adherence appears to be controlled by the level of antibody coating which in turn is determined by avidity of the antibodies and by the number of RBC membrane determinants. The failure of maternal antibodies to opsonize the newborn's RBC and thus cause hemolytic disease is very likely due to the low avidity of antibodies and to the weak expression of blood group determinants on the membranes of these RBC.     

The response of rabbits to the subclass specific epitopes of bovine IgG1 and IgG2

Thirty-two random breed New Zealand White rabbits were immunized with purified bovine IgG1, IgG2, unfractionated bovine IgG and a pepsin digest of bovine IgG using 75 μg and 2 mg doses of each immunogen. The amount of total precipitating antibodies, IgG1 subclass-specific antibodies (SSAb) and IgG2 SSAbs were measured by reverse single radial diffusion. Rabbits responded equally as well to IgG2 as to IgG1 subclass specific epitopes. The amount of SSAb varied from 13–69% of the total precipitating antibodies. Immunodiffusion analyses indicated that some subclass-specific determinants were located elsewhere on the heavy chain than on the papain-generated Fc. Considerable individual variation in responsiveness was noted although there was a tendency for higher doses of purified subclass proteins to produce a higher proportion of SSAbs. Administration of a second antigen booster uniformly resulted in a significant reduction in the amount of SSAb. Finally the enzyme susceptibility of IgG2 to pepsin provides a simple method for obtaining an immunogen for stimulating exclusively IgG2 SSAbs.     

禽脑脊髓炎病毒单克隆抗体的制备、鉴定与初步应用

为获得禽脑脊髓炎病毒(Avian Encephalomyelitis virus,AEV)VP1蛋白的单克隆抗体,通过原核表达AEV VP1蛋白,纯化后作为免疫原免疫BALB/c小鼠,并按常规方法制备杂交瘤细胞。经ELISA方法筛选阳性杂交瘤细胞,经过3次亚克隆获得2株杂交瘤细胞株,命名为4#、19#,并进行了抗体亚类的鉴定、Western-blot和IFA检测。结果显示：制备的株单克隆抗体亚型分别为IgG2b、IgG2a,Western-blot和IFA试验结果表明单克隆抗体均能与AEV发生特异性反应而与其他禽病常见病毒均无交叉反应。运用建立的IFA对单抗进行了初步运用,在外源病毒检测方面与经典方法符合率高。本研究成功制备了AEV单克隆抗体,为进一步建立AEV检测方法和深入研究AEV的生物学特性奠定了基础。     

IgG subclass responses in a longitudinal study of canine visceral leishmaniasis

Visceral leishmaniasis (VL), caused by Leishmania infantum, is an important disease of domestic dogs. Here, we present data on the IgG subclass antibody response to crude L. infantum antigen in a cohort of naturally infected Brazilian dogs. Specific IgG1-IgG4 responses could be detected in 98, 58, 70 and 82%, respectively of 57 dogs that were seropositive for specific IgG. Levels of all IgG subclasses were strongly inter-correlated. Levels of all IgG subclasses increased at the time of seroconversion, before reaching a plateau after several months. Levels of all IgG subclasses were higher in sick dogs than healthy dogs, and levels of all except IgG2 were higher in parasite-positive (by PCR) than parasite-negative dogs. However, levels of IgG2 relative to IgG1 were lower in sick or parasite-positive dogs compared to healthy or parasite-negative infected dogs. In contrast to previous studies, the results suggest that canine VL is associated with upregulation of specific antibody of all IgG subclasses, particularly IgG1, IgG3 and IgG4.     

Development and validation of monoclonal and polyclonal antibodies for the detection of immunoglobulin G of bottlenose dolphins (Tursiops truncatus).

Antibodies directed against species-specific immunoglobulin G (IgG) have a broad range of applications in serologic and immunologic research and in the development of clinical assays. Validated anti-IgG antibodies for marine mammal species are in short supply. The objective of this study was to produce and validate antibodies with specificity for IgG of the common bottlenose dolphin (Tursiops truncatus). Bottlenose dolphin IgG was purified using protein G. Two mouse monoclonal antibodies and a rabbit polyclonal antibody were developed from mice and rabbits immunized with bottlenose dolphin IgG. The specificity of the monoclonal antibodies and the polyclonal antibody for bottlenose dolphin IgG was first verified by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). For further validation, both monoclonal antibodies and the polyclonal antibody were incorporated in an indirect ELISA for the detection of the immune response of bottlenose dolphins to a vaccine antigen. Three bottlenose dolphins were immunized with a commercial Erysipelothrix rhusiopathiae vaccine, and serial blood samples were collected from all dolphins for measurement of levels of circulating antibodies. Seroconversion was observed in all 3 dolphins by use of both monoclonal antibodies and the polyclonal antibody. Circulating antibodies were detectable as early as 6 days after immunization in 1 dolphin. Peak antibody levels were detected 14 days after the immunization. The ability to detect seroconversion in all 3 immunized bottlenose dolphins firmly establishes the specificity of the monoclonal antibodies and the polyclonal antibody for IgG of the common bottlenose dolphin.     

A congenital persistent swine fever infection. II. Immune response to swine fever virus and unrelated antigens

After the disappearance of maternal antibodies, no antibodies were detected in five pigs that were congenitally infected with the Bergen strain of swine fever virus. The pigs lived for 2–11 months. One pig showed low levels of precipitating antibody during the last month of its life. The pigs had a normal immune response to sheep red blood cells and to pig parvovirus. These observations suggest the presence of immunological tolerance to swine fever virus in these pigs.Cell mediated immunity, measured by phytohaemagglutinin lymphocyte stimulation, appeared to be normal.     

Ginseng extract in aluminium hydroxide adjuvanted vaccines improves the antibody response of pigs to porcine parvovirus and Erysipelothrix rhusiopathiae

Ginseng, the dry extract prepared from the Panax ginseng C.A. Mayer-root contain immunomodulators named ginsenosides, which in the pig enhance the antibody response to viral and bacterial antigens. The enhancing effect of ginseng was demonstrated vaccinating pigs against porcine parvovirus (PPV) and Erysipelothrix rhusiopathiae infections, using commercially available vaccines. The potency of the licensed, aluminium hydroxide adjuvanted; vaccines were compared with those supplemented with ginseng. The antibody response to PPV was measured by the haemagglutination inhibition (HI) test whereas the mouse potency test and ELISA evaluated the immune response to E. rhusiopathiae. Antibodies to the 64-66 kDa glycoprotein of the E. rhusiopathiae were demonstrated by immunoblotting. The qualitative antibody responses were evaluated by means of ELISA(s) using monoclonal antibodies to swine IgG1 and IgG2. The addition of 2mg ginseng per vaccine dose, potentiate the antibody response of the commercial vaccines without altering their safety. Significantly higher (P<0.001) antibody titres were achieved to both PPV and to E. rhusiopathiae by the supplementation with ginseng. Aluminium hydroxide adjuvanted vaccines favoured the production of IgG1 antibodies. Interestingly, the vaccines supplemented with ginseng favoured IgG2. The vaccines used in the evaluations varied in their immunogenic potency. However, after the addition of ginseng the less immunogenic vaccine proved to be as potent as the better one without ginseng. Thus, the use of ginseng as a co-adjuvant provides a simple, safe and cheap alternative for improving the potency of aluminium hydroxide adjuvanted vaccines.     

IgG precipitating and non-precipitating antibodies in rabbits repeatedly injected with soluble and particulate antigens

The immune response of precipitating antibodies and non-precipitating antibodies of high affinity (co-precipitating) of the IgG class was analyzed in rabbits repeatedly injected with egg albumin (as a soluble antigen) B. abortus-egg albumin and polymerized egg albumin (as particulate antigens). The results showed that the levels of anti-egg albumin non-precipitating antibodies induced by the soluble antigen were never higher than 10-15% of total antibodies throughout the experimental time. When particulate antigens were injected, the levels of non-precipitating antibodies increased up to 30-70% of the total antibody levels. This phenomenon is related to the way in which the antigen is available to the immune system (particle or aggregated), and is independent of the response induced by the particulate carrier. Components from the cell wall or bacterial membrane that could act as coadjuvants do not participate in this phenomenon. The results obtained seem to indicate that possibly there was a suppression of the synthesis of precipitating antibodies, and this would produce a relative increase in the non-precipitating antibodies.     

Development of a hyena immunology toolbox

Animals that hunt and scavenge are often exposed to a broad array of pathogens. Theory predicts the immune systems of animals specialized for scavenging should have been molded by selective pressures associated with surviving microbial assaults from their food. Spotted hyenas (Crocuta crocuta) are capable hunters that have recently descended from carrion feeding ancestors. Hyenas have been documented to survive anthrax and rabies infections, and outbreaks of several other viral diseases that decimated populations of sympatric carnivores. In light of the extreme disease resistance manifested by spotted hyenas, our objective was to identify tools available for studying immune function in spotted hyenas and use these tools to document the hyena antibody response to immunization. Domestic cats (Felis catus) are the closest phylogenetic relatives of hyenas that have been studied in detail immunologically, and we hypothesized that anti-cat isotype-specific antibodies would cross react with hyena immunoglobulin epitopes. We used ELISA and Western blots to test isotype-specific anti-feline antibodies for specific cross-reaction to hyena Ig epitopes. Molecular weights of heavy (IgA, IgG, IgM) and light chains of hyena immunoglobulins were determined by protein electrophoresis, and as expected, they were found to be similar to feline immunoglobulins. In order to further validate the cross-reactivity of the anti-feline antibodies and document the hyena humoral response, eight spotted hyenas were immunized with dinitrophenol conjugated keyhole limpet hemocyanin (DNP-KLH) and serum anti-DNP responses were monitored by enzyme-linked immunosorbent assay (ELISA) for one year. The full array of isotype-specific antibodies identified here will allow veterinarians and other researchers to thoroughly investigate the hyena antibody response, and can be used in future studies to test hypotheses about pathogen exposure and immune function in this species.