Canine IgM Research Articles

A novel colorimetric strategy based on canine IgM for ultrasensitive and selective detection of Staphylococcus aureus

Conventional methods for Staphylococcus aureus (S. aureus) detection using mammalian immunoglobulins could yield false positive results, owing to the presence of the protein G-producing Streptococcus. In this study, because canine IgM can bind to protein A but not to protein G, we firstly designed a colorimetric platform based on canine IgM to identify S. aureus. Magnetic beads (MBs) functionalized with canine IgM acted as captors to enrich S. aureus, relying on the binding interaction between the Fc region of canine IgM and protein A in the bacterial cell wall, while horseradish peroxidase (HRP)-labeled rabbit anti-S. aureus IgG served as a tracer in the sandwich-type immunoassay, catalyzing the colorimetric reaction. Under optimal conditions, the proposed strategy achieved a satisfactory analytical performance with a broad linear range from 1.6 × 103 CFU/mL to 1.0 × 105 CFU/mL, a colorimetric minimum resolution of as low as 1.4 × 102 CFU/mL in 100 μL phosphate-buffered saline (PBS), and good selectivity in differentiating target from protein G-producing Streptococcus. Moreover, the proposed approach successfully identified S. aureus in various application scenarios (except for samples containing IgG), can be completed in less than 90 min, and was not dependent on any specialized equipment.

Staphylococcus pseudintermedius Sbi paralogs inhibit complement and bind IgM, IgG Fc and Fab

The success of staphylococci as pathogens has been attributed, in part, to their ability to evade their hosts’ immune systems. Although the proteins involved in evasion have been extensively studied in staphylococci affecting humans little characterization has been done with Staphylococcus pseudintermedius, an important cause of pyoderma in dogs. Staphylococcus aureus binder of immunoglobulin (Sbi) interferes with innate immune recognition by interacting with multiple host proteins. In this study, a S. pseudintermedius gene that shares 38% similarity to S. aureus Sbi was cloned from S. pseudintermedius strains representative of major clonal lineages bearing two paralogs of the protein. Binding of immunoglobulins and Fab and Fc fragments as well as interaction with complement was measured. S. pseudintermedius Sbi protein bound IgG from multiple species and canine complement C3, neutralized complement activity and bound to canine IgM and B cells. Evidence from this work suggests Sbi may play an important role in S. pseudintermedius immune evasion.

Production and Characterization of IgY against Canine IgG: Prospect of a New Tool for the Immunodiagnostic of Canine Diseases

This study describes the production of a new avian polyclonal antibody (IgY) against canine IgG, as another tool for the immunodiagnostic using IgY technology. The immunization protocol caused neither deaths nor pathologies, and no decline in egg laying capacity was detected. The total concentration of isolated IgY was constant, without significant difference (p> 0.05), with average of 97.55 mg of IgY/yolk. The IgY revealed a strong sensitivity and specificity in recognition against canine IgG by ELISA. After the immunization, there was a significant increase in the production of IgY specific from the first to the second month (p <0.05), reaching a stable peak without decrease in the production by the end of the analysis period (p > 0.05). The IgY demonstrated a suitable specificity in Western blot against the purified and serum canine IgG, not enabling recognition of canine IgM or IgG of other animal species. The specific IgY in the egg yolks of immunized hens proved to be a molecule with an appropriate purity and desired specificity against the immunizing antigen. Moreover, its constant production in large quantities during the four months analyzed indicated that IgY antibody production technology could be considered as an excellent alternative to the standard methods.

Production and Characterization of IgY against Canine IgG: Prospect of a New Tool for the Immunodiagnostic of Canine Diseases

This study describes the production of a new avian polyclonal antibody (IgY) against canine IgG, as another tool for the immunodiagnostic using IgY technology. The immunization protocol caused neither deaths nor pathologies, and no decline in egg laying capacity was detected. The total concentration of isolated IgY was constant, without significant difference (p> 0.05), with average of 97.55 mg of IgY/yolk. The IgY revealed a strong sensitivity and specificity in recognition against canine IgG by ELISA. After the immunization, there was a significant increase in the production of IgY specific from the first to the second month (p <0.05), reaching a stable peak without decrease in the production by the end of the analysis period (p > 0.05). The IgY demonstrated a suitable specificity in Western blot against the purified and serum canine IgG, not enabling recognition of canine IgM or IgG of other animal species. The specific IgY in the egg yolks of immunized hens proved to be a molecule with an appropriate purity and desired specificity against the immunizing antigen. Moreover, its constant production in large quantities during the four months analyzed indicated that IgY antibody production technology could be considered as an excellent alternative to the standard methods.

Purification and Characterization of Canine Serum Ferritin-binding Proteins

Ferritin-binding protein (FBP) is known to interact with circulating ferritins in mammals. Canine FBPs were purified from canine serum by affinity chromatography and were identified as IgM, IgG, and IgA by immunoblotting with alkaline phosphatase-labeled antibodies to canine IgM, IgG, and IgA heavy chains. Following further purification by application to a Sephacryl S-300 column, canine FBPs were separated into 81.3- and 27.7-kDa bands by sodium dodecyl sulfate-polyacryamide gel electrophoresis, and the 81.3-kDa band reacted with the anti-canine IgM heavy chain antibody. Purified canine FBP bound to canine liver ferritin, but not to canine albumin and transferrin. FBP showed greater binding to the expressed bovine ferritin H-chain homopolymer than to the expressed bovine ferritin L-chain homopolymer. The binding of FBP with canine liver ferritin was dose-dependently inhibited by anti-rat liver ferritin antibody, and the anti-ferritin antibody dissociated the bound FBP in a dose-dependent manner, even after binding FBP with liver ferritin. The canine ferritin H subunit peptide fragment with amino acid residues 148-155 (NH(2)-GDHVTNLR-COOH) in its C-terminal region was recognized by FBP. These results indicate that canine serum FBPs are autoantibodies to ferritin (IgM, IgG, and IgA) and that anti-ferritin autoantibody (IgM) recognizes the C-terminal region of ferritin H subunit.

Characterization of autoantibodies to ferritin in canine serum.

Ferritin-binding proteins circulating in mammalian blood are thought to be involved in the clearance of ferritin. The present study characterizes canine serum autoantibodies (IgM and IgA) that react with ferritin. Canine IgM and IgA bound to bovine spleen ferritin as well as canine liver ferritin. To examine the specificity of canine IgM and IgA to ferritin H and L subunits, we used canine heart ferritin and canine liver ferritin with H/L subunit ratios of 3.69 and 0.43, respectively. Canine IgM and IgA recognized both of the H- and L-subunit-rich isoferritins, showing that their binding activities to ferritin depend on the H-subunit content. Recombinant bovine H-chain ferritin homopolymer expressed in a baculovirus expression system bound more with IgM and IgA than the recombinant L-chain homopolymer expressed under the same conditions. These results suggest that canine IgM and IgA recognize H-subunit-rich isoferritins, and that H-subunit-rich isoferritins are cleared from the circulation more rapidly than L-subunit-rich isoferritins.

Characterization of ferritin and ferritin-binding proteins in canine serum.

Ferritin and ferritin-binding proteins in canine serum were characterized. A certain percentage of ferritin in canine serum, but no tissue ferritin, was precipitated by centrifugation at 16,000 x g for 30 min. The precipitated ferritin was found to contain two subunits corresponding to the H and L subunits of canine liver ferritin by immunoblotting, the H subunit being predominant. More ferritin was precipitated from canine sera which had been incubated with anti-rat liver ferritin antibody than from untreated sera, and the H chain also predominated. To evaluate the possibility that the autoantibody was responsible for the precipitation of canine serum ferritin, the ferritin-binding activities of canine antibodies were examined using liver ferritin-coated microtiter plates and alkaline phosphatase-labeled antibodies specific for canine IgM, IgA, and IgG heavy chains. The results showed that IgM and IgA, but not IgG, had considerable ferritin-binding activities. Given these results, we suggest that there is H-chain-rich isoferritin in canine serum, and that ferritin exists as an immune complex.

Staphylococcal protein A binding to canine IgG and IgM

Staphylococcal protein A (SpA) binds with high affinity to immunoglobulins from many species, making it a usefel reagent for immunoassays and immunoglobulin purification procedures. However, its use is limited by poor reactivity with some immunoglobulin subclasses including human IgG 3 and murine IgG 1. Reports of SpA's reactivity with canine immunoglobulins have been inconsistent. Because the most recent reports indicated a much greater reactivity of purified SpA with canine immunoglobulins than was suggested by our observations, we quantitatively reevaluated the binding of canine IgG and IgM to Cowan I strain SpA under the conditions of use in our laboratory. IgG and IgM were purified from pooled normal canine plasma by affinity chromatography with heavy chain specific polyclonal anti-IgG and anti-IgM antibodies. The purified IgG and IgM were assessed for SpA reactivity by affinity chromatography using a SpA-agarose column. The relative proportions of total chromatographed IgG or IgM in the flow-through (SpA-nonbindable) and eluate (SpA-bindable) fractions were determined by absorbance at 280 nm. The IgG and IgM in each immunoglobulin fraction were also nonspecifically adsorbed to microtitration plates and tested for reactivity with 125I-SpA using a solid phase immunoradiometric assay (IRMA). Approximately 18% of the affinity purified canine IgG and 33% of the affinity purified canine IgM did not bind to the SpA affinity column and were also unreactive with 125I-SpA using the IRMA. A second approach using a different polyclonal antibody to canine IgG yielded similar results: about 21% of the purified IgG was unreactive with SpA. Incomplete reactivity of SpA with canine IgG and IgM limits the usefulness of SpA in canine immunologic procedures.

Seroepidemiology of canine leptospirosis on the island of Barbados

Previous surveillance in Barbados documented the absence of infection with Leptospira serogroup Canicola in dogs. The aim of this study was to survey the current state of canine leptospirosis in Barbados, 10 years after the last survey. Sera from 78 unwanted dogs scheduled for euthanasia and 61 dogs suspected of having acute leptospirosis were tested by microscopic agglutination (MAT) and by an ELISA method adapted for canine IgM and IgG antibodies. The seroprevalence in unwanted dogs was 62% ( 48 78 ), at an MAT titre of ≥ 100. The majority of animals had low titres, suggestive of previous infection. Serogroup Autumnalis was the most common reactor (45%), followed by serogroups Icterohaemorrhagiae and Australis (each 16%) and Pomona (13%). Serogroup Ballum was uncommon in this group. The seroprevalence determined by MAT in acutely-ill dogs was 75% ( 46 61 ). The most common predominant serogroup was Icterohaemorrhagiae (36%) followed by serogroup Australis (13%), while serogroups Autumnalis and Ballum were also of little significance. Paired specimens were available from eight acutely-ill dogs. One animal was seronegative while five dogs showed evidence of seroconversion. An IgM-ELISA titre of ≥ 320 was used to confirm current infection in eight of these nine animals. Previous studies in Barbados showed a higher prevalence of serogroup Icterohaemorrhagiae than of Autumnalis, but the relative frequency of these two serogroups may be changing. The high seroprevalence in dogs is of public health concern because the close contact between dogs and man may provide the link between a reservoir in the environment and susceptible humans.

Lysis of Human Tumor Cell Lines by Canine Complement plus Monoclonal Antiganglioside Antibodies or Natural Canine Xenoantibodies

Because certain antiganglioside monoclonal antibodies can facilitate antibody-dependent cellular cytotoxicity against GD2+ganglioside-bearing human and canine tumor cells, we wished to determine if clinically relevant antiganglioside monoclonal antibodies (Mabs) could also fix canine complement to lyse tumor cellsin vitro.Using flow cytometry, human tumor cell lines (M21 melanoma and OHS osteosarcoma) were shown to highly express ganglioside GD2 and, to a lesser degree, GD3. In51Cr release assays, M21 cells were lysed with canine serum, as a source of complement, plus either Mab 14.G2a or its mouse–human chimera, ch 14.18, specific for GD2. Heating canine serum abrogated its lytic activity and addition of rabbit complement reconstituted M21 lysis. Similar results were obtained with M21 cells when Mab R24 (against GD3) and canine serum were used. OHS cells were also lysed with canine serum plus Mab 14.G2a and lytic activity was abolished by heating canine serum but reconstituted with rabbit complement. Alone, canine serum or Mabs were not lytic to M21 or OHS cells. Conversely, human neuroblastoma (LAN-5) and K562 erythroleukemia cells were lysed by canine serum alone which was shown by flow cytometry to contain naturally occurring canine IgM antibodies that bound LAN-5 and K562 cells. The lytic activity of canine serum for LAN-5 or K562 cells was abolished by heating and restored by addition of either human or rabbit complement. Thus, human tumor cell lines can be lysed with antiganglioside Mabs through fixation and activation of canine complement-dependent lytic pathways. Canine xenoantibodies also mediate complement-dependent cytotoxicity of some human tumor cell lines. Together, these results are significant because they demonstrate an antitumor effect of the canine immune system which is of potential importance for cancer immunotherapy in a promising animal model.

Evidence That Fibronectin-Immunoglobulin Complexes Occur Normally in Plasma

This study was done to ascertain whether complexes of plasma fibronectin (PFn) and immunoglobulins (Igs) are normally present in plasma. PFn preparations from plasmas of six random human donors were analyzed for IgG, IgM, and IgA by competitive ELISA procedures. To look for direct evidence of circulating complexes, two of the plasma samples were chromatographed on Sephacryl S-300. PFn prepared from a pool of three plasmas by cryoprecipitation was studied by crossed immunoelectrophoresis to detect PFn-Ig complexes. The Ig content of PFn ranged from 0.78% to 9.0% IgG; 0.25% to 4.9% IgM, and 0.15% to 0.71% IgA. The relative amounts were IgG > IgM > IgA in every sample, but the total Ig content of PFn varied with the plasma donor. The Sephacryl S-300 chromatogram of the plasma from which Ig-rich PFn was made showed distortion of the PFn peak by Ig peaks. PFn prepared by cryoprecipitation contained PFn-immunoglobulin complexes. Plasma from adult beagle dogs was used to prepare canine PFn. IgM was detected in canine PFn by SDS-Page and by double diffusion experiments against antiserum to canine IgM. By crossed immunoelectrophoresis, a PFn-IgM complex was detected in canine plasma. The cryoprecipitate recovered from a Sephacryl S-300 chromatogram of canine plasma contained equivalent amounts of IgM and PFn. These data suggest that complexes of Igs with PFn are normally present in plasma.

Prolongation of cardiac xenograft survival by double filtration plasmapheresis and ex vivo immunoadsorption.

A double filtration plasmapheresis (DFPP) technique and ex vivo immunoadsorption were applied to remove natural antibodies and avoid hyperacute rejection in discordant xenotransplantation. A swine heart was heterotopically transplanted into a dog's neck after DFPP and ex vivo immunoadsorption using a swine spleen or liver. Mean graft survival time was prolonged to 240 +/- 141 min in the group treated by combined DFPP and splenic adsorption, and 225 +/- 62 min in the group treated by combined DFPP and hepatic adsorption, whereas it was 9 +/- 5 min in the group without any treatment (p < 0.01). Mean removal rates of IgG and IgM were 85.5% and 93.3%, respectively. Anti-swine lymphocytotoxic antibodies and hemagglutination antibodies were also effectively removed. Deposits of canine IgM and C3 on the vascular endothelium of the graft were observed on immunofluorescence, which suggested that natural antibodies in the IgM fraction played an important role in xenohyperacute rejection.

Isolation of human and canine IgM utilizing protein A affinity chromatography.

Human and canine high molecular weight IgM's were isolated employing a system of G-200 column chromatography and passage through a protein A Sepharose 4B column. Polyacrylamide Gel Electrophoresis (PAGE) analysis of the isolated immunoglobulin revealed polypeptides corresponding to mu and light immunoglobulin chains of IgM which were identified immunochemically as IgM. Ultracentrifugation studies revealed that the isolated immunoglobulin co-migrated with 19S IgM markers. This simple and efficient procedure may serve as an alternative to classical isolation procedures of IgM from certain mammalian species.

Protein A reactivity of various mammalian immunoglobulins.

Serum samples and immunoglobulin fractions of eight mammalian species were applied to a Sepharose--protein A column. As with the human immunoglobulin subclasses IgG1, IgG2 and IgG4, all examined IgG classes and subclasses were bound to a greater or lesser extent to protein A. However, the binding of IgG1 of ruminants was very poor. Polyclonal IgM and IgA of the pig, the dog and the cat may be separated in protein A reactive and protein A non-reactive fractions. In addition, monoclonal canine IgM and IgA partially reacted with protein A. In combination with methods such as ammonium sulphate precipitation, ion exchange chromatography and gel-filtration, affinity chromatography with protein A is recommended for the rapid purification of certain Ig (sub)classes of a number of mammalian species.