Application of a direct flow cytometric erythrocyte immunofluorescence assay in dogs with immune-mediated hemolytic anemia and comparison to the direct antiglobulin test.

A direct flow cytometric erythrocyte immunofluorescence assay (FC) was developed and compared with the direct antiglobulin test (DAT) for detection of erythrocyte-bound immunoglobulin (IgG and IgM) and complement (C3) in dogs with immune-mediated hemolytic anemia (IMHA). Tests were performed on erythrocytes from 13 healthy nonanemic dogs and from 13 anemic dogs with IMHA. The FC and DAT were negative for erythrocyte-bound immunoglobulin in all healthy dogs. The FC was negative for erythrocyte-bound C3 in 12 healthy dogs and positive in 1 healthy dog, and the DAT was negative for C3 in all healthy dogs. Of the 13 IMHA dogs tested for erythrocyte-bound IgG, 12 were positive using the FC and 7 were positive using the DAT. Sensitivity for the detection of erythrocyte-bound IgG in the 26 dogs was 92% for FC and 53% for DAT. Specificity for detection of erythrocyte bound IgG for FC and DAT was 100%. The addition of IgM and/ or C3 did not increase the sensitivity for FC or DAT. In this group of dogs, the FC provided a more rapid, cost-effective, sensitive, objective method to quantitate erythrocyte-bound immunoglobulin and/or complement compared with the currently used DAT.     

Serum concentrations of the third component of complement in healthy dogs and dogs with protein-losing nephropathy

OBJECTIVE: To develop a method for determining the concentration of the third component of complement (C3) in canine serum, to establish a reference range for C3 in healthy dogs, and to evaluate dogs with protein-losing nephropathy (PLN) to determine whether PLN is associated with decreased serum C3 concentrations. ANIMALS: 30 healthy dogs and 49 dogs with PLN. PROCEDURES: Serum samples were obtained from healthy dogs at the time of examination, whereas serum samples were obtained from dogs with PLN at the time of diagnosis. All samples were frozen at -70 degrees C until analyzed. Serum C3 concentrations were determined by use of a sandwich ELISA. Concentrations were expressed as the number of dilutions in which C3 could be detected. RESULTS: C3 was detectable in healthy control dogs (range, 1,920,000 to 15,400,000 dilutions; median, 9,600,000 dilutions). This represented a range of four 2-fold serum dilutions. In addition, C3 was detectable in dogs with PLN (range, 1,460,000 to 30,070,000 dilutions; median, 7,680,000 dilutions), which represented a range of six 2-fold serum dilutions. There was no significant difference in C3 concentrations between the 2 groups. CONCLUSIONS AND CLINICAL RELEVANCE: C3 is a critical part of the immune defense system that has not been extensively examined in veterinary medicine. An ELISA was developed for measuring C3 concentrations, and a reference range for healthy dogs was established. Significant decreases in C3 concentrations were not detected in any dog with PLN. Additional studies will be required to definitively determine the importance of serum C3 concentrations in PLN.     

Activation of complement by bovine respiratory syncytial virus-infected cells

Because complement activation is probably involved in the pathogenesis of as well as in recovery from the disease induced by bovine respiratory syncytial virus (BRSV), we studied the activation of complement by BRSV-infected cells in vitro in a homologous system. Binding of C3 on the surface of infected cells was measured in a biotin-streptavidin amplified ELISA, and complement-mediated lysis was measured in a 51Cr release assay. Without antibody, infected cells activated and bound more C3 than uninfected cells. C3 activation that occurred in the absence of antibody was largely mediated by the classical pathway and induced lysis inefficiently. BRSV-specific antibody enhanced complement activation as measured by both C3 ELISA and cytotoxicity assay. In the presence of antibody, C3 activation was largely dependent on the alternative pathway and efficiently induced lysis. Both IgG1 and IgM antibodies enhanced C3 activation, but IgG2 and IgA did not enhance C3 activation in our experiments. Preincubating cells with IgA or IgG2 did not inhibit C3 activation enhanced by IgG1 or IgM. Murine monoclonal IgG1 antibodies against epitopes on the Fusion protein of the virus also enhanced C3 binding, but differed in their capacity to induce complement-mediated lysis.     

The diagnostic significance of the direct antiglobulin test (DAT) in anemic dogs

The direct antiglobulin test (DAT) was positivein 134 (36.1%) of 371 anemic dogs with internal diseases. Four principal types of reaction were recognized: IgG alone in 15 (11.2%), IgG + C' in 41 (30.6%), C' alone in 74 (55.2%) and IgM + C' in 2 (1.5%). Rarely, IgM and/or IgA reactions occurred in association with strong IgG + C' reactions. In 2 (1.5%) DAT-positive dogs the type of reaction was not clear. One or more symptoms of hemolysis, such as hemoglobinemia, indirect type hyperbilirubinemia, increased red cell osmotic fragility, and increased fecal urobilinogen excretion, were demonstrated in 84 DAT-positive dogs. These consisted of 10 of 15 dogs with IgG type DAT, 36 of 41 dogs with IgG + C' type DAT, 36 of 74 dogs with C' type DAT and 2 of 2 dogs with IgM + C' type DAT. Most dogs with IgG + C' type reactions had severe hemolysis, whereas "primary" or "associated" diseases were recognized in only 26 of 56 cases. IgG type incomplete warm antibody, reacting with pooled donor cells, was demonstrated in red cell eluates in each of 3 dogs with IgG type DAT and in 6 of 7 dogs with IgG + C' type reactions. This indicates that dogs with IgG or IgG + C' reactions usually have autoimmune hemolytic anemia. In dogs with C' type DAT, indications of hemolysis were frequently minimal or absent. Symptoms almost always indicated some "primary" disorder. Diagnoses mainly included infections, inflammatory and neoplastic (especially myelo- and lymphoproliferative)diseases. In only 7 (9.5%) of 74 dogs with C' type DAT no diagnosis other than (transient peracute) hemolytic anemia was made. The results of tests for antibodies in the serum and red cell eluates were always negative in dogs with C' type DAT. In one dog with hemolytic anemia and C' + IgM type DAT, there was a high titer of IgM cold agglutinins in the serum and in heat eluates. It is concluded that a positive DAT with anti-IgG antiserum is a strong indication of autoimmune hemolytic anemia but that a reaction of the C' alone type is a rather common phenomenon in canine internal diseases which is seldom associated with serious hemolysis.     

Canine heterophilic antibodies as a source of false-positive B-type natriuretic peptide sandwich ELISA results

BACKGROUND: Interference by heterophilic antibodies is a well-known cause of false-positive sandwich ELISA results in human medicine. They are considered rarely in veterinary species and have not been characterized but could become important as newer, highly sensitive sandwich immunoassay technologies are developed. OBJECTIVES: The goals of this study were to use a B-type natriuretic peptide (BNP-32) sandwich ELISA to determine the effect of heterophilic antibodies on test performance; to characterize canine heterophilic antibodies; and to develop and test a method for heterophilic antibody removal. METHODS: A sandwich ELISA was developed using a mouse IgG(1)K monoclonal and a rabbit polyclonal antibody to two synthetic peptides of canine BNP-32. The effects on false-positive results of heterophilic antibody depletion and blocking by various techniques were compared. The titers of canine heterophilic antibodies were compared with various blood antigens from other species and the relative amount of canine IgG was compared with that of IgM heterophilic antibody. RESULTS: Heterophilic antibodies in dog plasma were shown to be capable of causing false-positive ELISA results. They reacted with blood proteins from a variety of animal species at relatively low titers and consisted of both IgG and IgM. Protein A agarose antibody precipitation, in conjunction with mouse IgG(1)K blocking antibody, was effective in eliminating false-positive sandwich ELISA results while retaining adequate test performance. CONCLUSIONS: Canine heterophilic antibodies can interfere with sandwich ELISA assays and cause false-positive test results. An effective technique for their removal that has a potentially broad application was developed, and allows measurement of canine blood constituents at low picomolar concentrations.     

A latex test for canine rheumatoid factor

Canine rheumatoid factor (RF) has been reported in several canine diseases, particularly in arthritis. Although RF can be assayed using IgG sensitized erythrocytes, the test has a number of disadvantages. As an alternative, latex sensitized with canine IgG was investigated as an assay of canine RF. The canine IgG-latex could be easily produced, was stable, and could be standardized with commercial antisera. The reagent detected RF of the IgM anti-canine-aggregated-IgG type. A comparison of the titres obtained using the canine IgG-latex reagent with those obtained using a rabbit IgG-erythrocyte reagent showed no correlation, suggesting that the two assays may detect RF of different specificities.     

Antibody complement-dependent bacteriolysis in experimentally induced pasteurellosis in mice

Affinity-purified bovine immunoglobulin isotypes were bacteriolytic for Pasteurella haemolytica biotype A, serotype 1 (PHA-1). This bacteriolysis was specific and complement-dependent. The IgM and IgG1 were the most active isotypes in the classic complement cascade. These isotypes also induced bacteriolysis through the alternative complement cascade. The comparative bacteriolytic activities of IgG1 and IgM were equal within each cascade; however, the bacteriolytic activities of IgG1 and IgM were lower in the alternative cascade than in the classical cascade. The IgG2 was more bacteriolytic than IgA in the classic and alternative complement pathways. Bovine immunoglobulins passively protected C57BL/6 mice from experimentally induced pasteurellosis. There were no major differences in the protection among hyperimmune sera, purified IgM, or purified IgG. Mice were protected from PHA-1 by approximately 1.9 micrograms of IgG and 1.2 or 0.1 micrograms of IgM. Elimination of murine complement with cobra venom factor 3 reduced PHA-1 clearance in passively immunized C57BL/6 mice. The protective effect of IgM mediated resistance was highly dependent on an intact complement system. The intact complement cascade was associated with enhanced clearance of PHA-1 from the liver. Although PHA-1 was susceptible to antibody complement-mediated bacteriolysis in vitro, the dependence on an intact complement cascade was not absolute in experimentally induced murine septicemic pasteurellosis.     

FMMU白化豚鼠与花色豚鼠血清Ig和补体含量及总补体活性比较

测定比较了 2月龄的封闭群 FMMU白化豚鼠与花色豚鼠的血清免疫球蛋白含量、补体含量及活性。结果表明 :白化豚鼠血清 Ig G含量极显著低于花色豚鼠 ,而 Ig A、Ig M含量显著低于花色豚鼠。白化豚鼠 C3含量极显著低于花色豚鼠 ,C4含量显著低于花色豚鼠。血清总补体活性 ( CH50 )显著低于花色豚鼠。     

Evaluation of the enzyme-linked immunosorbent assay in the detection of cattle infected with Brucella abortus

One group of 51 cattle was vaccinated with B. abortus S19 (S19) and a further 51 cattle were vaccinated with B. abortus S45/20 (S45/20). Forty-eight cattle (24 from each group) and a control group of 12 cattle were subsequently challenged with B. abortus S544. The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgM antibodies in these groups. All cattle vaccinated with S19 had high levels of IgG and IgM, but the S45/20 vaccine produced detectable antibody in only a few cattle. In those cattle where the challenge induced infection, the mean levels of IgG and IgM were much higher than those of the uninfected cattle in the same groups. When the isolation of B. abortus was compared at slaughter with the serological results, the ELISA, when used to detect specific IgG, was more sensitive but less specific than the serum agglutination test, complement fixation test and indirect haemolysis test, and more sensitive and more specific than the Rose Bengal test.     

Detection of high levels of canine herpes virus-1 neutralising antibody in kennel dogs using a novel serum neutralisation test

It is widely held that only cells of canine origin support canine herpesvirus-1 (CHV-1) replication and, that cytopathic effect (CPE) develops relatively slowly. Here we show that mink fetal lung cells (NBL-7 cell line) are permissive for CHV-1 and can be used to produce a sensitive test for neutralising antibody by plaque reduction in the presence of complement. The test was applied to the investigation of CHV-1 virus neutralising antibody levels in three kennel populations. The results showed that 26 out of 28 dogs were neutralising antibody positive (titre >/=2), and, 11 out of 28 had titres of >/=1024. The serum samples were analysed by enzyme linked immunoassay (ELISA); 27 out of 28 were graded as ELISA IgG positive (titre >/=500) and 26 of 28 were graded as ELISA IgM positive (titre >/=50).     

Interaction of certain bovine immunoglobulins with complement in a single radial haemolysis system

Bovine IgG1, IgG2, and IgM initiated haemolysis by bovine complement. With guinea pig complement bovine IgG1 and IgM appeared effective, but bovine IgG2 was much less effective. Single radial haemolysis systems using guinea pig complement to measure bovine antibody are likely to detect predominantly IgG1 and IgM. However, in vivo IgG2 should activate bovine complement.     

Bovine IgM: does it fix guinea pig complement in the absence of bovine complement components?

Purified bovine isotypes IgM, IgG1, IgG2, and IgA (secretory), affinity purified with Brucella abortus, were tested in a complement fixation test (CFT) for their ability to activate guinea pig complement directly or in the presence of 'normal' bovine serum. Only IgG1 fixed guinea pig complement in the direct test and approximately 250 ng of antibody was required to activate 50% of 3 CH50 units with a standard amount of antigen. Addition of 'normal' bovine serum as an additional source of complement resulted in activation of guinea pig complement by IgM, IgG2 and secretory IgA at levels of approximately 1200, 700 and 2250 ng, respectively for 50% of 3 CH50 units. Addition of 'normal' bovine serum did not enhance complement activation by bovine IgG1.     

Evaluation of a dot ELISA kit for measuring immunoglobulin M antibodies to canine parvovirus and distemper virus

A dot ELISA for the detection of immunoglobulin M (IgM) antibodies to canine distemper virus (CDC) and canine parvovirus (CPV) was assessed. The titres of IgM antibodies to CDV and CPV in 100 dogs were measured by the Immunocomb ELISA kit and compared with the results derived from the immunofluorescence assay (IFA). There was a strong correlation between the results of the dot ELISA technique and the IFA (P < 0.001). The dot ELISA kit was also used to assess the changes in the levels of immunoglobulin G (IgG) and IgM antibodies to CPV and CDV in 10 puppies vaccinated with a polyvalent vaccine. High levels of IgM antibodies to CPV were first detected seven days after they were vaccinated, and after nine days all the pups had high titres of IgG antibodies to CPV. High levels of IgM antibodies to CDV were detected after nine days and the highest average titres were recorded after 12 days. IgG antibodies to CDV were present from nine days after vaccination.     

Heterogeneity of human lymphocyte Fc receptors: Studies using heat-aggregated and antigen-complexed IgG from human, rabbit, guinea pig, horse and goat

We have studied the ability of human peripheral blood lymphocytes (HuPBL)⁴ to interact with IgG from several animal species. Three functions or activities that are reported to depend on an interaction between complexed IgG and HuPBL receptors (R) for the Fc piece of IgG (FcγR) were compared: (1) antibody-dependent cell-mediated cytotoxicity (ADCC); (2) binding of heat-aggregated IgG (aggG); and (3) rosette formation with IgG-sensitized erythrocytes [RBC-A(γ)]. IgG (and IgM) antibodies to chicken erythrocytes (CRBC) were purified from the sera of the following species after injection with CRBC stroma: (1) horse (Ho); (2) goat (Go); (3) rabbit (Ra); and (4) guinea pig (Gp). Good IgG-agglutinating antibody titers were obtained from each injected species.

Using ⁵¹Cr-labeled CRBC targets and HuPBL effector cells, only Ra anti-CRBC IgG gave good ADCC at high dilutions. Ho and Go anti-CRBC (IgG) failed to give A C , and p anti-CRBC (IgG) gave approx. 30% of the level of kill as Ra. Ra Fab'₂ fragments of IgG antibody failed to produce ADCC.

Treatment of HuPBL with Ra anti-lymphocyte serum (ALS) almost totally ablated ADCC, whereas HoALS failed to alter ADCC. Pretreatment of HuPBL with aggG showed that Ra or Hu aggG gave essentially equal inhibition of ADCC, Gp gave approx. 30% of the degree of inhibition as Hu and Ra, and Ho or Go aggG had essentially no effect of ADCC. These results confirmed the following order of ability of IgG to interact with HuPBL ADCC killer (K) cells: (Hu )Ra > Gp Ho, Go. The data suggest that Gp IgG interacts with only a subpopulation (≈ 30%) of HuPBL K cells.

The binding of aggG to total HuPBL failed to strictly correlate with the ADCC results or with the results of rosette formation between total HuPBL and CRBC-A(γ). The observations suggest that there is a heterogeneity of FcγR between K and non-K cell subpopulations of HuPBL both in terms of the type of complexed IgG they are able to bind, and in terms of the species of origin of the IgG. The data also support contentions that FcγR that bind RBCA(γ) complexes differ from those that bind aggG.     

An evaluation of five serological tests for the detection of antibody to bovine herpesvirus 1 in vaccinated and experimentally infected cattle

More than 300 bovine sera from a previously reported vaccination and challenge trial were tested for antibodies to bovine herpesvirus 1 (BHV1) by five serological assays: enzyme-linked immunosorbent assay (ELISA) for IgM and IgG, passive haemagglutination (PHA), and two methods of virus neutralisation (VN). In a statistical comparison of ELISA (IgG), PHA and VN results, the assays showed highly significant correlations (P less than 0.01). The sensitivities of ELISA and 24-hour neutralisation tests were similar, in contrast to passive haemagglutination and one hour neutralisation which failed to detect BHV1 antibodies in some low titre sera.     

Autoimmune haemolysis in the dog: relationship between anaemia and the levels of red blood cell bound immunoglobulins and complement measured by an enzyme-linked antiglobulin test.

A direct enzyme-linked antiglobulin test (DELAT) was used to measure the levels of red blood cell (RBC) bound IgG, IgM, IgA and C3 in dogs with autoimmune haemolytic anaemia (AIHA). At presentation, one or more DELAT parameters was raised in each AIHA case, and the RBC were typically coated with immunoglobulin of more than one class, together with C3. There was no relationship between the levels of RBC-bound IgG, IgM or IgA and the severity of the anaemia, although a significant negative correlation (rs = -0.66, P < 0.02) was found between bound C3 and blood haemoglobin concentration. These results indicate that the level of sensitisation of erythrocytes with IgG alone is not a reliable predictor of the severity of haemolysis in different cases, and that the pathogenesis of AIHA can be complex, involving multiple immunoglobulin classes and complement in the destruction of RBC. A significant relationship (rs = 0.63, P < 0.02) was found between serum IgG concentration and haemoglobin levels, and it is suggested that this may be due to free IgG inhibiting the interaction of IgG-sensitised RBC with macrophages. Serial measurements from individual AIHA cases during treatment revealed that the levels of RBC-bound immunoglobulins fell simultaneously with improvements in anaemia. In one dog, a relapse was associated with increases in bound IgG and IgM. Transient relative reticulocytopenia at presentation was common, but was not related to the severity of the anaemia. However, in other cases there was a persistent failure to increase RBC production, which was associated with slower recovery.     

Class-specific and polyvalent enzyme-linked immunosorbent assays for detection of antibodies to Borrelia burgdorferi in equids

Class-specific and polyvalent ELISA were developed to detect IgM antibody or total immunoglobulins to Borrelia burgdorferi in equine sera. Analyses of 122 serum specimens, collected during 1985 from horses and ponies in tick-infested areas of Connecticut, revealed IgM antibody in 41 (34%) samples; titration end points ranged from 1:160 to 1:2,560. In polyvalent ELISA, 73 (16%) of 454 serum specimens contained IgM and/or IgG antibody. Seropositivity was highest (32%) for blood samples collected during May. Both ELISA procedures had comparable sensitivities.     

Evaluation of canine serum for the presence of antiretinal autoantibodies in sudden acquired retinal degeneration syndrome

OBJECTIVE: To determine the presence of serum antiretinal antibodies in sudden acquired retinal degeneration syndrome (SARDS) affected dogs and the size of the antigen to which these antibodies bind via the use of enzyme-linked immunosorbent assay (ELISA) and Western blot immunoassays. ANIMALS STUDIED: Serum was collected from 13 dogs affected by SARDS and five dogs with normal ocular examinations. PROCEDURES: All serum samples were subjected to ELISA with saline-soluble canine retinal tissue and Western blot analyses with SDS solubilized normal canine retinal tissue as the antigen. Antirecoverin (23 kDa) and antiheat shock cognate (65 kDa) antibodies were used as positive controls for both procedures. Affinity-purified goat antidog IgG and IgM labeled with horseradish peroxidase were used for all clinical samples and goat antirabbit IgG was used as the secondary antibody for the positive controls. RESULTS: ELISA demonstrated antibody reaction with all samples. Western blot immunoassays identified multiple bands in all canine serum samples, as well as in negative controls. Approximate sizes of the bands were 25 and 50 kDa, corresponding to IgG light and heavy chains, respectively. CONCLUSION: No antiretinal autoantibodies were identified in the serum of dogs affected by SARDS as compared to normal canine patients.     

Monoclonal precipitating antibodies to porcine immunoglobulin M

Fusion of splenic immunocytes from a porcine IgM-immunized BALB/c mouse with SP2/0 mouse myeloma cells resulted in 231 primary hybrids. Culture fluids of the primary hybrids were screened for antibody production by enzyme-linked immunosorbent assay (ELISA) against porcine IgM and by radial immunodiffusion versus porcine serum. Culture fluids of 10 of the primary hybrids were positive in IgM-ELISA and radial immunodiffusion. Six of these primary hybrids (1A11, 1D10, 2D7, 2E2, 3B11, and 5C9) were cloned, and ascitic fluids were produced using cloned primary hybrids. The monoclonal antibodies (Mabs) in ascitic fluids were characterized as to their reactivity with porcine immunoglobulin isotypes. All six Mabs had mouse IgG1, K isotype and were mu-chain specific as they formed single precipitin lines against porcine serum and porcine IgM and no lines against porcine IgG, IgA, and fetal porcine serum in immunodiffusion and immunoelectrophoresis. In indirect ELISA, all Mabs reacted with porcine serum, porcine IgM, and mu-chains but did not react with porcine IgG, IgA, or light chains. All six Mabs were species-specific and recognized either of two antigenic regions of mu-chain. These Mabs have been successfully used to detect IgM-containing cells in tissue sections, to detect IgM in serum, and to quantitate surface membrane IgM-bearing cells in peripheral blood.     

Bovine IgM and IgM response to Leptospira interrogans serovar hardjo as measured by enzyme immunoassay

The enzyme-linked immunosorbent assay (ELISA) was used to detect specific IgG and IgG antibodies in the sera of cattle infected or immunized with Leptospira interrogans serovar hardjo. IgM appeared first but was quickly followed by IgG which persisted longer than IgM. The levels of antibody detectable by ELISA and by the microscopic agglutination test (MAT) did not correlate, suggesting that the two techniques measured different antigen-antibody systems. The transient nature of the IgM response as measured by ELISA indicates potential usefulness as a serodiagnostic test for detecting current leptospiral infections.