Detection and Characterization of Paraproteinemia in Canine Chronic B-cell Lymphocytic Leukemia Using Routine and Free Light Chain Immunofixation.

BACKGROUND: Hyperglobulinemia is reported in 26% of canine chronic B-cell lymphocytic leukemia (B-CLL) cases. However, few cases have been characterized by protein electrophoresis and immunofixation (IF), and the incidence of a monoclonal protein (M-protein) is unknown using these techniques. OBJECTIVE: To characterize and determine the proportion of canine B-CLL cases with an M-protein using plasma protein electrophoresis (PPE), routine and free light chain (fLC) IF, and to assess if productive B-CLL cases express MUM1/IRF4 by cell tube block (CTB). METHODS: PPE, routine (targeting IgG, IgA, IgM, IgG4, and light chain) and fLC IF were performed using 48 dog B-CLL plasma samples from patients diagnosed via peripheral blood flow cytometry. CTB was performed on a separate cohort of 15 patients. RESULTS: Hyperproteinemia (>7.5 g/dL) was present in 17/48 cases (35%). An M-protein was detected in 32/48 cases (67%). Of these, 19/32 cases (59%) had only complete (monoclonal heavy and light chain) M-proteins detected, 10/32 cases (31%) had both complete and fLC M-proteins detected, and 3/32 cases (9%) had only an fLC M-protein detected. IgM was the most common clonal immunoglobulin isotype detected (23 cases). CD21+ cell counts were higher in cases with detectable M-protein. Plasma fLC IF suggested ß-γ region interference, likely caused by clotting proteins. All B-CLL cases consistently expressed PAX5 and did not express MUM1/IRF4. CONCLUSIONS: Most B-CLL cases had an M-protein and were not hyperproteinemic. Most cases with paraproteins had a complete IgM monoclonal gammopathy; a subset had documented fLCs. The prognostic significance of heavy and fLC presence should be evaluated.

In vitro evaluation of potential interference of lokivetmab with protein electrophoresis and immunofixation.

OBJECTIVE: In humans, misdiagnoses of monoclonal gammopathy after use of therapeutic monoclonal antibodies has been documented. This triggers concerns for similar misdiagnoses in animals treated with monoclonal antibodies. The aim of this study was to evaluate if lokivetmab interferes with serum protein electrophoresis and immunofixation electrophoresis in dogs. MATERIAL AND METHODS: Residual sera from 25 client-owned, healthy blood donor dogs from 2 veterinary hospitals in Germany were used. The residual sera were analysed with serum protein electrophoresis and immunofixation electrophoresis before and after being spiked with lokivetmab at a concentration of 10 µg/ml (corresponding to the mean peak serum concentration after a subcutaneous injection of 2 mg/kg lokivetmab). RESULTS: No monoclonal gammopathy was observed on serum protein electrophoresis and all proteins had a normal distribution pattern without any pathologic bands on immunofixation electrophoresis. The absolute γ-globulin values of spiked samples, however, were significantly higher than in the native sera although they remained within the reference interval. No other globulin fractions were significantly different. CONCLUSION AND CLINICAL RELEVANCE: This study suggests that lokivetmab at a dose of 2 mg/kg is not detected as a monoclonal peak on serum protein electrophoresis or immunofixation electrophoresis, and thus is unlikely to lead to a misdiagnosis of other diseases that are characterised by monoclonal gammopathies.

Diagnostic performance of routine electrophoresis and immunofixation for the detection of immunoglobulin paraproteins (M-Proteins) in dogs with multiple myeloma and related disorders: Part 1 - Current performance.

BACKGROUND: Routine electrophoresis [agarose gel electrophoresis (AGE) and capillary zone electrophoresis (CZE)] and species-specific immunofixation (IF) can be used alone or in combination to detect immunoglobulin paraprotein (M-protein) and diagnose secretory myeloma-related disorders (sMRD). OBJECTIVE: We aimed to evaluate the performance of AGE, CZE, CZE plus IF (CZE-IF), and AGE plus IF (AGE-IF) for detecting canine serum M-proteins. METHODS: One hundred canine cases that had AGE, CZE, and routine IF performed on serum, and where B-cell lineage neoplasia (such as B-cell lymphoma and plasma cell tumors) had been diagnosed or excluded, were evaluated. Routine IF protocols targeted IgG-FC, IgA, and IgM heavy chains and light chains. IgG4 IF and free light chain IF were also performed. B-cell lineage neoplasms with an M-protein detected, using any available method, were classified as sMRD. Datasets from AGE, CZE, IF, CZE-IF, and AGE-IF (electrophoretograms, gel images, and fraction concentrations) were composed and reviewed. The sensitivity, specificity, and Youden's index for M-protein detection were determined for each dataset. RESULTS: The combination of AGE-IF or CZE-IF was more sensitive (82.9%) than CZE alone (72.0%) or AGE alone (64.6%) and more specific (66.1%, 48.3%, 51.7%, respectively). Immunofixation could be used alone to detect M-proteins (sensitivity 82.9%, specificity 61.9%), but there were technical challenges that complicated the performance and evaluation of the test. Myeloma with free light chains only was found in 5/41 cases of sMRD. CONCLUSIONS: Adding routine IF to routine electrophoresis increases the ability to accurately identify M-proteins; however, there is still room for further diagnostic performance improvements.

Diagnostic performance of routine electrophoresis and immunofixation for the detection of immunoglobulin paraproteins (M-Proteins) in dogs with multiple myeloma and related disorders: Part 2-Toward improved diagnostic performance.

BACKGROUND: The diagnostic performance of routine electrophoresis (agarose gel electrophoresis [AGE] and capillary zone electrophoresis [CZE]) and species-specific immunofixation (IF) for the detection of immunoglobulin paraproteins (M-proteins) and diagnosis of secretory myeloma-related disorders (sMRD) can be improved. Available canine IF targets were IgG-FC, IgA, IgM, light chain (LC), IgG4, and free LC (fLC) antibodies. OBJECTIVE: We aimed to review specific features associated with the presence of M-proteins in canine serum samples and the common features causing inaccurate reporting of M-proteins to improve the diagnostic performance of routine electrophoresis and IF for the detection of M-proteins. METHODS: Features found in AGE, CZE, routine IF, IgG4 IF, and fLC IF of 100 canine serum samples from Part 1 of this study were evaluated by simple and multivariate logistic regression to identify factors associated with the presence of M-proteins. Cases falsely called negative or positive for M-proteins were reviewed to identify the common features that could be used to increase the diagnostic performance of SPE and IF for M-protein detection. RESULTS: The presence of hypogammaglobulinemia or any peak taller than albumin was associated with an M-protein. Total protein concentrations, globulin concentrations, or peaks wider than albumin were not associated with an M-protein. Free LC sMRD cases were not diagnosed by SPE and routine IF. Cases with infectious and inflammatory etiologies had a restricted polyclonal gammopathy with multiple γ-globulin restrictions resulting in some false-positive results. SPE combined with all available IF results and the specific features identified in this study had an estimated sensitivity of 95.1% and specificity of 81.4%. CONCLUSIONS: The identified criteria of this study increase the diagnostic performance of the electrophoretic evaluation for M-proteins.

Protein characterization using electrophoresis and immunofixation; a case-based review of dogs and cats.

Protein electrophoresis and immunotyping can be a useful adjunct to the standard biochemical techniques for characterizing serum and urine proteins. This paper reviews currently available and commonly used methods for diagnostic protein electrophoresis, including both agarose gel and capillary zone electrophoretic techniques and total protein assessments. Immunofixation and immunosubtraction methods for identification of immunoglobulin location and class are also presented. Practical application of quality assurance and quality control strategies in compliance with American Society of Veterinary Clinical Pathology (ASVCP) best practices are discussed. Commonly encountered serum and urine electrophoretic diagnostic patterns, including electrophoretically normal, acute-phase protein responses, polyclonal gammopathies, restricted polyclonal/oligoclonal gammopathies, paraproteinemias (monoclonal or biclonal gammopathies), and Bence-Jones proteinurias are also reviewed using relevant case material. Cases in which immunofixation electrophoresis are particularly useful are highlighted, and methodologies to more accurately quantify serum monoclonal proteins (M-proteins), monitoring tests commonly used in human medicine, are discussed.

Detection of Aspergillus-specific antibodies by agar gel double immunodiffusion and IgG ELISA in feline upper respiratory tract aspergillosis.

Feline upper respiratory tract aspergillosis (URTA) is an emerging infectious disease. The aims of this study were: (1) to assess the diagnostic value of detection of Aspergillus-specific antibodies using an agar gel double immunodiffusion (AGID) assay and an indirect immunoglobulin G (IgG) ELISA; and (2) to determine if an aspergillin derived from mycelia of Aspergillus fumigatus, Aspergillus niger and Aspergillus flavus can be used to detect serum antibodies against cryptic Aspergillus spp. in Aspergillus section Fumigati. Sera from cats with URTA (group 1: n = 21) and two control groups (group 2: cats with other upper respiratory tract diseases, n = 25; group 3: healthy cats and cats with non-respiratory, non-fungal illness, n = 84) were tested. Isolates from cats with URTA comprised A. fumigatus (n = 5), A. flavus (n = 1) and four cryptic species: Aspergillus felis (n = 12), Aspergillus thermomutatus (Neosartorya pseudofischeri, n = 1), Aspergillus lentulus (n = 1) and Aspergillus udagawae (n = 1). Brachycephalic purebred cats were significantly more likely to develop URTA than other breeds (P = 0.013). The sensitivity (Se) of the AGID was 43% and the specificity (Sp) was 100%. At a cut-off value of 6 ELISA units/mL, the Se of the IgG ELISA was 95.2% and the Sp was 92% and 92.9% for groups 2 and 3 cats, respectively. Aspergillus-specific antibodies against all four cryptic species were detected in one or both assays. Assay Se was not associated with species identity. Detection of Aspergillus-specific antibodies by IgG ELISA has high Se and Sp for diagnosis of feline URTA.

Evaluation of Gastrothylax crumenifer antigenic preparation in serodiagnosis of paramphistomiasis in sheep.

An evaluation of Gastrothylax crumenifer crude antigen preparation viz., Somatic Antigen (SAg), Excretory Secretory Antigen (ESAg) and Egg Antigen (EAg) in serodiagnosis of disease was undertaken. Test sera samples were obtained from 30 Paramphistomiasis Positive and 30 Gastrothylax free sheep slaughtered at Hazratbal Kashmir. The referral antigenic preparation were evaluated against Paramphistomiasis positive sera, via., control negative sera, using double immunodiffusion test (DID), (IEP) Immunoelectrophoretic assay and ELISA. The performance of referral antigens, as assessed from percent sensitivity and specificity, revealed an increasing trend from DID (Double immunodiffusion-An immunological technique used in the detection, identification and quantification of antibodies and antigens) to IEP (immunoelectrophoresis-A general name for a number of biochemical methods for separation and characterization of proteins based on electrophoresis and reaction with antibodies), followed by ELISA, detecting higher number of sheep positive for paramphistomiasis. In ELISA the ESAg and SAg were evaluated as most reactive antigens with no significant difference and EAg was the least antigenic. In IEP, EAg had the higher sensitivity (60%) and analogous specificity of SAg and ESAg. The formation of the preceptin lines in the proximity to EAg containing wells (cathode end) in IEP was suggestive of higher molecular weight of G. crumenifer specific protein molecules with slower rate of migration. Purification and characterization of G. crumenifer and identification of specific antigenic molecules, particularly in EAg has been suggested for qualitative improvement of diagnostic value of the antigens in the tests used here in.

Clonality and virulence traits of Escherichia coli associated with haemorrhagic septicaemia in turkeys.

Fifty-five clinical isolates of avian pathogenic Escherichia coli (APEC) from seven outbreaks of acute haemorrhagic septicaemia in turkeys were characterized by serotyping, plasmid profiling including restriction analysis with HindIII, ribotyping with EcoRI and HindIII, multilocus sequence typing (MLST) and virulence profiling. A clonal relationship was demonstrated for each outbreak according to serotype, plasmid profiling, ribotyping, and MLST. In addition, isolates demonstrated highly similar virulence profiles, as all isolates were positive for F11 pili and possessed genes encoding aerobactin (iucD), increased serum survival (iss), temperature-sensitive haemagglutinin (tsh) and colicin V plasmid operon genes (cva/cvi). However, only 20% of the isolates produced colicin V and 42% exhibited serum resistance. All strains with O group O111 and a single O18ac strain (demonstrating non-clonal DNA profiles) were positive for enteroaggregative heat-stabile toxin (EAST1), while isolates of a single outbreak all possessed the enteroaggregative toxin gene (astA). All isolates were negative for genes encoding verocytotoxins (vtx/stx), iron-repressible protein (irp2), P-fimbria (papC), invasion plasmid antigen (ipaH), attaching and effacing gene (eae), enterohaemolysin (ehxA), and enterotoxins LT, STIa (ST(p)) and STIb (ST(h)). In conclusion, highly similar virulence profiles were demonstrated for isolates of E. coli associated with a single well-defined lesion type of colibacillosis in turkeys; acute haemorrhagic septicaemia. The isolates obtained, however, demonstrated a different phylogenetic background, underlining the importance of using well-defined strain collections for characterization of APEC pathotypes.

Bovine hypodermosis--a global aspect.

Cattle hypodermosis (warble fly infestation) is a notorious veterinary problem throughout the world. Larvae of Hypoderma species cause a subcutaneous myiasis of domesticated and wild ruminants. This disease is caused by, Hypoderma bovis, Hypoderma lineatum in cattle whereas, Hypoderma diana, Hypoderma actaeon, and Hypoderma tarandi, affect roe deer, red deer, and reindeer, respectively. Adults of the cattle grub are commonly known as heel flies, warble flies, bomb flies or gad flies. The biology of hypodermosis is complex because it passes through ecto- as well as endoparasitic stages in the life cycle. The parasitic stage of hypodermosis lasts about 1 year in domesticated as well as in the wild animals, while in the adult stage, a free-living fly lasts only for few days. The diagnosis of hypodermosis is of prime importance for planning treatment and the eradication program. Generally, there are two methods that are routinely used for diagnosis of hypodermosis, i.e., the direct clinical examination and immuno diagnosis by the use of pooled serum and/or milk sample. For the control of hypodermosis, different preparations are available and their use in most of the countries is limited to an individual level but never cover the whole cattle population of a country. Re-infestation in the herd occurs due to the untreated animals that remain the reservoir of the disease. The disease causes huge economic losses in animal production due to the effect of this disease on meat, milk, and the leather industry. It can also affect the general health status as well as the immune system of the body of the diseased animals. As regards the control measures of the disease, different methods have been efficiently practiced and consequently this disease is controlled at national level in many European countries.

Increase of canine leishmaniasis in a previously low-endemicity area in Tunisia.

An epidemiological study of canine leishmaniasis (CanL) was carried out in nine districts of Sfax, in the southern central part of Tunisia. Sera from 250 dogs were tested by two serological methods: the indirect immunofluorescence antibody test and the counter-immunoelectrophoresis. Seven to eight months later, before the next season of transmission, seropositive dogs from the first test were re-examined and a second sampling was performed. Infection status was assessed by serology and by other methods. PCR, in vitro culture and direct examination were applied on blood and other samples (bone marrow, liver, lymph node, spleen and cutaneous biopsies). The seroprevalence of the infection in dogs was 6%. Infection was then confirmed by at least one other method. The PCR is the method which agreed most with serology, all seropositive dogs were found PCR-positive. The sensitivity of the direct examination and the culture was only 33% and 55% respectively as compared with serology. A similar value of seroprevalence has been observed previously in Sousse, in the northern central part of Tunisia. The present report suggests a significant increase of CanL in the Sfax area and confirms that the disease is continuing to move southwards in Tunisia.

Tissue-specific expression and regulation of the haemolymph clottable protein of tiger shrimp (Penaeus monodon).

The clottable protein (CP) involved in Penaeus monodon haemolymph coagulation has previously been characterized and cloned. Polyclonal antibodies against purified CP were also prepared from rabbit serum. By Western blot analyses, we showed occurrence of CP in the shrimp central nervous system, gill, and lymphoid organ. Results of RT-PCR further indicated that the central nervous system, gill, and lymphoid organ transcribed more CP, heart and hepatopancreas transcribed less, while the haemocytes and the muscle did not. We further analyzed the CP distribution within shrimp lymphoid organ by immunohistochemical method, CP was found to localise in stromal cells of lymphoid organ rather than in the developing haemocytes. In addition, concentrations and regulation of the plasma CP under normal and artificially traumatic conditions were studied with rocket immunoelectrophoresis. The average plasma CP concentration in normal intermolt shrimps was elevated from 3 mg ml(-1) to above 12 mg ml(-1) after successive blood-withdrawing for a week. The production and secretion of CP apparently were increased more than 4 folds to compensate its loss. Our result also suggested that the shrimp sinus gland endocrine system is not directly required for the expression and up-regulation of CP.

Production of polyclonal antisera against feline immunoglobulin E and its use in an ELISA in cats with experimentally induced asthma.

Serum samples from six cats with experimentally induced asthma were used to purify feline IgE using gel filtration and affinity chromatography. The resultant IgE, evaluated for purity by immunoelectrophoresis (IEP) and reactivity by Prausnitz-Kustner (PK) testing, was used to develop polyclonal rabbit anti-feline IgE antisera. Using reverse cutaneous anaphylaxis (RCA), the antisera were determined to be specific for feline IgE. The polyclonal rabbit anti-feline IgE antiserum was then validated in an allergen-specific ELISA. Serum samples from an additional five asthmatic cats sensitized with Bermuda grass allergen (BGA) were evaluated prior to sensitization, after parenteral sensitization, and after aerosol sensitization and challenge. A significant increase in serum BGA-specific IgE was noted over time.

Purification, characterization, antibacterial activity and N-terminal sequencing of buffalo-milk lysozyme.

Lysozyme from buffalo milk was purified to homogeneity and its N-terminal amino acid sequence, biochemical properties and antibacterial spectrum were determined. The purification procedure, comprising ion-exchange chromatography using CM-cellulose and size-exclusion chromatography using Sephadex G-50, conferred 8622-fold purification and 39.3% recovery of lysozyme. The purified enzyme migrated as a single band on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and native PAGE. Immunological purity of lysozyme preparation was confirmed by immuno-electrophoresis. Molecular weight of buffalo-milk lysozyme as determined by SDS-PAGE was 16 kDa and its amino acid composition was determined by reverse phase high performance liquid chromatography (HPLC). The sequence of 23 amino acid residues at the N-terminal end showed 56.5% homology with bovine milk lysozyme and 30.4% with equine milk lysozyme. The specific activity of buffalo milk lysozyme was ten-times that of bovine milk lysozyme. Buffalo-milk lysozyme was active over a wide range of pH and its activity was strongly influenced by molarity of the medium. Antibacterial activity of buffalo-milk lysozyme was determined against 11 species of bacteria; out of seven Gram-positive bacteria tested, four were inhibited, while Gram-negative bacteria were resistant.

Biclonal gammopathy associated with immunoglobulin A in a dog with multiple myeloma.

A 10-year-old neutered male Airedale Terrier was evaluated for inappetance, weight loss, and lameness. Multiple myeloma was diagnosed based on bone marrow plasmacytosis, multiple lytic bone lesions, and hyperglobulinemia with a clonal gammopathy on serum protein electrophoresis. Splenic plasmacytosis, and retinal lesions consistent with hyperviscosity syndrome also were found. Temporary responses to 2 different chemotherapy protocols (melphalan and prednisone, and cyclophosphamide and prednisone) were seen, with remission of clinical signs and a decrease in the biclonal gammopathy but no resolution of the splenic mass. Eventual return of clinical signs led to euthanasia at 175 days postdiagnosis. Necropsy examination confirmed multiple myeloma involving bone marrow and spleen, and glomerulonephritis. An immunoglobulin-A (IgA) gammopathy was demonstrated by immunoelectrophoresis; biclonality was ascertained by immunofixation electrophoresis. The clonal components consisted of intact Ig with heavy chain of the alpha class and light chain of an undetermined class. To our knowledge, this is the first report of undimerized biclonal gammopathy in a dog caused by a single heavy chain class involving IgA.

Immunoglobulin G class identification from wild ungulates by cross-reactivity with antisera to domestic animals.

Seven species of Spanish ungulates were tested for the presence of homologous immunoglobulin G (IgG) with a gel-diffusion test using bovine, ovine, caprine and porcine IgG antisera. Homologous ovine and caprine IgG were detected in sera from chamois (Rupicapra pyrenaica), Spanish ibex (Capra pyrenaica hispanica), mouflon (Ovis orientalis musimon), red deer (Cervus elaphus), fallow deer (Dama dama) and roe deer (Capreolus capreolus). Homologous porcine IgG was detected in wild boar (Sus scrofa) serum. Immunoelectrophoretic assays were performed to compare the electrophoretic mobility of IgG from domestic and wild species.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) to detect isometamidium chloride in Oncorhynchus spp.

An antigen-capture enzyme-linked immunosorbent assay (ELISA) was developed to detect and measure isometamidium chloride in the plasma of Oncorhynchus tshawytscha and O. mykiss. Isometamidium-ovalbumin conjugate and anti-isometamidium antibodies were used to coat polystyrene plates. The peroxidase saturation technique was used to optimize the coating antigen concentration; it demonstrated low affinity of the isometamidium-ovalbumin conjugate but high affinity of the anti-isometamidium antibodies for polystyrene surface sites. The optimal conditions of antiisometamidium antibodies to coat plates was at pH 7.3 and a 1:1000 dilution (0.0012 mg ml(-1) protein). The ELISA was sensitive as it detected 0.0006 mg ml(-1) of isometamidium in fish plasma. Isometamidium diluted with saline could not be detected at concentrations less than 0.05 mg ml(-1). The results indicate that this ELISA is much more sensitive when isometamidium is bound to plasma than unbound isometamidium in saline.

Effects of sarcoptic mange on serum proteins and immunoglobulin G levels in chamois (Rupicapra pyrenaica) and Spanish ibex (Capra pyrenaica).

Three groups of chamois (Rupicapra pyrenaica) and three groups of Spanish ibex (Capra pyrenaica) were established to study the effects of sarcoptic mange on serum proteins and immunoglobulin G (IgG) levels. The first group of chamois consisted of 22 healthy Pyrenean chamois (R. pyrenaica pyrenaica) from a non-infested area, the second group consisted of 20 healthy Cantabrian chamois (R. p. parva) from an area where sarcoptic mange has been reported since 1994 and the third group consisted of 16 Cantabrian chamois from the same area but naturally infested by Sarcoptes scabiei. The first group of Spanish ibex was 39 healthy animals from a sarcoptic mange non-infested area, the second group was 23 healthy animals from a sarcoptic mange infested area and the third group consisted of 20 animals from the same area but naturally infested with the parasite. Blood samples were taken after killing the animals as part of hunting programmes. Values for total proteins, gamma-globulin and IgG were higher in infested and healthy chamois from the infested area compared to healthy chamois from the non-infested area, and IgG levels were higher in infested chamois compared to healthy-exposed chamois. Values for alpha2-globulin were higher in healthy Cantabrian chamois. In Spanish ibex, albumin, alpha2-globulin and IgG levels were lower in the healthy Spanish ibex from the non-infested area than in healthy animals from an infested area. The differences found in the chamois were indicative of the establishment of a humoral antibody response in the animals in contact with the disease. As the IgG levels were not significantly different between healthy and infested Spanish ibex from the same area, a different pattern of chronic infection with humoral response to the disease was suggested.

Purification and characterization of liver ferritins from different animal species.

The ferritins were purified from liver homogenates of buffalo, camel, cattle, sheep and shark by thermal denaturation, ammonium sulphate fractionation, Sephacryl S-300 gel filtration and DEAE-blue gel affinity chromatography. The yield and iron content of affinity-purified liver ferritins ranged from 0.008 to 0.052 mg/g and 3.17% to 11.4% respectively. As they are glycoproteins, the ferritins contained variable amounts of neutral carbohydrates. Except for shark ferritin, the ferritins all exhibited immunological cross-reactivity with anti-buffalo liver ferritin and anti-horse spleen ferritin by immunodiffusion and immunoelectrophoresis. Gel electrophoresis, gel filtration and ultracentrifugal analysis indicated the presence of a monomeric ferritin in all cases. SDS-gel electrophoresis of shark ferritin gave a protein band of 18 kDa. Ovine, buffalo and bovine ferritin comprised two protein subunits, the H (20 and 21 kDa) and the L types (18 and 19 kDa). Oligomeric ferritin subunits with molecular weights of 27, 37 and 55 kDa were also found for bovine and buffalo ferritin. SDS-PAGE of camel ferritin revealed a complex pattern with four prominent bands of 61, 51, 44 and 39 kDa. Two fast-migrating components of 15 and 16 kDa were also found in the purified liver ferritins, including reference preparations. The PO4(3-)/Fe ratios of purified shark (0.10) and bovine ferritin (0.12) were similar to that of standard equine spleen ferritin (0.11). However, the ratio was higher in ovine (0.17), camel (0.22) and bovine (0.26) ferritins. The amino acid compositions, molecular weights and sedimentation coefficients of the different liver ferritins were similar.

Immunorheophoresis for the diagnosis of infectious bursal disease.

The immunorheophoresis (IR) technique was used for the detection of infectious bursal disease antigen from bursae collected from field cases and experimentally infected chickens. When these results were compared with that of the agar gel immunodiffusion (AGID) test, they showed excellent agreement as determined by kappa value. However, the time taken for the appearance of the precipitin lines was reduced from 14-24 hr in the AGID test to 3-5 hr in the IR technique.

Development of an automated turbidimetric immunoassay for quantification of bovine serum immunoglobulin G.

OBJECTIVE: To develop an automated turbidimetric immunoassay (TIA) for measurement of bovine IgG. SAMPLE POPULATION: 24 bovine serum samples. PROCEDURE: IgG concentration was determined by use of the TIA, and results were compared with those of the radial immunodiffusion (RID) method. Variables were determined, using commercially available reagents and a clinical biochemical analyzer. For the TIA, polyclonal goat anti-bovine IgG (Fc specific) serum, bovine IgG calibrator serum, and polyethylene glycol reaction buffer were used. Sample concentrations were determined by the instrument, using the linear regression method of least squares. The accuracy of this assay was validated by referencing to a purified bovine IgG standard and by recovery of control standards. Parallelism was documented by assay linearity and serial sample dilution linearity. Interference resulting from hemolyzed samples was examined. RESULTS: The TIA method correlated positively (r = 0.9957) and significantly (P < 0.05) with the RID method, yielding a regression equation with slope of 0.78708 and y-intercept of 1.02102. Bias attributable to hemolysis was not observed. CONCLUSIONS: The TIA method is automated, accurate, and precise for bovine serum IgG quantification. CLINICAL RELEVANCE: This assay provides sample results in approximately 10 minutes and may be used as an alternative to the manual RID method.