Production and Characterisation of Murine Monoclonal Antibodies to Feline Erythrocyte A and B Antigens

Anti-A antiserum from blood type B cats, the current reagent used to detect blood type A cats, is expensive, labour intensive to produce, and can vary in sensitivity between preparations. In contrast, monoclonal antibodies are produced easily in large quantities and pure form. We produced six IgM class murine monoclonal antibodies, four specific for feline blood type A and two that detect feline blood type B, by injection of mice with liposomes incorporating type A or B erythrocyte membrane antigens. Specificities of each monoclonal antibody were characterised by high performance thin layer chromatography of feline erythrocyte membrane glycolipids and by immunoblotting of feline erythrocyte membrane proteins separated by SDS-PAGE. The anti-A monoclonal antibodies specifically detected feline blood type A by direct agglutination of blood-typed samples from many cats. Each anti-A monoclonal antibody agglutinated some, but not all, feline blood type AB samples. Two anti-A monoclonal antibodies appeared identical and recognised [NeuGc]₂G_D3, the major glycolipid antigen of type A blood. The other two also appeared identical to each other and recognised a slower migrating glycolipid band, which may be [NeuGc]G_T3. The two anti-B monoclonal antibodies detected feline blood type B by direct agglutination and both recognised [NeuAc]₂G_D3, the major glycolipid antigen of type B blood. None of the monoclonal antibodies recognised erythrocyte membrane glycoproteins specific for either feline type A or type B blood. The ability of the anti-A monoclonal antibodies produced in this study to specifically detect feline blood type A makes them useful replacements for anti-A antiserum for blood typing of cats. The inability of each anti-A antibody to agglutinate blood from every type AB cat suggests a difference between the A antigen of some type A and some type AB cats.     

A Retrospective Study of Feline Pancytopenia

To better define the incidence and causes of feline pancytopenia we reviewed cases of pancytopenia submitted to the University of Minnesota, Veterinary Teaching Hospital over an 18-month period. Pancytopenia was defined as a combination of anaemia (packed cell volume < 26%), neutropenia (segmented neutrophils < 3000/μl), and thrombocytopenia (platelet counts < 200 000/μl). Of 2011 complete blood counts reviewed, pancytopenia was detected in 56 (2.8%). Associated clinical disorders included drug-associated disorders (n = 9) infectious diseases (n= 14) immune-mediated haematological diseases (n= 3), chronic renal failure (n = 3), idiopathic aplastic anaemia (n= 3), and other causes (n= 5). Results of this study indicate that feline pancytopenia has multiple causes and that the prognosis is dependent on the cause of the pancytopenia. Differentiation of the various causes of pancytopenia requires a systematic approach that includes evaluation of infectious, drug-induced, and immune-mediated causes, and examination of bone marrow.     

A Retrospective Study of Feline Pancytopenia

To better define the incidence and causes of feline pancytopenia we reviewed cases of pancytopenia submitted to the University of Minnesota, Veterinary Teaching Hospital over an 18-month period. Pancytopenia was defined as a combination of anaemia (packed cell volume < 26%), neutropenia (segmented neutrophils < 3000/μl), and thrombocytopenia (platelet counts < 200 000/μl). Of 2011 complete blood counts reviewed, pancytopenia was detected in 56 (2.8%). Associated clinical disorders included drug-associated disorders (n = 9) infectious diseases (n= 14) immune-mediated haematological diseases (n= 3), chronic renal failure (n = 3), idiopathic aplastic anaemia (n= 3), and other causes (n= 5). Results of this study indicate that feline pancytopenia has multiple causes and that the prognosis is dependent on the cause of the pancytopenia. Differentiation of the various causes of pancytopenia requires a systematic approach that includes evaluation of infectious, drug-induced, and immune-mediated causes, and examination of bone marrow.     

Cerebral Blood Flow Velocity,Erythrocyte Deformability and Alcohol Intake

The aim of the study was to determine if there is a relationship between low blood flow velocity in the cerebral arteries and erythrocyte deformability in heavy alcohol drinkers. The study comprised 47 heavy alcohol drinkers (mean age 47 years). All of them drank daily more than 84 g of alcohol (84–400 g). Blood flow velocity (V _mean) in intracranial arteries was determined by transcranial Doppler. Erythrocyte membrane biophysical properties were estimated using the method of cation-osmotic haemolysis (COH). The present study revealed a significant decrease in V _mean in all examined arteries, with p= <0.01 in the middle (MCA) and posterior (PCA) cerebral arteries and p= <0.05 in the anterior cerebral artery (ACA) when compared with age-matched controls. Cation-osmotic haemolysis in the low ionic strength of the incubating medium (15.4 mmol/l NaCl) as well as in the high ionic strength (123.2–154.0 mmol/l NaCl) was significantly decreased (p<0.001–0.01). This means that changes in both parts of the erythrocyte membrane (actin–spectrin complex and membrane lipid bilayer) are the cause of decreased erythrocyte deformability. We conclude that one of the factors which can cause low blood flow velocity (a possible risk factor for stroke) is decreased cation-osmotic haemolysis of erythrocytes.     

Haemorheological Characterisation of a Model of Diet-induced Hypercholesterolaemia in Rats

This investigation examined the haemorheological changes that occurred in rats during and after administration of high-fat diets containing 10% lard and 1% cholic acid, both with and without 2% cholesterol for a period of 2 weeks. Rats fed the high-fat diet enriched with cholesterol showed markedly impaired fluidity of whole blood, increases of blood viscosity, plasma low-density lipoprotein (LDL)-cholesterol, VLDL-cholesterol, and impaired skin microcirculation shortly after the start of this diet regimen. Additionally, increased liver mass, and lipid accumulation in hepatocytes were observed after two weeks. In contrast, rats fed the high-fat, cholesterol-free diet had less pronounced haemorheological disorders, increased liver mass and lipid accumulation in the liver than rats given the cholesterol-enriched diet. Histopathologically, there were no marked changes in blood vessels or liver in rats fed these diets for 2 weeks, except for fatty liver changes. Dietary induced hypercholesterolaemia therefore contributes to the haemorheological disorder, impaired microcirculation and fatty liver change.     

Phenotypic differences within the AB blood type of the feline AB blood group system

Flow cytometry immunolabeling, tube agglutination tests, and thin-layer chromatography immunostaining with two different anti-A monoclonal antibodies (anti-A mAb1 and anti-A mAb2) and one anti-B mAb were used to demonstrate differences in expression of the A and B antigens among erythrocytes from type A and four different type AB cats. Although the flow cytometric patterns of reactivity and agglutination scores for erythrocytes from types A and B cats detected with the anti-A and anti-B mAbs were consistent, reactivity among erythrocytes of different type AB cats was variable. By flow cytometric analysis, 99.9% of type A erythrocytes, no type B erythrocytes, 2.5–4.0% of erythrocytes from type AB cats 1, 3, and 4, and 60.7% of erythrocytes from type AB cat 2 had detectable A antigen when anti-A mAb1 was used. In contrast, 86.4% of type A erythrocytes, no type B erythrocytes, 20.2–38.0% of erythrocytes from type AB cats 1, 3, and 4, and 68.5% of erythrocytes from type AB cat 2 had detectable A antigen when anti-A mAb2 was used. In addition, 86.9% of type B erythrocytes, no type A erythrocytes, 83.1–96.8% of erythrocytes from type AB cats 1, 3, and 4, and 73.0% of erythrocytes from type AB cat 2 had detectable B antigen when the anti-B mAb was used. Agglutination scores of type AB cats were comparable to the percent binding on flow cytometry. Thin-layer chromatography immunostains confirmed differences in the amount of A antigen between erythrocyte glycolipids of type A and AB cats and those of type AB cats 1 and 2. These results suggest that at least two different phenotypes exist within the feline AB blood type, which differ in the amount of A antigen expressed on the erythrocyte surface.     

单克隆抗体相对亲和力和特异性的测定

在单克隆抗体的制备中,亲和力和特异性的测定是非常重要的。实验证明单抗的特异性不是绝对的,我们也发现在免疫酶组化染色过程中,当单抗浓度达到一定水平时会出现交叉反应,即抗3型(或7型)腺病毒单抗与7型(或3型)腺病毒出现反应。因此有必要测定单抗特异性,以便筛选高特异性的单抗。     

Q热立克次体单克隆抗体的产生及其特性的研究

Q热立克次体存在相变异的现象。随着相变异的出现,Q热立克次体的生物学特性、理化性质和抗原性等方面均有变化。这种变化的发生与Q热立克次体的表面结构和抗原组分的改变有密切关系。为了在分子水平上研究Q热立克次体,阐明相变异的机制或研究Q热立克次体的致病性、血清免疫学     

Generation and Characterization of Monoclonal Antibodies Specific to Surface Antigens of Human Trophoblast Cells

PROBLEM : To generate and utilize specific monoclonal antibodies for routine fetal cell isolation from the maternal circulation. METHODS : Monoclonal antibodies specific to human trophoblast cell surface antigens were generated and characterized. After cell fusion, antibodies secreted by hybridomas were screened by enzyme-linked immunosorbent assay and immunohistochemical assays. RESULTS : By using cultured BeWo choriocarcinoma cells or the membrane fraction of human placenta as the immunogen, seven (BW-108, 110, 123, 124, HP-15, 16 and 17) antibodies specific to the surface antigens of trophoblast were produced. They were shown to have little cross-reactivity to other human tissues. Among the antibodies raised against human sperm, HSA-10 was also found to cross-react with human trophoblast, but not detected in other tissues. When immobilized to magnetic beads, these antibodies were shown to react only with BeWo cells in suspension, but not blood cells and ovarian carcinoma cell line, OC-3-VGH. CONCLUSION : Therefore, these antibodies may have potential application in fetal trophoblast cell isolation from the maternal circulation for prenatal genetic diagnosis.     

Production and Characterization of Monoclonal Antibodies against the Extracellular Domain of CA 125

Carcinoma antigen 125 (CA 125) is overexpressed in ovarian cancer and antibodies against it are widely employed for diagnostic purposes. The rarity of CA 125 antigenic domains and its highly glycosylated structure, however, is a problem that may prevent immunized mice from developing a diversified population of anti-CA 125 antibodies. In this study a prime-boost strategy, which potentially could augment the humoral immune responses against rare and poorly immunogenic determinants, was used for immunization of mice and monoclonal antibodies (mAbs) were produced by hybridoma technology. Reactivity of mAbs was then assessed by ELISA, western blotting, immunoprecipitation, immunohistochemistry and immunofluorescence staining of OVCAR-3 cell line. Altogether, 10 clones were produced, 3 of which had IgG isotype and the rest were IgM. Two-third of clones recognized cognate antigen in fixed and living cells and had strong immunoreactivity in IHC staining. In Western blotting, our antibodies recognized CA 125 as high molecular weight antigen mostly migrated in the 3% stacking gel. Immunoprecipitation of OVCAR-3 cell lysate by mAbs resulted in a very similar migration pattern that reconfirmed their specificities. The mAbs produced in this study are invaluable tools in diagnosis and research fields for assessment of CA 125 expression in cancerous ovarian tissues.     

日本乙型脑炎病毒单克隆抗体的产生及其特性鉴定

我国有日本乙型脑炎、森林脑炎及登革热等病流行,其病原体在抗原性上有明显的交叉反应,长期以来用动物免疫血清不易鉴别。单克隆抗体技术问世提供了新的病毒分析方法,但用于日本乙型脑炎病毒抗原分析方面的研究尚少,且无一致结论。因此,不论为提高检验诊断的准确性或评价现行疫苗的效果,都有必要建立各种分泌日本乙型脑炎病毒单克隆抗体的杂交瘤细胞系,以深入研究该病毒的抗原特性。     

Relationship Between the Reference Value of the Erythrocyte Sedimentation Rate of the Presenile Population and of Geographical Factors in China

In order to provide a scientific basis for a strictly medical comparison (a ‘unified standard’) of local reference values for erythrocyte sedimentation rate (ESR) among the presenile population in China, the reference values for ESR of healthy persons have been collected, the assays having been performed by the method of Wintrobe. The relationship between the reference value for ESR in the presenile population and geographical factors has been examined. Altitude was found to be the most important factor affecting the reference value for ESR in the presenile population; ESR reference values decrease as the altitude gradually increased, the relationship being highly significant. The method of stepwise regression analysis was used to deduce two regression equations, so that if the geographical factor value of a particular area in China is known, the national reference value for ESR in the presenile population can be calculated by means of these equations. Based on geographical factors, China can be divided into six districts: Qingzang, Southwest, Northwest, Southeast, North and Northeast.     

肝癌单抗—蓖麻毒素A链结合物的制备及其细胞毒性质

本文应用异型双功能基化合物SPDP与单克隆抗体反应,产生单克隆抗体-PDP,然后再与暴露巯基的RTA结合,制备肝癌单抗-RTA结合物。ELISA及毒素重组试验鉴定表明:结合物在10~(-9)mol/L浓度时与靶细胞仍有较好的结合能力,结合物中RTA保持良好活力。体外细胞毒结果表明:结合物对靶细胞具有良好的导向杀伤作用,在10~(-9)mol/L浓度时对人肝癌细胞株BEL7404的杀伤率为41.2％,对正常细胞SL_7的杀伤力很弱。     

Monoclonal antibody against CD45RB for the therapy of rejection and autoimmune diseases

 Rejection continues to be the single largest impediment to successful organ transplantation. Current therapy, which must be taken for a lifetime is nonspecific and has significant side effects including infection and cancer. There is a need to develop improved means of immunosuppression. The current goal of transplantation immunology is to induce a prolonged state of nonreactivity to the allograft but preserving an otherwise intact immune system (tolerance). We have recently reported that a monoclonal antibody against CD45RB is a potent immunosuppressive agent, and that it induces donor specific tolerance in the mouse. In this contribution we briefly review our understanding of the molecular basis for the activity of this therapy and update results in various transplant and autoimmune disease animal models. The clinical relevance and future development of this novel therapy is also discussed. Received: 15 May 1997 / Accepted: 25 September 1997     

Comparative Nuclear Magnetic Resonance Studies of Diffusional Water Permeability of Red Blood Cells from Different Species. XII. Dog (<Emphasis Type="BoldItalic">Canis familiaris</Emphasis>) and Cat (<Emphasis Type="BoldItalic">Felis domestica</Emphasis>)

The diffusional water permeability (P _d ) of dog and cat red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulphonate (PCMBS). The values of P _d were in the case of cat RBC ∼3.0 × 10⁻³ cm/s at 15 °C, 3.5 × 10⁻³ cm/s at 20 °C, 4.2 × 10⁻³ cm/s at 25 °C, 4.4 × 10⁻³ cm/s at 30 °C and 5.9 × 10⁻³ cm/s at 38 °C. In case of dog RBC the values of P _d were higher ∼3.8 × 10⁻³ cm/s at 15 °C, 4.6 × 10⁻³ cm/s at 20 °C, 5.0 × 10⁻³ cm/s at 25 °C, 5.9 × 10⁻³ cm/s at 30 °C and 7.9 × 10⁻³ cm/s at 37 °C. Systematic studies of the effect of PCMBS on water diffusion indicated that in the case of dog RBCs the maximal inhibition was reached in 15–30 min with 1 mm PCMBS, whereas in the case of cat RBCs in 60 min with 1 mm PCMBS or in 30 min with 2 mm PCMBS. The values of maximal inhibition in the case of cat RBC were in the range of 55–60% at 15 °C, 60–68% at 20 °C and 25 °C, 50–60% at 30 °C and 50–55% at 37 °C. In the case of dog RBC the corresponding values were higher, 75–80% at 15 °C, 70–80% at 20 °C and 25 °C, 65–70% at 30 °C and 55–60% at 37 °C. The basal permeability to water was estimated to be ∼1 × 10⁻³ cm/s −2 × 10⁻³ cm/s in the range of temperatures of 25–37 °C. The activation energy of water diffusion E _a,d was ∼19 kJ/mol for the dog RBC and ∼23 kJ/mol for the cat RBC. After incubation with PCMBS the values of E _a,d increased, reaching 40 kJ/mol in conditions of maximal inhibition of water exchange. The membrane polypeptide electrophoretic pattern of dog and cat RBCs has been compared with its human counterpart. Dog and cat RBCs contained higher amounts of spectrin (band 1 and 2) and lower amounts of bands 4.4, 4.2, band 5 and band 7 compared to human RBCs. Band 4.9 was decreased only in the cat RBCs, whereas band 6 was decreased only in the dog RBCs. Correspondence and offprint requests to: Gheorghe Benga, Department of Cell and Molecular Biology, ‘Iuliu Haţieganu’ University of Medicine and Pharmacy, 6 Pasteur St, 3400 Cluj-Napoca, Romania. Tel:/Fax: 40–64–194373; e-mail: GBenga@personal.ro; gbenga@umfcluj.ro     

A、B血型单克隆抗体临床应用的研究

为了探索人血型分型试剂工业化生产的可能性，应用人Ａ、Ｂ血型单克隆抗体，进行了抗体特性及临床血型分型的研究。经血凝特性试验，测得Ｄ２８６－Ｅ１２单克隆抗体仅对Ａ血型特异，６－１－Ｇ１１单克隆抗体仅对Ｂ血型特异。由盐水试管法测得它们的血凝滴度均为１：５１２。以血库常规人抗Ａ、抗Ｂ血清为标准对照，采用盐水试管法，对３７０４例血液样品进行了临床血型分型试验。结果显示，Ａ、Ｂ血型单克隆抗体分型试剂与标准人血清１００％相符。证明：Ｄ２８６－Ｅ１２和６－１－Ｇ１１两株杂交瘤分泌的Ａ、Ｂ血型单克隆抗体可作为非常优越的血型分型试剂。具有工业化生产血型单克隆抗体的潜力。     

Studies on the Relationship Between Haemoglobin Types of Adult Dromedary Camels and the Concentrations of Haemoglobin, Copper, Ceruloplasmin and Iron

The relationship between haemoglobin types of adult dromedary camels and the concentrations of haemoglobin, copper, ceruloplasmin and iron were studied. Blood samples were taken from the jugular vein of 100 clinically healthy Iranian dromedary camels according to their age (1–2, 2–4, 4–6 and >6 years) and sex. Haemoglobin electrophoresis showed two haemoglobin types: HbA (95.2%) and HbA₂ (4.8%). Age and sex had no significant effect on haemoglobin types nor on the concentrations of haemoglobin, copper, ceruloplasmin and iron. There was no correlation between the concentrations of haemoglobin, copper, iron, ceruloplasmin, HbA and HbA₂.     

Effects of Duration of Storage and Storage Temperature on Cell Counts of Bovine Blood Samples as Determined by an Automated Haematology Analyser

Analysis of blood cells is an important part of many scientific investigations in the field of cattle herd health. Over the last 30 years, automated blood analysis has all but replaced manual counting of blood cells using counting chambers. The present study investigated the effects of prolonged storage and storage temperatures on cell counts as determined by a haematology analyser. Blood samples from 20 clinically healthy cows were repeatedly analysed with a Cell-Dyn 3500 (Abbott Diagnostika, Delkenheim), within 24 hours after collection and after storage at either 4° C or 20° C. The counts of most blood cells were more stable in samples stored at 20° C than those stored at 4° C. For at least 8 h, the counts of all analysed cell types, with the exception of lymphocytes, remained within ±3 standard deviations that were calculated for fresh samples, provided that the blood was stored at 20° C. Correspondence and offprint requests to: Dr Ulrich Bleul, Klinik für Fortpflanzungskunde, Universit?t Zürich, Winterthurerstrasse 260, 8057 Zurich, Switzerland.     

Mathematical Model of Time Needed for the Immune System to Detect and Kill Cancer Cells in Blood

Disfunction of the immune system has generally been blamed for the development of cancers however, the immune system needs time to detect and kill cancer cells. To date, the chance of random collision between immune cells and cancer cells in blood has drawn less attention. In this study we used a random principle to analyse the possibility of collision between immune and cancer cells in blood. With the criterion of p>0.95, the results show that an immune cell needs, for example, five random collisions in order to hit a cancer cell when there are one cancer cell, one immune cell and one normal cell in our consideration; and 14 999 999 random collisions in order to hit a cancer cell when there are one cancer cell, one immune cell and 4 999 999 normal cells in consideration. Furthermore, we analyse how different proliferation rates of cancer cells affect the random collisions between the detecting/killing immune cells and cancer cells.     

Blood Biochemistry Values of Green Turtles, Chelonia Mydas , With and Without Fibropapillomatosis

Baseline blood biochemistry values were obtained for two foraging aggregations of clinically healthy wild, juvenile green turtles (Chelonia mydas) inhabiting Kaneohe Bay, Island of Oahu, and the Kona Coast, Island of Hawaii. Mean reference values were compared to values obtained from green turtles of similar size affected with fibropapillomas (FP) collected at Kaneohe Bay. Statistically significant differences were identified for total protein values, blood urea nitrogen, and enzyme values between healthy turtles and turtles with FP. In addition, turtles with severe FP were hypoproteinaemic, hypoalbuminaemic, hypoferraemic, azotaemic, and presented inverse calcium/phosphorus ratios, low cholesterol and triglyceride values, indicating the chronicity and severity of FP. It is concluded that blood reference values should be established for green turtles at the population level and by geographic area considering disease status, age, sex, and seasonal variations.