Mouse monoclonal antibodies reacting with M blood group-related antigens

Four mouse monoclonal antibodies were produced with specificities related to human blood group M antigen. The antibodies react in direct hemagglutination systems, and their specificities were investigated by their reactions with variant and enzyme-modified red cells. The effects of temperature and pH on their hemagglutination reactions also were investigated, and all four were murine IgG antibodies. Physicochemical and serologic investigations showed them to be four distinct antibodies, and each one could be used for M blood grouping under appropriate conditions.     

E-receptors mediate the attachment of activated T-lymphocytes to human red blood cells: evidence from studies with monoclonal antibodies and simple sugars

Lectin-stimulated human T-lymphocytes display the capacity of forming stable E-rosettes with sheep red blood cells (SRBC) and of forming rosettes with allogeneic and autologous erythrocytes (RBC). Fractionation experiments indicated that the same subpopulation of lymphocytes displays both of these characteristics. Exposure of T-lymphocytes stimulated with concanavalin A (Con A) or phytohemagglutinin (PHA) to T11 monoclonal antibody (directed against the E-receptor) completely blocked the formation of rosettes with human RBC. Monoclonal antibodies to other T-cell surface markers (Leu 4, T4, T8 and OKT9) had no such effect. The formation of human RBC rosettes and stable E rosettes by PBL stimulated for 2 to 8 days by Con A was inhibited to a similar extent by dilutions of T11 monoclonal antibody. Lectin-stimulated PBL were exposed to various sugars to determine their effect on human RBC rosette formation. Rosette formation was inhibited over 80% with 2-deoxy-D-glucose and by 50-70% with D-mannose, while the other sugars tested had either less effect or none. The present findings support the concept that E-receptors on the surface of human T lymphocytes play a role in the recognition of carbohydrate containing "self" structures.     

A study of HLA (Bg) on red cells and platelets by immunoblotting with monoclonal antibodies

HLA class I antigens (Bg) on red cells (RBCs) are expressed by some normal donors and by many patients with systemic lupus erythematosus (SLE). To identify the membrane components previously detected by hemagglutination with HLA class I-specific monoclonal antibodies (MoAbs), RBC membrane preparations were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with the HLA class I MoAbs. Two components were obtained that reacted with the MoAbs: a heavy chain of 45 kDa and a light chain termed beta2-microglobulin (beta2-M) of 11 kDa. The effect of chloroquine and acid elution in stripping HLA antigens is shown to be due to the removal of beta2-M, as only that component was detected in eluates from reactive RBCs. Neither antibody elution method affected the heavy chain expression assessed by immunoblotting. It is concluded that HLA class I antigens on RBCs are integral membrane components of the type normally found and wisely distributed on many nucleated cells. Platelets, which have stronger HLA class I antigen expression, were also studied, and their membrane preparations yielded heavy chain and beta2-M molecules; the effect of chloroquine treatment was harder to assess than that of acid elution, owing to the sensitivity with which both components are detected in immunoblotting. In eluates obtained from acid treatment only beta2-M is detected.     

A strong antibody reacting with enzyme modified E positive red blood cells

A high titer antibody was discovered in a healthy young man of blood group A1 R2r. The antibody strongly agglutinated all E positive red blood cells including his own, which had been modified by papain, ficin and bromelin, but only very weakly when modified by trypsin. The antibody was shown to be an IgM antibody. It did not react with unmodified red blood cells.     

Studies on the binding of an alloimmune and two murine monoclonal antibodies to the platelet glycoprotein IIb-IIIa complex receptor

The platelet-binding characteristics of three different antibodies that completely block adenosine diphosphate (ADP)-induced platelet fibrinogen binding and react with glycoproteins IIb, IIIa, or both, were studied. Two of the antibodies are murine monoclonal antibodies (10E5 and 7E3), and the third is an alloimmune antibody produced by a patient with Glanzmann's thrombasthenia who has received multiple transfusions (E.S.). The two monoclonals differ in that 7E3, but not 10E5, binds to dog platelets as well as to human platelets. In mutual competition studies, 7E3 did not interfere with 10E5 binding, indicating that both antibodies could bind to the complex simultaneously. E.S. IgG produced only minor inhibition of 10E5 binding, but nearly complete inhibition of 7E3 binding, suggesting that its epitope lies closer to the 7E3 epitope than the 10E5 epitope. Ethylenediaminetetraacetic acid (EDTA) pretreatment of intact platelets at 22 degrees C and pH 7.75 did not affect 10E5 binding, but significantly inhibited 7E3 binding. Similar treatment of intact or solubilized platelets at high pH and temperature produced splitting of the glycoprotein IIb-IIIa (GPIIb-IIIa) complex and loss of both 10E5 and 7E3 binding. The 10E5 bound equally well to unactivated and activated platelets, and even rapid fixation of whole blood produced only a modest decrease in 10E5 binding. Preincubation of platelets with either E.S. IgG or 10E5 partially prevented EDTA from splitting the complex, and both monoclonal antibodies were able to bind to such complexes despite the EDTA treatment. These studies indicate that the binding of antibodies to several different epitopes on the GPIIb-IIIa complex can result in similar functional properties in blocking fibrinogen binding and platelet aggregation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Surface modification with protein A for uniform binding of monoclonal antibodies

We describe optimal conditions for immobilization of two monoclonal antibodies to progesterone for solid-phase assays. Polystyrene surfaces are refined with Protein A to achieve uniform, reproducible, stable, and sterically accessible immobilization of immunoglobulins (IgG). To this end, we optimized the amount of immobilized Protein A, the pH of the medium for immobilization, the concentration of antibody, and the polystyrene surface. We also investigated three carriers for solid-phase assays: 12 X 75 mm polystyrene test tubes, Macrowells (Skatron, Inc.; suitable for processing with multiple pipettors), and microwell strips (Immulon II, Dynatech Inc.). Immunoglobulin does not appreciably dissociate from any of these solid matrices, even if the assay procedure takes several hours. Therefore, we postulate that more than one molecule of immobilized Protein A binds to IgG, or that there is an additional interaction between the antibody and the polymer surface.     

Immunochemical and molecular characterization of regulatory idiotopes expressed by monoclonal antibodies exhibiting or lacking beta 2-6 fructosan binding activity

Hybridomas secreting antibodies bearing the ABPC48 (A48) regulatory idiotype (Id) were generated from BALB/c mice treated at birth or as adults with minute amounts of anti-A48-Id antibodies. The majority of these antibodies were recognized by the syngeneic monoclonal anti-A48-Id and anti-UPC-10-Id antibodies, IDA10 and 10-1, respectively. In Northern blotting experiments, most of these hybridomas were shown to use VH (heavy chain variable region) genes related to the 441-4 germline VH gene that encodes the A48 VH region. Hybridization was detected between polyadenylated H chain mRNA, isolated from the majority of the hybridomas, and the VH probe. Southern blots confirmed these results by showing a rearrangement of VH-related sequences to the JH (H chain joining segment) clusters on these same hybridomas. The antibodies from all of the hybridomas that derived from neonatal mice and half of those derived from adult mice showed specificity for fructosan determinants that, in most cases, was different from the beta 2-6 fructosan linkage specificity of A48. Surprisingly, several of the non-fructosan-binding hybridomas generated from the adult mice and the MOPC-173 myeloma demonstrated a clear specificity for the beta 1-6-D-galactan determinant. Of four galactan-binding myeloma proteins studied. XRPC 44 alone shared idiotypy with the UPC-10 myeloma. These findings suggest a possible clonal crossreactive regulation mediated by regulatory idiotopes. The crossreactive regulation concept is discussed.     

Production and characterization of a cytotoxic monoclonal antibody reacting with rat islet cells.

We have produced a murine monoclonal antibody (F43 A1D2) that binds to the cell surface of both rat islet tumor cells (RINm clone 5F and RINm clone 14B) and normal rat islet cells. This antibody is cytotoxic in the presence of complement for RIN tumor cells as well as A, B, and D pancreatic polypeptide rat islet cells. Antibody A1D2 does not bind to rat thymus cells, pancreatic acinar cells, or fibroblasts. Antigen A1D2 (termed ICSAn-1 [islet cell surface antigen 1]) is a trypsin-sensitive glycoprotein with a molecular mass of approximately 24,000 daltons. In addition to purification of its islet cell antigen, antibody A1D2 can be used to identify and isolate living islet cells from pancreatic digests.     

Immunochemical and functional characterization of anti-idiotypic antibodies to a mouse anti-CD4 monoclonal antibody

Summary Immunization of BALB/c mice with the mouse anti-CD4 monoclonal antibody (mAb) HP2/6 resulted in the production of anti-idiotypic antibodies. Analysis of the kinetics of the development of anti-idiotypic antibodies showed a homogeneous response among the immunized animals. Cross-blocking assays performed with anti-CD4 mAbs OKT4, OKT4c and OKT4d showed that syngeneic anti-idiotypic antiserum elicited with mAb HP2/6 recognizes idiotope(s) expressed only on the immunizing mAb. The idiotope(s) is (are) located within or closely related to the antigen-combining site of mAb HP2/6. Hybridization with the myeloma cell line NSO of splenocytes from a BALB/c mouse hyperimmunized with mAb HP2/6 generated the anti-idiotypic mAbs F11-2113, F11-2302 and F11-2444 which recognize idiotope(s) outside the antigen-combining site of mAb HP2/6. Although the anti-idiotypic mAbs cross-inhibit each other in their binding to mAb HP2/6, they differ in the ability to elicit anti-anti-idiotypic antisera. Furthermore, mAb F11-2113 enhances CD4 down-regulation in the presence of mAb HP2/6 to a larger extent than mAbs F11-2302 and F11-2444. The latter results suggest an additional mechanism by which anti-idiotypic antibodies may induce functional abnormalities of CD4⁺ T cells in human immunodeficiency virus-infected T cells.     

Quantitative studies on the D antigen of red cells with the Du phenotype

Rh-positive red cells (RBCs) that fail to agglutinate with commercial anti-D reagents by the tube method are considered to have the Du phenotype. The quantities of D epitopes on such RBCs have been measured previously in one kindred. The authors report on the number of D epitopes on Du RBCs of 23 unrelated individuals, as calculated by Scatchard's analysis. Cell-bound anti-D was measured by an automated antiglobulin consumption technique. On the average, RBCs of the Rh phenotype CcDue had a mean of 1568 +/-1220 (n = 12) D epitopes per cell. The relatively large range of values in this group implies a heterogeneous genetic background. The lowest number of D epitopes, 285 per cell, was observed on the RBCs of one individual who was apparently homozygous for C. In this case, the D antigen was detected only by adsorption/elution tests. RBCs of the phenotype cDuEe had a mean of 775 +/- 378 epitopes per cell (n = 8), and those from two individuals with phenotype cDue had 2840 and 1560 D epitopes, respectively. Thus, on the average, RBCs with the Du phenotype bear about 10 to 20 times less D antigen than normal Rh-positive RBCs. It is suggested that the low D antigen density of Du red cells may account for their poor immunogenicity.     

Further observations and characterization of monoclonal antibodies reacting with B cell alloantigens associated with rheumatic fever and rheumatic heart disease

Elevated levels of B lymphocytes with a unique surface alloantigen have been reported to be characteristic of patients with acute rheumatic fever or rheumatic heart disease. Mouse monoclonal antibodies (mAbs) to this alloantigen have been proposed as being useful in identifying individuals at risk for the development of these sequelae of group A streptococcal infection. However, previous studies have suggested that the discriminating ability of the mAbs was highest when the mAbs were made by using lymphocytes from the same ethnic population. To confirm and extend this observation, additional mouse mAbs were developed and their properties defined. These three mAbs-PG-12A, PG-13A, and PG-20A-reacted with B cells from more than 90% of North Indian patients with acute rheumatic fever or rheumatic heart disease. Each of these three new mAbs identified the highest levels of reactive B cells in patients with active acute rheumatic fever. Lower levels of positive reacting lymphocytes were found in individuals with quiescent chronic rheumatic heart disease, and markedly reduced percentages of reactive cells were observed in normal control subjects. The proportion of reactive lymphocytes in individual patients varied according to which of the three was tested, suggesting the possibility of a spectrum of "rheumatic" epitopes in susceptible individuals. The data further suggested that enhanced discriminatory ability for identifying "at-risk" susceptible patients could be obtained by testing with a combination of mAbs. If reduction in the incidence of acute rheumatic fever can be facilitated by early identification of susceptible individuals, accurate and sensitive detection of a marker antigen would result in more cost-effective public health measures. Additional population studies are required to more precisely define and confirm these detection techniques.     

阻断血管性血友病因子与胶原结合的抗人vWF-A3单抗的鉴定和功能研究

目的 制备阻断血管性血友病因子(von Willebrand因子)A3区(vWF-A3)与胶原结合的抗vWF-A3单抗,并对其进行生化鉴定和功能研究.方法 用重组vWF-A3蛋白免疫BALB/c小鼠,经标准的方法制备单抗.用ELISA法鉴定单抗特异性,经vWF胶原结合抑制试验筛选获得抑制性单抗,用免疫印迹技术确定单抗与重组(r)vWF-A3和还原性人vWF识别的抗原分子.经胶原结合试验确定单抗对vWF与胶原结合的影响.结果 获得一组共30株抗vWF-A3单抗,其中两株确定为抑制vWF与胶原结合的单抗,分别命名为SZ-123和SZ-125,两株单抗分别与rvWF-A3和vWF强反应,而不与rvWF-A1、A2反应.免疫印迹分析显示SZ-123和SZ-125分别识别相对分子质量为27×103的rvWF-A3、250×103还原性人vWF单体和170×103的vWF酶切片段.单抗SZ-123和SZ-125不仅剂量依赖性地抑制纯化的人血浆vWF与人胎盘和牛皮肤Ⅲ型胶原结合,而且分别抑制人、猕猴或Beagle犬血浆vWF与人胎盘和牛皮肤Ⅲ型胶原结合.结论 抗vWF-A3单抗SZ-123和SZ-125是两株抑制性单抗,有望成为具有治疗潜力的抗血栓药物.     

Interferon: A greatly simplified immuno enzyme determination with two monoclonal antibodies

Human leukocyte interferon (HuIFN-alpha) can be determined by a substantially simplified enzyme-immunological method, based on the solid phase sandwich principle. In a single step, the interferon in the sample or the standard solution (tests and standards are run concomitantly) is bound to a polystyrene bead coated with monoclonal (mouse) anti-interferon, and at the same time it is labelled with a second monoclonal (mouse) interferon antibody, which is coupled to horse radish peroxidase. After this immunological incubation, the non-bound material is removed by washing, and the quantity of peroxidase bound to the bead is measured enzymically. The resulting colour intensity is determined photometrically, and it is directly proportional to the interferon concentration in the sample. The detection limit of the test can be as low as 100 I.U./l interferon, depending on the assay conditions. The variation coefficient within series was 3-6%. Serum samples can be used directly in the test without pretreatment. Depending on the required sensitivity, the incubation may be performed for periods between 30 minutes and 24 hours.