Comparisons of Pooled Polyclonal Rabbit Anti-Human C3d with Four Monoclonal Mouse Anti-Human C3ds

Labelled polyclonal IgG anti-C3d and monoclonal IgM and IgG anti-C3d antibodies (MAs) were employed at increasing antibody excess to measure the number of C3d molecules on human red blood cells (RBC) coated by complement in vitro and in vivo. Values for the number of C3d sites per cell determined with polyclonal anti-C3d were at least 4-fold higher than when MAs were used. The results suggest that the molar combining ratio for polyclonal anti-C3d with a single RBC-bound C3d fragment is more likely greater than 4.0 than 1.0 as previously assumed. Antiglobulin agglutination studies compared polyclonal and monoclonal anti-C3d antibodies against C3d-coated RBC from 27 patients with autoimmune hemolytic anemia. All four MAs showed striking prozones, requiring their use over a 25-fold higher range of dilutions than polyclonal anti-C3d. Polyclonal anti-C3d produced stronger agglutination reactions than any of the IgG MAs. Only the IgM MA produced agglutination as strong as, or stronger than, polyclonal anti-C3d. While IgM MA always gave the strongest MA agglutination reactions, no consistent ranking of the three IgG MAs was observed. Agglutination was not enhanced when all IgG MAs were combined; addition of IgG MAs to IGM MA reduced the strength of agglutination seen with IgM alone, suggesting blocking of IgM binding by competing IgG anti-C3d.     

泡沫细胞相关基因4兔抗人多克隆抗体的制备

目的探讨制备泡沫细胞相关基因4多克隆抗体的方法。方法将从人胎肝文库经聚合酶链反应扩增获得的泡沫细胞相关基因4基因全长cDNA序列，通过生物信息学分析，预测泡沫细胞相关基因4编码氨基酸序列的二级结构、抗原决定簇、功能结构域．并进行了多序列比对，并根据蛋白质的亲疏水性、二级结构、偶联难度及实验难度等，确定泡沫细胞相关基因4抗原13肽PKLVKEEVFWRNY，采用固相多肽合成法合成该抗原片段．偶联后经过基础免疫，6次加强免疫，经追踪检测，于第四次免疫后10～14天颈动脉放血，分离血清，冷冻抽干，-20℃保存而制备了泡沫细胞相关基因4兔抗人多克隆抗体。结果用固相多肽合成法成功制备了泡沫细胞相关基因4兔抗人多克隆抗体，该抗体经间接酶联免疫吸附测定法检测其效价为1：16000，Western blot检测可见40kDa处有目的带，用免疫组织化学方法从HepG2细胞中检测到泡沫细胞相关基因4蛋白表达，证实该抗体具有较好的反应性和特异性。结论固相多肽合成法省时、省力、制备的抗体效价高；泡沫细胞相关基因4兔抗人多克隆抗体的制备，为进一步研究泡沫细胞相关基因4蛋白在动脉粥样硬化中的功能作用奠定了基础。     

Mouse Monoclonal Antibodies with Anti-A, Anti-B and Anti-A,B Specificities; Some Superior to Human Polyclonal ABO Reagents

A series of fusions using mouse myeloma cells and spleen cells from mice immunized with blood group substances or human red cells was performed with the aim of obtaining ABO antibodies suitable for routine blood grouping. Seven strongly agglutinating antibodies against antigens in the ABO system were obtained from 18 fusions. These antibodies were tested extensively in manual and automated routines, and six of them were found to be as good as or better than commercial polyclonal test sera of human origin.     

In vitro Functional Activity of IgG1 and IgG3 Polyclonal and Monoclonal anti-D

Background and objectives: IgG anti-D is generally restricted to IgG1 and IgG3; it mediates red cell destruction through interactions with IgG Fc receptors (FcγR) on effector cells. The relative ability of these two IgG subclasses of anti-D to mediate haemolysis in vitro by monocytes and K cells was investigated. Materials and methods: Anti-D was affinity purified from 5 preparations of prophylactic anti-D immunoglobulin, and IgG subclasses quantified by ELISA; mean levels were 86.5% IgG1, 1.4% IgG2, 11.6% IgG3 and 0.4% IgG4. IgG1 and IgG3 polyclonal anti-D were further purified separately from some of the anti-D by removal of either IgG3 using magnetic beads coated with anti-IgG3, or of IgG1 using protein A. These preparations were compared with monoclonal anti-D (BRAD-3 and BRAD-5) for their ability to lyse red cells in antibody-dependent cell-mediated cytotoxicity (ADCC) assays. Results: Monocyte-mediated lysis of red cells coated with IgG3 anti-D was approximately twice that of cells coated with IgG1 anti-D at similar sensitization levels, and anti-D preparations containing 10% or more IgG3 gave similar lysis. By contrast, in the K cell ADCC, IgG1 anti-D was 2–4 times more haemolytic than IgG3 anti-D. Polyclonal IgG1 and IgG3 anti-D promoted about 20% more lysis than BRAD-5 (IgG1) and BRAD-3 (IgG3), respectively, in the K cell ADCC, although no difference was observed between polyclonal and monoclonal anti-D in the monocyte ADCC. Conclusions: These experiments demonstrated a functional dichotomy between these two subclasses of anti-D; IgG3-coated red cells were lysed preferentially by monocytes mediated predominantly through FcγRI interactions, whereas haemolysis of IgG1-sensitized cells was mediated mainly by FcγRIII on K cells.     

In vivo instability of red-blood-cell-bound C3d and C4d

Until now, there have been no measurements of the in vivo stability of red-blood-cell-bound C3d and C4d subfragments of the third and fourth components of human complement. We have recently described a radiolabeled antiantiglobulin method for measuring RBC-bound C3d and have demonstrated that small amounts of C3d are present on RBC of all normal subjects tested. In the present study, the method was applied to follow the increments above baseline of RBC-bound C3d and C4d produced by autotransfusing 3 normal volunteers with 160-200 ml of RBC strongly coated in vitro by C3d and C4d. Posttransfusion measurements were carried out over 21-34 days. Immediate and long-term in vivo survival of the transfused RBC was unimpaired by C3d and C4d coating. Of the bound C3d antigen, 85%-95% disappeared from circulating RBC in 5-8 days; the remainder disappeared more slowly, with half-times in the range of 8-29 days. C4d antigen disappeared substantially more slowly, describable by a single exponential function in 2 of the 3 subjects, with half-times in the range of 12-31 days. Recognition of the in vivo instability of RBC-bound C3d helps in interpreting steady-state and changing levels of RBC C3d coating in a variety of alloimmune and autoimmune disorders.     

Preparation of Red Cells Coated with C4 and C3 Subcomponents and Production of Anti-C4d and Anti-C3d

Abstract. Methods of preparing red cells coated only with C4b, C3b or C3d have previously been described by others; the present observations supply further information about the optimal conditions for preparing such cells and also describe conditions for the preparation of cells carrying only C4d. In addition, the preparation of specific anti-C4d and anti-C3d sera is described.     

Complement fragment C 3d in the plasma of patients with chronic polyarthritis

Activation of the complement system via immune complex formation plays an important role in the perpetuation of chronic inflammatory disorders. The serum levels of C3 or C4 do not necessarily reflect the actual complement catabolism. By measurement of the long-lived C3 split product "C3d" direct information about complement activation can be obtained. C3d was measured with a modified immunoelectrophoresis technique in EDTA-plasma of 106 patients with rheumatoid arthritis. 36% of them showed significantly elevated C3d levels (mean: 8.93 micrograms/ml). Significant linear correlations were found for C3d with ESR, Lansbury-Index and plasma viscosity.     

Quantification by ELISA of erythrocyte-bound C3 fragments expressing C3d and/or C3c epitopes in patients with factor I deficiency and with autoimmune diseases

A sensitive ELISA assay for quantifying erythrocyte (E) bound C3 fragments was developed. The assay employs a double-antibody sandwich technique, using polyclonal anti-C3d or anti-C3c antibodies to quantify C3 fragments, expressing C3d and/or C3c epitopes in washed, detergent-solubilized E. The assay detected 50-120 molecules of C3d per E in healthy individuals. Antigens reacting with anti-C3c antibodies were also detected on E from normal individuals, but the density of C3c-epitopes was 0.9-2.4 times lower than that of C3d-epitopes. In 2 patients with congenital factor I deficiency significantly increased density of E-bound C3c- as well as C3d-antigen was observed. Plasma infusion in one of the patients induced a loss of E-bound C3c-antigens, indicating cleavage of E-bound C3b to iC3b and further to C3c and E-bound C3d. Loss of C3c-antigens also occurred following in vitro treatment with normal human serum of E from one of the patients. Two thirds of 22 patients with systemic lupus erythematosus (SLE) and of 18 patients with rheumatoid arthritis had significantly increased density of E-bound C3d, the highest density being 490 C3d molecules/E in an SLE patient. The density of E-bound C3d correlated with the plasma-C3d concentration, indicating that the coating of E with C3d reflects the degree of complement activation.     

单克隆多克隆抗体酶联免疫吸附法测定血清脂蛋白(a)的比较

应用淋巴细胞杂交瘤技术，建立了抗人血清脂蛋白（ａ）杂交瘤细胞株，对制备的单克隆抗体进行了特异性鉴定和抗原位点测定。建立了多克隆双抗体夹心酶联免疫吸附法、载蛋白（ａ）、载脂蛋白Ｂ双位点酶联免疫吸附法、单克隆单株、混合株双抗体夹心酶联免疫吸附法对脂蛋白（ａ）进行了检测，并对结果进行了分析。认为样本、参考品间载脂蛋白（ａ）的多态型不同可引起结果间的差异。     

Effect of Four Medications Associated with Gastrointestinal Motility on Oddi Sphincter in the Rabbit

Aim: Modulatory drugs of gastrointestinal (GI) motility are a possibility for use to relieve the main clinical presentation of sphincter of Oddi (SO) dysfunctions which are not easily distinguished from those occurring in high prevalence functional GI disorders. The aim of this study was to investigate the effects of GI motility modulators including pinaverium, domperidone, trimebutine, and tegaserod on the contractile activity of SO stimulated by carbachol in the rabbit. Methods: The contraction responses precontracted by carbachol (0.1 μM) of in vitro rabbit SO rings were evaluated before and after the addition of a series concentration (10^?13 to 10^?3 M) of pinaverium, domperidone, trimebutine, and tegaserod. Results: Pinaverium induced a concentrationdependent relaxation of isolated SO rings (10^?13 vs. 10^?7 vs. 10^?13 M = 16.6 ± 4.8 vs. 47.1 ± 5.5 vs. 81.2 8 6.2%, p<0.001 by ANOVA) precontracted with carbachol (0.1μM). Tegaserod did not significantly effect (10^?13 vs. 10^?7 vs. 10^?3 M = 2.3 ± 2.2 vs. 6.7± 2.1 vs. 10.1 ± 2.3%, p>0.05 by ANOVA) SO motility, but domperidone seemed to stimulate SO contractions (1010^?12 vs. 1010^?7 vs. 1010^?3 M = ?2.2 ± 1.5 vs. ?13.9 ± 2.0 vs. ?21.0 ± 2.7%, p<0.05 by ANOVA). At low doses (10^?13 to 10^?7 M), trimebutine stimulated SO contraction (?8.7 ± 1.4 vs. ?9.3 ± 2.0%); however, high doses (10^?6 to 10^?3 M) of trimebutine inhibited SO motility (?-5.9 ± 1.7 vs. 14.5 ±2.0%, p<0.05 by ANOVA). Conclusion: Pinaverium totally inhibits contractions induced by carbachol and tegaserod has no effect on carbachol-induced contractions. Domperidone stimulates contractions induced by carbachol. Trimebutine could either stimulate or inhibit SO contractions depending on its dosage.     

Plasma concentrations of fibronectin and C3d in patients with amoebic liver abscess.

By means of simple and specific ELISA techniques, the plasma concentrations of soluble fibronectin and C3d, a breakdown product of C3 complement, were determined in patients with amoebic liver abscesses (ALA) and in healthy controls. The mean plasma fibronectin concentrations in 23 patients with ALA and in 20 controls were found to be 441 +/- 89 mg/l and 442 +/- 66 mg/l, respectively. The difference between these two values was not statistically significant. The mean C3d value in 21 patients with ALA, however, was found to be 84 +/- 14 AU/l which was significantly different from the value of 12 +/- 4.7 AU/l noted in 20 healthy persons. Plasma concentrations of these two proteins are discussed in relation to their possible implications in the immunopathogenesis of amoebic liver abscess.     

Use of Monoclonal Anti-C3 Antibodies to Characterise the Fragments of C3 that Are Found on Erythrocytes

Using monoclonal antibodies to C3 it has been shown that the red blood cells of patients with cold haemagglutinin disease carry on their cells C3d,g (alpha-2D-globulin) rather than C3d. C3d,g seems to be the final product of in vivo C3 activation in fluid phase and on red cells. The cleavage of C3dg to C3d and C3g does not appear to occur in vivo either in the fluid phase or on red cell bound C3bi. In vitro C3-coated red cells prepared by antibody or low ionic strength techniques produce cells with C3d and C3bi as the predominant C3 fragment, whereas the Fruitstone technique in which coating occurs by the alternative pathway has principally C3b. The activity of C3 cleaving enzymes in whole serum is strongly influenced by the ionic conditions of the serum.     

Monoclonal rheumatoid factor-IgG immune complexes. Poor fixation of opsonic C4 and C3 despite efficient complement activation

Monoclonal IgM rheumatoid factor forms complexes with IgG in essential mixed cryoglobulinemia. We demonstrate that such complexes fix C3 and C4 poorly, although efficient fluid-phase C3 conversion can occur. Fixation of small amounts of C4 may be sufficient to generate a C3 convertase, but may prevent subsequent fixation of C3 by competing for binding sites on the complex. These complexes bind inefficiently to normal erythrocyte complement receptor type 1 (CR1) in vitro, and are undetectable on erythrocytes of patients with essential mixed cryoglobulinemia in vivo. Clearance of such phlogistic complexes from tissues by CR1-bearing cells may be inefficient.     

Treatment of Type 1 Diabetes with Anti-CD3 Monoclonal Antibody

Reviews in Endocrine and Metabolic Disorders -     

Studies on the Macroglobulins of Human Serum IV: The Frequency of Light Chain Types K and L in Polyclonal and Monoclonal γM.

125 M-components of class γM in 123 sera have been typed with regard to light chain determinants K and L. The typing of most sera required initial isolation of 19 S-fractions by thin layer gel filtration. 100 components were typed kappa and 25 lambda, giving a ratio of 4 to 1. Polyclonal γM from young adults contained kappa and lambda determinants in the ratio of 2 to 1. Possible explanations for this discrepancy are discussed.     

Dual interaction of factor H with C3d and glycosaminoglycans in host-nonhost discrimination by complement

The alternative pathway of complement is important in innate immunity, attacking not only microbes but all unprotected biological surfaces through powerful amplification. It is unresolved how host and nonhost surfaces are distinguished at the molecular level, but key components are domains 19–20 of the complement regulator factor H (FH), which interact with host (i.e., nonactivator surface glycosaminoglycans or sialic acids) and the C3d part of C3b. Our structure of the FH19–20:C3d complex at 2.3-Å resolution shows that FH19–20 has two distinct binding sites, FH19 and FH20, for C3b. We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan binding to FH20, thus providing a feedback control for preventing excess C3b deposition and complement amplification. This explains the molecular basis of atypical hemolytic uremic syndrome, where mutations on the binding interfaces between FH19–20 and C3d or between FH20 and glycosaminoglycans lead to complement attack against host surfaces.     

HLA Class I (Bg) Antigens on Red Cells of SLE Patients: A Serological Study with Polyclonal and Monoclonal Antibodies

The enhanced HLA class I (Bg) on red blood cells (RBC) of many patients with systemic lupus erythematosus has allowed a significant correlation to be made between their HLA-B types and haemagglutination reactivity with lymphocytotoxic anti-HLA-B sera stimulated by pregnancy alone. Therefore the class I expression on these RBC relates to classical, rather than non-classical, class I gene products. Studies of class I expression on RBC by means of monoclonal antibodies (MAb) to epitopes on the heavy polypeptide chain and beta 2-microglobulin (beta 2m) have suggested that the complete extracellular structure is present. The specific effect of chloroquine in 'stripping HLA' from RBC had been assumed to support the concept that HLA class I was adsorbed from plasma. However, from our data, we conclude that HLA class I is an intrinsic membrane component. We suggest that the action of chloroquine is to remove beta 2m alone, which prevents normal class I expression and also results in conformational changes to the class I heavy chain, but that it is not capable of removing the membrane-bound heavy chain.     

Mapping of the C3d receptor (CR2)-binding site and a neoantigenic site in the C3d domain of the third component of complement.

The C3d domain of C3 contains the site that binds to the C3d receptor (CR2) which is expressed on B lymphocytes. It also contains a neoantigenic determinant that is recognized by monoclonal antibody (mAb) 130 and is expressed when C3b is cleaved to iC3b and subsequently to C3dg or C3d. mAb 130 inhibits the binding of C3d to CR2. In this study, the locations of the CR2-binding site and of the neoantigen recognized by mAb 130 within the C3d domain were investigated. Treatment of human C3d with CNBr generated two major fragments with Mrs of 12,500 and 8600. Binding studies showed that only the Mr 8600 fragment was capable of binding to both CR2 and mAb 130. Amino-terminal sequence analysis of the Mr 8600 fragment and comparison with the amino acid sequence derived from human C3 cDNA [de Bruijn, M. H. L. & Fey, G. H. (1985) Proc. Natl. Acad. Sci. USA 82, 708-712] placed it between residues 1199 and 1274 of the C3 sequence. Several peptides were synthesized according to the derived C3 sequence of amino acid residues 1209-1236. Based on their differential binding to CR2 and mAb 130, we localized the CR2-binding site and mAb 130 neoantigenic site, respectively, to residues 1227-1232 and 1217-1232 of the C3 sequence.     

Four cases with intrafamilial clustering of hepatitis C virus infection.

Four hepatitis C patients with intrafamilial clustering of hepatitis C virus (HCV) infection are reported. Antibodies to C100-3 antigen, capsid protein of HCV and GOR epitope were tested to detect histories of HCV infection. Transmission of HCV from mother to children, from father to children, and from wife to husband was implicated. Of all family members studied, three were positive for all antibodies, one for only antibody to capsid protein, two for antibodies to capsid protein and GOR epitope but negative for antibody to C100-3 antigen and one vice versa.