Effect of Brazilian, Indian, Siberian, Asian, and North American ginseng on serum digoxin measurement by immunoassays and binding of digoxin-like immunoreactive components of ginseng with Fab fragment of antidigoxin antibody (Digibind)

We compared Brazilian, Indian, Siberian, Asian, and North American ginseng for potential interference with 3 digoxin immunoassays: fluorescence polarization (FPIA), microparticle enzyme (MEIA), and Tina-quant (Roche Diagnostics, Indianapolis, IN). We supplemented aliquots of a drug-free serum pool with ginseng extracts representing expected in vivo concentrations and overdose. We observed apparent digoxin-like immunoreactivity with FPIA, modest immunoreactivity with MEIA, and no apparent digoxin immunoreactivity with the Tina-quant with all ginsengs except Brazilian, which showed no immunoreactivity with any assay. When aliquots of serum pools prepared from patients receiving digoxin were supplemented with ginsengs, we observed falsely elevated digoxin values with FPIA, falsely lower digoxin values (negative interference) with MEIA, and no interference with the Tina-quant. Digoxin-like immunoreactive components of various ginsengs have moderate protein binding; monitoring free digoxin concentrations does not eliminate such interference. We also observed that Digibind (Burroughs Wellcome, Research Triangle Park, NC) can bind free digoxin-like immunoreactive components of ginsengs; such effects can be monitored by measuring apparent free digoxin concentrations. Indian, Asian, and North American ginsengs interfere with serum digoxin measurement by FPIA and MEIA; the Tina-quant is free of such interference. Digibind can bind free digoxin-like immunoreactive components of ginseng.     

Digoxin and digoxin-like immunoreactive factors (DLIF) modulate the release of pro-inflammatory cytokines

Objective:   Cardiac glycosides such as digoxin and their endogenous counterpart digoxin-like immunoreactive factor (DLIF) may possess anti-inflammatory properties. Methods:  Pro-inflammatory cytokines from human peripheral blood mononuclear cells (PBMC) were measured by ELISA using specific antibodies. Immunocytochemistry was used to localize NF-kB. Results:   Non-stimulated PBMC constitutively secreted minimum amounts of cytokines. LPS (1 mg/L) stimulation lead to steep increases in TNF-alpha, IL-6 and IL-8 concentrations with peak rises at 8 h. An 8 h delay was observed for IL-10. Increases in IL-10 were sustained for18 h period. Significant inhibition (P > 0.05) of TNF-alpha, IL-6 and IL-8 at non-toxic of digoxin concentration (< 100 nM) and DLIF (10 nM digoxin equivalent (de)) was observed whereas no such effect was seen for IL-10. Inhibition of the degradation of activated NF-kB in the PBMC was observed with the indicated concentrations of digoxin, DLIF or Pyrrolidine dithiocarbamate (PDTC). Conclusion:   Digoxin and DLIF inhibit the release of proinflammatory cytokines from PBMC via NF-kB-dependent pathway suggesting an anti-inflammatory effect. Received 30 December 2007; returned for revision 22 April 2008; received from final revision 24 April 2008; accepted by G. Wallace 22 May 2008 The first two authors contributed equally to this work.     

Neutralization of bacterial exotoxin (tetanus toxin) by catfish IgM antibody.

A bacterial exotoxin neutralization response by fish EgM antibody has not been demonstrated previously in any fish species. Channel catfish, Ictalurus punctatus, were immunized intraperitoneally with alum-adsorbed tetanus toxoid. Catfish immune serum demonstrated 1.28 antitoxin units (a.u.) of antitoxin neutralization and gave an indirect haemagglutination (IHA) titre of 1:65,536. After 2-mercaptoethanol (2ME) reduction of immune serum, no antitoxin neutralization remained by an IHA serum titre of 1:4096 was present. After Sephadex G-200 gel filtration of the catfish immune serum, the 14S antibody gave 0.32 a.u./ml and an IHA titre of 1:256. The 7S antibody gave no antitoxin neutralization but an IHA titre of 1.512 was found. After 2ME reduction, neither the 14S or 7S globulins demonstrated antitoxin neutralization, but minimal IHA titres of 1:16 and 1:4, respectively, were still found. The catfish immune serum and the 14S and 7S globulins did not precipitate tetanus toxoid by immunodiffusion in 1% agar gel.     

Suppression of total digoxin concentrations by digoxin-like immunoreactive substances in the MEIA digoxin assay. Elimination of negative interference by monitoring free digoxin concentrations

Digoxin-like immunoreactive substances (DLIS) cross-react with antidigoxin antibody and falsely elevate immunoassay-measured total digoxin concentrations. The fluorescence polarization immunoassay (FPIA) for digoxin showed high cross-reactivity with DLIS, but a new microparticle enzyme immunoassay (MEIA) had low cross-reactivity. The concentration of digoxin in the presence of DLIS was falsely lowered (negative interference) when measured by MEIA. We prepared the following serum pools: 2 normal (no DLIS), 2 from patients with uremia, and 3 from patients with liver disease (high DLIS). No patients received digoxin or digitoxin. When normal pools were supplemented with known concentrations of digoxin, total and free concentrations measured by both assays were comparable, but when liver and uremic pools containing high DLIS were supplemented with digoxin, the measured total digoxin concentrations were lower by MEIA and higher by FPIA. However, by taking advantage of 25% protein binding of digoxin and high protein binding of DLIS, free digoxin levels were not affected by DLIS. In 2 patients receiving digoxin but without volume expansion, total and free digoxin concentrations measured by both assays were comparable; in the 2 volume-expanded patients, only free digoxin concentrations were comparable. Monitoring free digoxin concentration can eliminate negative interference of DLIS in the MEIA for digoxin.     

Neutralization of infectious ME (Maus-Elberfeld) virus-antibody complexes by anti-γ-globulin antibody

Neutralization of MB (Maus-Elberfeld) virus by rabbit anti-ME virus antiserum has been studied by the plaque reduction method. A certain amount of virus scored as infectious even after prolonged incubation with antibody in excess. This persistent virus fraction was readily neutralized by sheep anti-rabbitγ-globulin antiserum, complement, and unheated calf serum. The results of these experiments are interpreted to mean that the persistent fraction of MB virus consists of virus which has retained its infectivity in spite of attached antibody. The persistent virus was neutralized by anti-γ-globulin antiserum even after adsorption of the virus to susceptible cells, indicating that it was still complexed with antibody. Virus free of antibody or virus aggregates could not be demonstrated in the persistent fraction.     

Crystallization of a complex between the Fab fragment of a human immunoglobulin M (IgM) rheumatoid factor (RF-AN) and the Fc fragment of human IgG4.

Rheumatoid factors (RF) are the characteristic autoantibodies found in patients with rheumatoid arthritis. They recognize epitopes in the Fc region of immunoglobulin G (IgG) and are often of the IgM isotype. In order to analyse the nature of RF-Fc interactions, we have crystallized a complex between the Fab fragment of a human monoclonal IgM rheumatoid factor (RF-AN) and the Fc fragment of human IgG4. The stoichiometry of the complex within the crystals was found to be 2:1 Fab:Fc. The crystals diffracted X-rays to 0.3 nm resolution, and the space group was C2, with cell dimensions a = 16.03 nm, b = 8.19 nm, c = 6.42 nm, beta = 98.3 degrees. We have also determined the sequence of the variable region of the RF-AN light chain, not hitherto reported. This belongs to the V lambda III-a subgroup and is closely related to the germline gene Humlv318, from which it differs in three amino acid residues. This is the first reported crystallized complex between a human autoantibody and its autoantigen.     

Structure of the Fab fragment of the anti-murine EGFR antibody 7A7 and exploration of its receptor binding site

The EGF receptor is an important target of cancer immunotherapies. The 7A7 monoclonal antibody has been raised against the murine EGFR, but it cross-reacts with the human receptor. The results from experiments using immune-competent mice can therefore, in principle, be extrapolated to the corresponding scenario in humans. In this work we report the crystal structure of the 7A7 Fab at an effective resolution of 1.4?. The antibody binding site comprises a deep pocket, located at the interface between the light and heavy chains, with major contributions from CDR loops H1, H2, H3 and L1. Binding experiments show that 7A7 recognizes a site on the EGFR extracellular domain that is not accessible in its most stable conformations, but that becomes exposed upon treatment with a tyrosine kinase inhibitor. This suggests a recognition mechanism similar to that proposed for mAb 806.     

丹参提取物F对大鼠乙酸-消炎痛胃溃疡愈合的影响

以大鼠乙酸－消炎痛胃溃疡为动物模型，用甲氰咪胍作为对照药，观察了丹参提取物Ｆ对溃疡愈合过程的影响及其与胃粘膜微循环、粘膜细胞再生、胃壁结合粘液分泌的关系。结果表明，丹参提取物Ｆ组的溃疡指数和溃疡边缘毛细血管网眼面积明显低于对照组和低于甲氰米胍组，而溃疡边缘粘膜血流量、细胞标记率及胃壁结合粘液量明显高于对照组，亦高于甲氰咪胍组。提示：丹参提取物Ｆ能促进大量乙酸－消炎痛胃溃疡的愈合，其作用优于甲氰咪胍。其机制可能与改善胃粘膜微循环、边速粘膜细胞的再生、促进粘液分泌有关。     

Production of an anti-severe acute respiratory syndrome (SARS) coronavirus human monoclonal antibody Fab fragment by using a combinatorial immunoglobulin gene library derived from patients who recovered from SARS

A combinatorial human immunoglobulin gene library was constructed from the peripheral lymphocytes of two patients who recovered from severe acute respiratory syndrome (SARS). The library was screened for the production of Fab antibody fragments to a recombinant spike protein of SARS-associated coronavirus (SARS-CoV). One Fab clone, AS3-3, reacted with the spike protein in an enzyme-linked immunosorbent assay. The dissociation constant of AS3-3 was 1.98 x 10(-8) M. Immunofluorescent microscopy revealed that it reacted with SARS-CoV-infected cells. The library seems to be a potent tool for the production of human antibodies to SARS-CoV.     

嵌合抗CD20抗体片段F(ab'')2的表达及活性

目的 :为了简化生产步骤 ,提高抗体蛋白的生物活性 ,探索在工程菌体内直接进行抗CD2 0F(ab’) 2 的高效率分泌性表达。方法 :采用单因素考察法优化培养条件 ,使蛋白G柱和S2 0 0 HR分子筛柱分离纯化目的蛋白 ,用MTT法检测抗CD2 0F(ab’) 2 抑制Daudi细胞体外生长的活性。结果 :发现用论文所选定的最适培养条件 ,抗CD2 0F(ab’) 2 的产量有了明显提高 ,从1 9～ 2 .2mg L达到了 3 7～ 4 3mg L ,F(ab’) 2 在表达产物中所占的比例也从 9 7%～ 13 2 %提高到了 38 1%～ 4 6 8% ;使用S2 0 0 HR分子筛柱对蛋白G柱亲和纯化后的产物进一步分离纯化 ,可以使F(ab’) 2 的纯度达到 85 %以上 ;MTT法检测结果证明F(ab’) 2 抑制Daudi细胞生长的IC50 值为 14 6 μg ml,而Fab’为 39 5 μg ml。 结论 :实现了抗CD2 0F(ab’) 2 在工程菌体内的高效率分泌性表达 ,而且所表达的抗CD2 0F(ab’) 2 比抗CD2 0Fab’具有更强的抑制Daudi细胞体外生长的能力     

Thermodynamics of the interaction of epsilon-dinitrophenyl-L-lysine and the subunits (Fab) of bovine colostral immunoglobulin G1 anti-dinitrophenyl antibody.

Investigation of the binding of epsilon-DNP-1-lysine to the subunits (Fab') of bovine colostral IgG1 anti-DNP over a wide range of temperatures yielded non-linear van't Hoff plots with curvatures which were indicative of large positive heat capacity changes. Thermodynamic functions which were calculated using a non-linear least-squares procedure revealed an enthalpy-entropy compensation mechanism for binding. While the enthalpy factor was the driving force for the hapten-subunit interaction(s) at low temperatures, the entropy factor assumed greater importance with increasing temperatures. In addition, the enthalpy-entropy compensation plot for the interaction of epsilon-DNP-1-lysine with bovine colostral Fab' anti-DNP, intact anti-DNP IgG1 and rabbit IgG anti-DNP revealed a constant compensation temperature (T degrees c) of 27 degrees which might be considered as indicative of a single kind of protein-solvent conformation.     

Flexibility of the major antigenic loop of foot-and-mouth disease virus bound to a Fab fragment of a neutralising antibody: structure and neutralisation

The interaction of foot-and-mouth disease virus (FMDV) serotype C (clone C-S8c1) with a strongly neutralising monoclonal antibody (MAb) 4C4 has been studied by combining data from cryoelectron microscopy and x-ray crystallography. The MAb 4C4 binds to the exposed flexible GH-loop of viral protein 1 (VP1), which appears to retain its flexibility, allowing movement of the bound Fab. This is in striking contrast to MAb SD6, which binds to the same GH-loop of VP1 but exhibits no movement of the bound Fab when observed under identical conditions. However, MAbs 4C4 and SD6 have very similar neutralisation characteristics. The known atomic structure of FMDV C-S8c1 and that of the 4C4 Fab cocrystallised with a synthetic peptide corresponding to the GH-loop of VP1 were fitted to the cryoelectron microscope density map. The best fit of the 4C4 Fab is compatible only with monovalent binding of the MAb in agreement with the neutralisation data on 4C4 MAbs, Fab2s, and Fabs. The position of the bound GH-loop is related to other known positions of this loop by a hinge rotation about the base of the loop. The 4C4 Fab appears to interact almost exclusively with the G-H loop of VP1, making no other contacts with the viral capsid.     

Activation of the alternative complement pathway by the immune precipitate formed with F(ab')2 fragment of human IgG antibody

The complement fixing ability of the F(ab')2 fragment of human IgG was studied using an immune precipitate (Ippt) formed between tetanus toxoid and the F(ab')2 of high-titer IgG antibody against tetanus toxin. A major subclass of the specific IgG antibody against tetanus toxin, which was separated by affinity column chromatography, was identified as IgG1. On incubation of normal human serum (NHS) with the Ippt formed at equivalence, a dose-dependent consumption of CH50, C3 and C5 activities was observed without significant loss of the early acting complement components. A similar consumption of CH50, C3 and C5 activities was found in NHS reacted with Ippt formed at any antigen/antibody ratio. The Ippt formed at antibody excess was more efficient in complement consumption than the Ippt formed at antigen excess. An apparent consumption of C3 and C5 activities was also noted in C4-deficient guinea pig serum treated with Ippt. When Ippt was incubated with Mg2+--EGTA-treated NHS, both C3 and C5 convertases of the alternative pathway were generated on the Ippt. From these results, it was concluded the the F(ab')2 of human IgG antibody, especially IgG1 antibody, when it formed an Ippt with antigen, could activate the alternative complement pathway.     

Neutralization of the biological activity of cytokines and other protein effectors by antibody: theoretical formulation of antibody titration curves in relation to antibody affinity

Patients treated with interferons, other cytokines, or various biologically active proteins may form neutralizing antibodies, which can adversely affect clinical outcome. It is therefore important to understand how antibodies neutralize such soluble protein antigens and how best to quantitate such antibodies. By applying the mass action law to antigen-antibody reactions, we previously developed a mathematical model applicable in two situations: first, for antibodies having low affinity for the antigen concerned (the Constant Proportion (CP) case), and, second, for antibodies having high affinity (the Fixed Amount (FA) case). The results allowed calculation of neutralization titers which were independent of the particular assay method used. Neutralization by antibodies of intermediate affinity, however, requires different mathematical treatment because the mode of neutralization does not fit the two cases mentioned above. In this paper, theoretical neutralization curves were derived, based on the same mathematical model, for antibodies of intermediate affinity. We show that the slope of the neutralization curve relating residual active antigen to the concentration of antibodies is determined by the antibody association constant and the molar concentration of the effector antigen. It is therefore possible to infer the magnitude of the association constant from the observed neutralization curve. We show that values obtained for the neutralization titer of antibodies of intermediate affinity by the use of the formula previously described for the Fixed Amount and Constant Proportion cases may deviate from the theoretically sound values; the magnitude of the deviation can be estimated by applying the formulas described herein. These relationships should apply generally to antibody neutralization reactions with all biologically active soluble protein effector molecules that have a single and nonrepetitive epitope.     

Comparison of methods for the generation of immunoreactive fragments of a monoclonal antibody (B72.3) reactive with human carcinomas

Immunoglobulin fragments, whether of polyclonal or monoclonal antibodies, offer a number of advantages over the intact immunoglobulin. The generation of immunoreactive fragments from monoclonal antibodies (MAb) is not always a straightforward task. Both pepsin and papain can be used to digest MAb to a bivalent molecule with a Mr of 100,000. However, pepsin pepsin digestions does not always result in immunoreactive fragments and a stable consistent product by papain digestion is often difficult to obtain. MAb B72.3 is an example of both situations. MAb B72.3 reacts with a glycoprotein (TAG-72) with a molecular weight greater than 10(6). MAb B72.3 has been shown to exhibit a high degree of selective reactivity with colon, breast and ovarian carcinomas and has been used for radioimmunodiagnosis in model systems and in clinical trials. A third enzyme, bromelain, in the same family of sulfhydryl proteases as papain, has been used to generate a fragment of MAb B72.3, with a Mr of approximately 100,000. The bromelain-generated fragment of MAb B72.3 retained 100% immunoreactivity as measured in competitive solid-phase radioimmunoassays and could be generated with consistent results from one preparation to another. Both the bromelain- and papain-generated fragments were radiolabelled with 125I without significant loss of the MAb's reactivity to tumor extracts. Differences were observed between the bromelain- and papain-generated fragment when compared in vivo. Fragmentation of MAb B72.3 with bromelain has yielded a superior bivalent fragment for radioimmunolocalization.     

Random and directed migration of trout (Salmo gairdneri) leukocytes: activation by antibody, complement, and normal serum components

Purified peripheral blood leukocytes from rainbow trout (Salmo gairdneri) were used in experiments to determine whether these cells are capable of random or directed leukotaxis in response to immunologically important mediators. Leukocyte migration was assessed by the use of microporous filter penetration assays and migration-under-agarose tests. Leukocyte migration rates were enhanced in filter penetration assays by the presence of antigen-antibody-complement complexes, and chemotactic migration was observed in migration-under-agarose tests as a response to whole trout serum. Trout leukocytes thus altered normal migratory activities in response to chemical changes in their immediate environment. The role of complement in chemotaxis may be similar in fish and mammals.     

Isolation of human Fab antibodies specific for the low-affinity IgE receptor (CD23) by selecting a hierarchical antibody library system against B lymphoblastic IM-9 cells

The development of human antibodies specific for certain B cell markers is required to generate therapeutic antibody leads with improved therapeutic indices against B-cell lymphomas. To meet this demand, we selected a primary human antibody library, HuDVFab-8L, against human B lymphoblastic IM-9 cells via a ‘Biopanning and Rapid Analysis of Selective Interactive Ligands (BRASIL)’ cell panning approach. Six Fab clones that specifically bound to IM-9 cells were successfully isolated. Among these clones, two clones (IM-L6-E and IM-L8-G), were found to be specific for CD23 (Fc?RII). Affinity maturation of these Fab clones was then performed in a hierarchical manner by constructing secondary antibody libraries through combining heavy (H) chains of two Fabs with the human kappa L chain sublibrary HuNL-D3 followed by biopanning against the CD23 antigen. Clone IM-L6-5, one of the affinity maturated Fab derivatives from IM-L6-E, has a binding affinity of k_D ≈ 30 nM to soluble CD23. In addition, IM-L6-5 Fab is able to bind to an inducible form of CD23 expressed on U937 cells upon IL-4 stimulation, and inhibits binding of human IgE to CD23. Since the Fab IM-L6-5 is derived from a fully human naïve origin, we believe that IM-L6-5 can be utilized for the development of a therapeutic mAb which may have an improved therapeutic index over lumiliximab, a primatized anti-CD23 mAb, for the treatment of CLL or allergic diseases.     

Monoclonal antibody to fibrin D-dimer (DD-3B6) recognizes an epitope on the gamma-chain of fragment D

Laboratory determination of fibrinolysis has been facilitated by diagnostic tests that use monoclonal antibody DD-3B6 to measure fibrin D-dimer levels in plasma. When DD-3B6 is reacted with soluble fibrin fragments, it is specific for fragment D-dimer. The authors have used immunoblot analysis of DD-3B6 binding to purified fragment D and fragment D-dimer to localize the binding site of the antibody. Although DD-3B6 recognizes only fragment D-dimer in solution, it binds to both immobilized fragment D-dimer and fragment D in immunoblots. When immunoblots were performed using protein which was electrophoresed under reducing conditions, DD-3B6 bound to the gamma-chain of fragments D1, D2, and D3. Therefore, the epitope recognized by DD-3B6 resides between amino acids 86 and 302 in the gamma-chain of fragment D. This epitope is masked in soluble non-cross-linked or nondegraded fibrin, but becomes expressed after cross-linked fibrin has been cleaved by plasmin.