Production and characterization of monoclonal antibodies against Naja naja atra cobrotoxin.

Twelve monoclonal antibodies against cobrotoxin from Naja naja atra venom were tested for cross-reactivity with eight different snake toxins, binding to linear epitopes, prevention of cobrotoxin binding to acetylcholine receptor (AchR) in vitro, and protection in mice concomitantly given a lethal dose of cobrotoxin. The antibodies were highly specific, as evidenced by little reactivity with other snake toxins. None of the monoclonal antibodies bound to reduced cobrotoxin or synthesized 8-mer regions spanning the whole molecule, thus suggesting the recognition of conformational epitopes. The in vitro binding of toxin to AchR was competitively inhibited (23-79%) with a 1.66:1 mole ratio of antibody:AchR. Preincubation of monoclonal antibody with toxin before adding AchR (3:1 mole ratio of AchR:antibody) inhibited the in vitro binding of toxin to AchR by 20-80%. Monoclonal antibodies added after the preincubation of toxin with AchR did not dissociate the toxin-AchR complex. An antibody:toxin mole ratio of 2.5:1, with 6 micrograms of cobrotoxin, delayed the time to death of mice 3.7-23.8-fold over control mice. The monoclonal antibodies that most effectively prevented in vitro binding of toxin to AchR also provided the longest delay in time to death in mice.     

Lack of the blocking effect of cobrotoxin from Naja naja atra venom on neuromuscular transmission in isolated nerve muscle preparations from poisonous and non-poisonous snakes

Neuromuscular transmission in the Chinese cobra Naja naja atra was not affected by its own toxin (cobrotoxin) at a concentration of as high as 100 microM, while in the frog a concentration of less than 0.1 microM cobrotoxin depressed the amplitude of the end-plate potential to less than 10% of its original value within 30 min or directly blocked the nerve-evoked muscle action potential. The lack of effect of cobrotoxin was also observed in the nerve-muscle preparations from a pit viper (Agkistrodon blomhoffii brevicaudus) and three species of non-poisonous snakes (Elaphe dione, Elaphe bimaculata and Elaphe rufodorsata). On the other hand, the snake preparations were sensitive to the blocking effect of D-tubocurarine and less sensitive to that of atropine, indicating that the nicotinic acetylcholine receptor is responsible for transmission between the nerve and the skeletal muscle of the snake. We suggest that the snake nicotinic cholinergic receptor lacks the cobrotoxin binding-site.     

诱生型一氧化氮合酶抗体的制备与初步应用

目的制备诱生型一氧化氮合酶 (iNOS)特异性抗体并应用于iNOS检测。方法将iNOSN端片段装入原核表达载体pET 2 8a(+) ,在大肠杆菌BL2 1中表达 ,两步法纯化目的蛋白 ,使用纯化蛋白制备iNOS多克隆抗体。获得的抗血清用于小鼠巨噬细胞及小鼠脑缺血模型中iNOS的检测。结果得到具有较高表达量的融合蛋白 ,经两步纯化获得iNOSN端融合蛋白纯品 ,将此蛋白免疫家兔 ,得到iNOS多克隆抗体 ,Westernblot分析表明该抗体不与nNOS、eNOS交叉反应 ,而能检测组织、细胞中的iNOS。结论原核表达iNOSN端片段制备的iNOS抗体具有很好的反应性及特异性     

PURIFICATION AND PROPERTIES OF NON-PRECIPITATING ANTIBODIES TO COBROTOXIN*

The heterogeneity of precipitating and non-precipitating antibodies to cobrotoxin was demonstrated by their elution pattern on cobrotoxin-Sepharose and chromatography on DEAE-cellulose column. Gel filtration patterns on a Sepharose 6B column revealed that the soluble complexes formed from non-precipitating antibody and HNB-cobrotoxin at a different molar ratio all emerged in the void volume, indicating that the molecular weight of the soluble complex is around 4,000,000 or larger. Unreacted free non-precipitating antibody coincided with the peak of IgG and was proved to be free from HNB-cobrotoxin. The molar ratio of antibody to antigen for the soluble complex was found to be 0.79 to 0.97 indicating that 1.58–1.94 molecules of non-precipitating antibody are bound to HNB-cobrotoxin instead of three molecules as in the case of precipitating antibody.     

No specific reactivity to E. coli glutamic acid decarboxylase from sera of newly-diagnosed insulin dependent diabetic patients

The 65 kD isoform of Glutamic Acid Decarboxylase (GAD), is one of the major autoantigens in human type 1 diabetes mellitus. This enzyme shares amino acid identity, in select regions already determined as antigenic with its counterpart from E. coli. We tested the reactivity of diabetic and normal sera and an E. coli GAD-specific monoclonal antibody (2D9) to E. coli GAD by solid phase and competition ELISA, as well as immunoblotting to check for cross-reactivity of autoantibodies to the two antigens. Specific antibodies for E. coli GAD are present in diabetics and normal subjects without any differences in frequency and titer. The reactivity of such antibodies in ELISA could be blocked in a dose-dependent manner by the addition of excess antigen in the liquid phase. Furthermore, the monoclonal antibody against E. coli GAD does not recognise human recombinant GAD65 in an ELISA. We conclude that there is no basis for cross-reactivity between the two antigens, and antibody reactivity to GAD65 in man cannot arise from cross-reactivity to the E. coli enzyme.     

Enzyme-linked immunosorbent assay, immunofluorescent assay, and recombinant immunoblotting in the serodiagnosis of early Lyme borreliosis

Serum samples from Bulgarian patients with physician-diagnosed erythema migrans (EM) (n=105) were examined using Borrelia burgdorferi ELISA (Boehring, Germany) after previous absorption with Treponema phagedenis. For IgM antibody detection sera were additionally pretreated with anti-IgG serum (RF absorbent). Serum samples of 93% of persons from healthy control group were IgM negative and all were IgG negative. Out of 105 patients with EM, 49% were IgM positive and 14 % were borderline. IgG ELISA showed positive results for 17% and borderline for 6% of the patients. Positive and borderline serum samples were examined further by immunofluorescent assay (IFA) and immunoblot test with recombinant B. burgdorferi proteins from strain PKo (B. afzelii) - p100, flagellin, OspA and OspC, and internal flagellin fragments from strains PKo and PBi (B. garinii) [B.Wilske, V.Fingerle, P. Herzer et al. 1993. Med. Microbiol. Immunol. 182:255]. IFA detected IgM antibodies against B. burgdorferi in 47 % of the positive and in none of the borderline by IgM ELISA serum samples as well as IgG antibodies in 83% of the positive and in 50% of the borderline by IgG ELISA samples. Presence of specific antibodies was confirmed by immunoblot in 71 % of the IgM ELISA postive and in 67 % of the IgG ELISA positive sera. In addition, anti-B. burgdorferi antibodies were detected in 60 % of the borderline by IgM ELISA serum samples. IgM serum reactivity was directed mainly against OspC antigen and flagellin and IgG antibodies were directed mainly against flagellin and p100. These findings clearly showed advantages of the ELISA test based on previous pretreatment of sera and capable to detect specific antibodies in more than half of patients with early Lyme borreliosis despite the well-known delayed immune response. IFA was less sensitive than ELISA in detection of anti-B. burgdorferi antibodies. An additional examination of ELISA borderline sera by immunoblot revealed more positive results. Serum reactivity to a single OspC antigen seems to be a sufficient criterion for positive IgM immunoblot.     

A comparison of the antagonisms by neostigmine and diaminopyridine against the neuromuscular block caused by cobrotoxin and (+)-tubocurarine

Cobrotoxin was about 11-fold more potent than (+)-tubocurarine on a weight basis in blocking neuromuscular transmission in mouse isolated phrenic nerve-diaphragm preparations. Neostigmine and diaminopyridine increased the concentrations of cobrotoxin for 70% inhibition of indirect contraction by 290 and 320%, and increased those of (+)-tubocurarine by 180 and 230%, respectively. More than additive increases were obtained when neostigmine and diaminopyridine were used simultaneously. Cobrotoxin, however, was only 6-fold more toxic than (+)-tubocurarine after intraperitoneal injection in mice. The lethal dose of (+)-tubocurarine was increased by 80% when both antidotes were used together, but only by 15-20% when used alone. In contrast, the lethality of cobrotoxin was not decreased by these drugs. Unexpectedly, the time to death after treatment with cobrotoxin was shortened when mice were pretreated with these antidotes.     

Purification of anticobrotoxin antibody by affinity chromatography

C. C. Yang, M. F. Lin and C. C. Chang. Purification of anticobrotoxin antibody by affinity chromatography. Toxicon15, 51–62, 1977.—Cobrotoxin was immobilized on Sepharose through its free amino groups without altering its antigenic activity. Modification of Arg-residues did not change the coupling capacity of cobrotoxin to Sepharose; however, the antigenic activity markedly decreased when Arg-30 and Arg-36 were modified.Rabbits hyperimmunized with cobrotoxin in Freund's complete adjuvant produce non-precipitating as well as precipitating antibodies. By affinity chromatography of supernatants obtained from precipitin reaction at the maximum precipitation on a column of cobrotoxin-Sepharose, the non-precipitating antibodies were separated from the antisera. Both precipitating and non-precipitating antibodies were similar with regard to their molecular size and elution pattern on cobrotoxin-sepharose column.Specific neutralizing capacities of the non-precipitating antibody and its papain fragment increased 23- and 27·6-fold, respectively, over that of the antisera. These results may lead to a substantial improvement in the therapy of victims of snake bites.     

North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens.

We report the cloning of sphingomyelinase D (SMD) cDNA from Loxosceles reclusa, Loxosceles boneti and Loxosceles laeta into bacterial expression systems, as well as optimization of expression conditions so as to obtain soluble and active recombinant enzymes. The recombinant mature SMDs, tagged with a histidine tail at the N- or C-termini, were compared in terms of toxicity and enzymatic activity, and were used as immunogens for the production of monovalent antisera in rabbits and F(ab')(2) preparations in animals used for commercial antivenom production (horses). We performed studies on in vitro inhibition of enzymatic activity of natural venom preparations by antibodies generated against the tagged proteins. We also present and discuss the results of studies on the specific and para-specific in vivo protective potential of the rabbit and equine antibody preparations against the recombinant proteins themselves and natural venom preparations. Our conclusions support the feasibility of using recombinant SMDs for production and evaluation of polyvalent anti-Loxosceles antivenoms, and we offer data on the potential of paraspecific neutralization in the context of the antigenic groupings and the molecular phylogeny of those active SMDs for which amino acid sequence information is available.     

Marginal involvement of pyroglutamyl aminopeptidase I in metabolism of thyrotropin-releasing hormone in rat brain

On thyrotropin-releasing hormone (TRH) metabolism, pyroglutamyl aminopeptidase II (PAP-II), a zinc-dependent ectoenzyme primarily located in the central nervous system, is believed to play a predominant role. Recently we cloned pyroglutamyl aminopeptidase I (PAP-I) which is known for specifically removing a L-pyroglutamate (L-pGlu) residue from the amino terminus of proteins and peptides including TRH. To investigate possible contribution of PAP-I toward TRH metabolism, we conducted biochemical and immunohistochemical characterization using recombinant rat, mouse and human PAP-Is and an antibody raised against rat PAP-I. The Km values toward TRH by the recombinant PAP-Is were about 0.05 mM, being similar value to the reported value of recombinant PAP-II. The L-pGlu-cleaving activities toward TRH in rat brain homogenate were inhibited by a PAP-II specific inhibitor 1,10-phenanthroline, but not inhibited by the antibody against rat PAP-I. Immunohistochemical study in rats revealed heterogeneous distribution of PAP-I in the pituitary, the target tissue of TRH, but the distribution was cytosolic. Taken together, these results suggested that PAP-I might not be dominantly involved in the degradation of TRH in rats. Additionally, we found that PAP-I was localized in the renal proximal tubules. Further investigations are needed for elucidating the function of PAP-I in these restricted sites.     

Role of amino and carboxyl groups of cobrotoxin in the conformational stability and the interaction with acetylcholine receptor

To study the functional involvements of the common interaction of the Leu-1 α-amino group and Asp-58 in cobrotoxin, the lysine ε-amino groups of cobrotoxin were initially guanidinated with o-methylisourea. The α-amino group of Leu-I was then modified with TNBS after the guanidination of cobrotoxin. Both modified derivatives displayed no significant changes in the secondary structure and antigenicity of cobrotoxin, whereas the binding affinity for nicotinic acetylcholine receptor (nAChR) was pronouncedly decreased when Leu-1 was modified. Six out of seven free carboxyl groups and the remaining buried Glu-21 carboxyl group of cobrotoxin were modified with glycine methyl ester in the absence and presence of guanidine HCl, respectively. Alternation in the β-sheet secondary structure of cobrotoxin was observed with the carboxyl-group modified derivatives, which caused a decrease in the binding activity of the toxin molecule to the antibody and nAChR. Moreover, modification of the Glu-21 carboxyl group of cobrotoxin further reduced the nAChR binding activity, while the antigenicity remained unchange. Thus, our results conclude that the Glu-21 residue and the common interaction of the terminal Leu-1 α-amino group and the Asp-58 carboxyl group are related to the nAChR-binding activity of cobrotoxin, and the free carboxyl groups in cobrotoxin are conformation-essential. © Munksgaard 1995.     

Glutaraldehyde cross‐linking alters the environment around Trp29 of cobrotoxin and the pathway for regaining its fine structure during refolding

Abstract: Cobrotoxin, purified from the venom of Naja naja atra (Taiwan cobra), was subjected to modification with glutaraldehyde in order to prepare intra‐ and intermolecule cross‐linked derivatives. Monomeric and dimeric derivatives were separated from polymeric derivatives by gel filtration. The results of amino acid analysis and sequence determination revealed that only Lys residues were selectively modified by glutaraldehyde. Glutaraldehyde cross‐linking was accompanied by a change in the gross conformation of cobrotoxin as revealed by circular dichroism spectra of the modified derivatives. Compared with cobrotoxin, Trp²⁹ of monomeric and dimeric derivatives was in an apolar microenvironment. This was in agreement with acrylamide quenching studies showing that the spatial position of the Trp indole ring became buried in the interior of the molecule after glutaraldehyde cross‐linking. Moreover, the Trp of modified derivatives was less accessible for iodide than that observed with cobrotoxin. Notably, disulfide reduction could not completely unfold the structure of glutaraldehyde‐modified derivatives as evidenced by the results of acrylamide quenching studies and enzyme‐linked immunoassay. Study of the characteristic changes in Trp fluorescence after the initiation of refolding suggested that the fine structure around Trp²⁹ of cobrotoxin and glutaraldehyde‐modified derivatives was formed differently. These results suggest that glutaraldehyde cross‐linking leads to a change in the microenvironment of cobrotoxin Trp²⁹ and alters the pathway of its fine structure formation during the refolding of cobrotoxin.     

以CpG为佐剂的人IL-24多克隆抗体的制备

目的：采用在原核表达系统表达的人IL-24蛋白及人IL-24真核重组质粒免疫新西兰兔，制备兔抗人IL-24多克隆抗体。方法：利用IPTG诱导hIL-24在大肠杆菌中表达，纯化hIL-24重组蛋白，纯化后的蛋白经SDS—PAGE分析，同时提取hIL-24真核重组质粒，用以免疫新西兰兔，并以CpG为佐剂制备多克隆抗体。Western-blot鉴定抗体的特异性，ELISA法测定抗体效价。结果：人IL-24经IPTG诱导后可在大肠杆菌中大量表达，表达量占细菌总蛋白的30％，纯化后的蛋白纯度高；原核表达的重组蛋白和真核重组质粒免疫新西兰兔，通过抗体效价测定，获得了抗血清效价达1：640的多克隆抗体。结论：原核表达的hIL-24蛋白和hIL-24真核重组质粒均能刺激家兔产生抗体．其多克隆抗体的效价较高。     

An enzyme immunoassay to determine the levels of specific antibodies toward bacterial surface antigens in human immunoglobulin preparations and blood serum

Human polyvalent intravenous immunoglobulin (IVIG) preparations are used as a complementary aid to the proper antimicrobial treatment of severely septic patients in intensive care units (ICUs) and/or as a prophylactic agent to immunocompromized hosts, particularly prone to bacterial infections. There is skepticism about the usefulness of IVIGs since it is not known whether their administration ensures the enhancement of humoral immune responses by providing a sufficient amount of specific antibodies towards the specified bacterial pathogen to be treated. In this report, a simple and reproducible enzyme-linked immunosorbent assay for determining the content of specific antibodies against bacterial surface antigens in commercially available IVIG preparations is described. The method is also easily applied to determine the amount of bacterial antibodies in blood serum. The levels of specific antibodies toward Gram positive and negative pathogenic isolates often encountered in ICUs were estimated in two IVIG (Sandoglobulin® and Gamimmune®) preparations. Significant differences regarding the content of antibodies to certain clinically bacterial isolates were identified not only between the two IVIG preparations tested, but also among various lots from each IVIG preparation. No significant variation (P≤0.001) among the bottles derived from the same lot was determined in both preparations. The variation in the levels of specific antibodies in IVIG preparations may be attributed to differences between the donor pools as well as the manufacturing procedure. Application of the method to patients with primary immune deficiencies showed that infusion of highly reactive IVIG preparations enhanced significantly their humoral response toward various pathogens. The results of this study suggest that the content determination of pathogen-specific antibodies in IVIG preparations before administration may be of great importance for treating bacterial infections.     

Prevalence and Isotypic Complexity of the Anti-Chinese Hamster Ovary Host Cell Protein Antibodies in Normal Human Serum

Host cell-derived protein impurities may be present at low levels in biopharmaceutical products. Antibodies to host cell proteins are present in individuals with no known exposure to these products. In this study, antibodies to drug process-specific Chinese hamster ovary host cell-derived proteins (CHO-HCP) were measured in unexposed individuals using a validated enzyme-linked immunosorbent assay. Samples that tested positive for anti-CHO-HCP reactivity were further characterized to determine the isotypes and IgG subclasses expressed in each positive individual. The specificity of the detected anti-CHO-HCP antibody isotypes was confirmed by the competitive inhibition assay and the uncoated plate specificity testing. These antibody characterization experiments revealed that the prevalent anti-CHO-HCP antibody subclasses were predominantly IgG1 (present in 66.6% of individuals) and IgG2 (60%), with 33.3% positive for IgG3 while IgG4 was detected in low abundance. Forty percent (40%) of the individuals with IgG type reactivity were also identified as IgM-positive. Anti-CHO-HCP-specific IgE was not detected in the assays used in this study. Some individuals exhibited single isotype anti-CHO-HCP reactivity; others contained a combination of multiple antibody isotypes. Data presented in this study demonstrate the isotypic complexity and the high prevalence of anti-CHO-HCP antibodies in human serum with no known exposure to CHO cell-derived therapeutic biologics.     

The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics

Erwinia carotovora L-asparaginase was conjugated via the epsilon-amino groups of its lysine residues with colominic acid (CA) (polysialic acid) of average molecular mass of 10 kDa by reductive amination in the presence of NaCNBH3. Polysialylation using 50-, 100- and 250-fold molar excess CA relative to the enzyme led to an increasing proportion of the enzyme's in-amino groups (5.8, 7.6 and 11.3%, respectively) being conjugated to CA. Polysialylated and native (intact) asparaginase were used to immunize mice intravenously. Results (total IgG immune responses) indicate that all preparations elicited antibody production against the enzyme moiety but not against the CA of the conjugates. Moreover, antibody titres appeared highest for the native enzyme and were generally reduced as the degree of polysialylation increased. In other experiments mice pre-immunized with native or polysialylated asparaginase, with anti-asparaginase antibodies in their blood, were injected intravenously with the corresponding enzyme preparations. Results revealed that polysialylation reduces the antigenicity of asparaginase thus leading to circulatory half-lives (t 1/2 beta) that were 3-4-fold greater than that of the native enzyme, and similar to those observed in naive, non-immunized mice. Our data suggest that polysialylation of therapeutic enzymes and other proteins may be useful in maintaining their pharmacokinetics in individuals with antibodies to the therapeutic proteins as a result of chronic treatment.     

A non-radioactive receptor assay for snake venom postsynaptic neurotoxins

A non-radioactive assay was developed for detecting the binding of postsynaptic neurotoxins to acetylcholine receptor (AchR) from Torpedo californica. Enzyme linked immunosorbent assay (ELISA) wells coated with long or short chain neurotoxins specifically bound to purified AchR while crotamine or two different cardiotoxins did not. Bound receptor was detected by antibody against AchR. Specificity was determined by dose-response experiments and competition studies using carbamylcholine chloride, acetylcholine chloride, or Naja naja atra cobrotoxin mixed with receptor.     

Recombinant polyclonal antibodies: therapeutic antibody technologies come full circle

Current antibody therapeutics can be grouped into two generations, each distinguished by a unique feature of the immune system: diversity and specificity. Antibodies from human blood (immunoglobulin) represent the first generation, and are characterized by the natural diversity of human antibody responses. The second generation consists of recombinant monoclonal antibodies (mAbs), which are characterized by high specificity toward a single, often well-described antigen. The natural immune response comprises a plurality of specificities, many of which do not compete for binding, whereas molecules in a mAb all compete for binding to the same epitope. Thus, the epitope is more likely to become a limiting factor for mAb binding to complex targets compared with a polyclonal antibody. Also, epitope-escape by mutation or natural variation is less likely to be a problem for polyclonal antibodies. Technologies attempting to develop truly human recombinant antigen-specific polyclonal antibodies, such as the Sympress technology, are closing a natural circle between the first generations of antibody technologies.     

Affinity and potency of proinhibitory antipeptide antibodies against CYP2D6 is enhanced using cyclic peptides as immunogens.

A series of antipeptide antibodies directed against CYP2D6 were produced by immunizing rabbits with peptides that were sterically unrestrained (linear) or conformationally restricted by cyclization. A variety of sites within the region comprising residues 254 to 290 of CYP2D6 were targeted. In immunoblotting studies, each of the antibodies against the linear and cyclic peptides recognized only a single immunoreactive band of 54 kDa in human liver microsomal fraction and bound to recombinant CYP2D6, but not recombinant CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2E1, or CYP3A4. However, the relative intensity of immunoreactive bands was considerably stronger for those antibodies raised against cyclic peptides. Similarly, in an enzyme-linked immunosorbent assay, antibodies raised against cyclic peptides bound 10 to 100 times more strongly to recombinant CYP2D6 than antibodies raised against the corresponding linear peptides. None of the antibodies raised against linear peptides had any effect on debrisoquine 4-hydroxylase activity of human hepatic microsomal fraction; however, anticyclic peptide antibodies targeted against residues 254 to 273, 261 to 272, and 257 to 268 of CYP2D6 inhibited enzyme activity by a maximum of 60, 75, and 91%, respectively. In contrast, despite binding strongly to CYP2D6, an anticyclic peptide antibody directed against residues 278 to 290 did not inhibit enzyme activity. The epitope of the proinhibitory anticyclic peptide antibody directed against residues 257 to 268 of CYP2D6 included Thr-261 and Trp-262, and indicates a role for these residues in enzyme inhibition. In conclusion, immunization with peptides conformationally restricted by cyclization to mimic loop regions of CYP2D6 resulted in strongly binding antibodies that when targeted appropriately were able to inhibit CYP2D6-catalyzed activity.