The kinetic properties and sensitivities to inhibitors of lactate dehydrogenases (LDH1 and LDH2) from Toxoplasma gondii: comparisons with pLDH from Plasmodium falciparum.

Toxoplasma gondii differentially expresses two forms of lactate dehydrogenase in tachyzoites and bradyzoites, respectively, designated LDH1 and LDH2. Previously it was demonstrated that LDH1 and LDH2 share a unique structural feature with LDH from the malarial parasite Plasmodium falciparum (pLDH), namely, the addition of a five-amino acid insert into the substrate specificity loops. pLDH exhibits a number of kinetic properties that previously were thought to be unique to pLDH. In the present study, kinetic properties of LDH1 and LDH2 were compared with those of pLDH. LDH1 and LDH2 exhibit broader substrate specificity than pLDH. For both LDH1 and LDH2, 3-phenylpyruvate is an excellent substrate. For LDH2, 3-phenylpyruvate is a better substrate even than pyruvate. By comparison, pLDH does not utilize 3-phenylpyruvate. Both LDH1 and LDH2 can utilize the NAD analog 3-acetylpyridine adenine dinucleotide (APAD) efficiently, similar to pLDH. LDH1 and LDH2 are inhibited competitively by a range of compounds that also inhibit pLDH, including gossypol and derivatives, dihydroxynaphthoic acids, and N-substituted oxamic acids. The lack of substrate inhibition observed with pLDH is also observed with LDH2. By comparison, LDH1 differs from LDH2 in exhibiting substrate inhibition in spite of an identical residue (M163) at a cofactor binding site that is thought to be critical for production of substrate inhibition. For gossypol and gossylic iminolactone, but not the other gossypol derivatives tested, the in vitro inhibition of T. gondii LDH activity correlated with specific inhibition of T. gondii tachyzoite growth in fibroblast cultures.     

重组人心肌肌钙蛋白Ⅰ单克隆抗体的制备及鉴定

目的制备重组人心肌肌钙蛋白I（rhcTnI）的单克隆抗体,并对其单克隆抗体的特性进行鉴定。方法利用纯化后的rhcTn I作为免疫原,常规免疫Balb／c小鼠．取其脾细胞与同系骨髓瘤细胞Sp2／0进行常规融合。用甲基纤维素半固体培养基法获得单克隆抗体杂交瘤细胞株,利用ELISA、Western blot等方法对单克隆抗体的特性进行鉴定。结果筛选出3株稳定分泌抗rhcTnI的单克隆抗体杂交瘤细胞株,分别命名为4B3、7D5和3E6。 3株单克隆抗体的免疫球蛋白亚类分别为IgGl（4B3、7D5）和IgG2b（3E6）。3株单克隆抗体与rhcTnI发生特异性结合,而与大肠杆菌菌体蛋白、乳酸脱氢酶（LDH）、肌酸激酶（CK）、肌酸激酶同工酶（CK-MB）无特异性交叉反应,3株单克隆抗体的亲和力常数分别为1．76×10^9L／mol、1．43×10^9L/mol和1．04×10^9L/mol。结论获得了效价高、特异性好的抗rhcTnI的单克隆抗体,为cTnI诊断试剂盒的研制奠定了基础。     

Diagnosis of malaria by detection of plasmodial lactate dehydrogenase with an immunodot enzyme assay

We have previously demonstrated, using polyclonal and monoclonal antibodies, that the lactate dehydrogenase (LDH) of malaria parasites is immunologically distinct from the host enzyme. The polyclonal antibodies, produced against the affinity purified plasmodial LDH (pLDH) in rabbits, showed specificity to LDH of malaria parasites. In the present study, these anti-pLDH polyclonal antibodies were used to develop an immunodiagnostic test (immunodot enzyme assay of plasmodial LDH) based on the detection of parasite LDH in patient blood. The immunodot enzyme assay of plasmodial LDH was evaluated using blood samples from patients with malaria or other infections. Out of 502 microscopically positive malaria blood samples, 497 blood samples showed positive immunodot assays of pLDH while all the 423 microscopically negative cases were found negative by our test. The blood samples from other infections and non-endemic controls were negative by the immunodot enzyme assay of pLDH. This LDH based test was also found negative in blood samples of cured patients 7 days after chloroquine treatment. The test is simple to perform, can be read visually, econimal, highly specific with a sensitivity of ∼99% and is thus suitable for accurate diagnosis of malaria in field conditions.     

DIAGNOSIS OF MALARIA BY DETECTION OF PLASMODIAL LACTATE DEHYDROGENASE WITH AN IMMUNODOT ENZYME ASSAY

We have previously demonstrated, using polyclonal and monoclonal antibodies, that the lactate dehydrogenase (LDH) of malaria parasites is immunologically distinct from the host enzyme. The polyclonal antibodies, produced against the affinity purified plasmodial LDH (pLDH) in rabbits, showed specificity to LDH of malaria parasites. In the present study, these anti-pLDH polyclonal antibodies were used to develop an immunodiagnostic test (immunodot enzyme assay of plasmodial LDH) based on the detection of parasite LDH in patient blood. The immunodot enzyme assay of plasmodial LDH was evaluated using blood samples from patients with malaria or other infections. Out of 502 microscopically positive malaria blood samples, 497 blood samples showed positive immunodot assays of pLDH while all the 423 microscopically negative cases were found negative by our test. The blood samples from other infections and non-endemic controls were negative by the immunodot enzyme assay of pLDH. This LDH based test was also found negative in blood samples of cured patients 7 days after chloroquine treatment. The test is simple to perform, can be read visually, econimal, highly specific with a sensitivity of ～99% and is thus suitable for accurate diagnosis of malaria in field conditions.     

Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus

As Plasmodium rely extensively on homolactic fermentation for energy production, Plasmodium falciparum lactate dehydrogenase (PfLDH)--the key enzyme in this process--has previously been suggested as a novel target for antimalarials. This enzyme has distinctive kinetic and structural properties that distinguish it from its human homologues. In this study, we now describe the expression, kinetic characterisation and crystal structure determination of the LDH from Plasmodium berghei. This enzyme is seen to have a similar kinetic profile to its P. falciparum counterpart, exhibiting the characteristic lack of substrate inhibition that distinguishes plasmodial from human LDHs. The crystal structure of P. berghei lactate dehydrogenase (PbLDH) shows a very similar active site arrangement to the P. falciparum enzyme. In particular, an insertion of five amino acid residues in the active site loop creates an enlarged volume in the substrate binding site, and characteristic changes in the residues lining the NADH cofactor binding pocket result in displacement of the cofactor relative to its observed position in mammalian and all other LDH structures. These results imply the special features previously described for PfLDH may be shared across the Plasmodium genus, supporting the universal application of therapeutics targeting this enzyme.     

广州管圆线虫单抗结合抗原定位分布及在免疫诊断上的应用

目的 研制广州管圆线虫单克隆抗体诊断循环抗原提高诊断的特异性.方法 将广州管圆线虫分泌性抗原免疫小鼠,免疫鼠脾细胞与骨髓瘤细胞融合为杂交瘤细胞,用广州管圆线虫阳性患者血清筛选阳性杂交瘤细胞,培养阳性的杂交瘤细胞分离制备单克隆抗体命名为12D5和21B7,用免疫组织化学的方法分析12D5和21B7单抗结合抗原在广州管圆线虫体内的分布,并用筛选的双12D5和21B7单抗进行抗体夹心ELISA检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病人血清循环抗原(CAg),用其他寄生虫抗原鉴别单抗的特异性,并与抗体检测比较其敏感性和特异性.结果 经鉴定单抗12D5为IgG1,21 B7为IgM,两株单抗同时识别广州管圆线虫成虫相对分子质量为55 × 103的蛋白,两个单抗针对的抗原分布在虫体肠表面,12D5和21 B7双抗体夹心ELISA法对实验感染的广州管圆线虫的大鼠血清中CAg检出率为100%(48/48),广州管圆线虫感染病人血清CAg检出率为100%(32/32),与日本血吸虫、肝吸虫、肺吸虫、旋毛虫、蛔虫、包虫病人血清无交叉反应,与健康人血清无反应;而用抗原检测32个广州管圆线虫感染病人的抗体检出率为75%(24/32),同时抗体检测与其他寄生虫出现一定的交叉反应.结论 12D5和21 B7单抗结合的抗原为肠相关抗原,双抗体夹心ELISA法对感染广州管圆线虫人和动物血清中CAg检测的特异性强,敏感性高,优于抗体检测试剂,并能够确定现症感染.

Abstract：

Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.     

Epitope mapping of the alpha-toxin of Clostridium perfringens.

A panel of monoclonal antibodies specific for the Clostridium perfringens alpha-toxin was produced by the fusion of X63.Ag8-653 cells with splenocytes from mice immunized either intrasplenically or intraperitoneally with an alpha-toxoid. The toxin-binding activity of each monoclonal antibody was evaluated. The monoclonal antibodies were also screened for their toxin-neutralizing potential in vitro, as determined by the inhibition of phospholipase C and hemolytic activities. In vivo inhibition of toxicity was assessed by the survival of mice challenged with preincubated alpha-toxin-antibody mixtures. Only one monoclonal antibody (3A4D10) was protective in vivo and neutralizing in both in vitro assays. Since 3A4D10 could inhibit both activities, the evidence suggests that these are colocated in the same area of the toxin molecule. This paper identifies a significant continuous linear binding region for 3A4D10 at positions 193 to 198 in the primary amino acid sequence of alpha-toxin.     

6株抗rhlL－8单克隆抗体识别表位的鉴定及双单克隆抗体夹心法ELISA检测IL－8方法的建立

采用酶标抗体竞争结合试验对６株抗ＩＬ－８单克隆抗体识别的表位进行了鉴定。按识别表位的不同可将６株单克隆抗体分为两组，一组包括２Ｇ２、４Ｃ３和４Ｅ１，识别相同或相近的表位；另一组包括４Ｄ７、５Ｃ９和５Ａ４，识别相同或相近的表位。在表位鉴定的基础上，选择识别不同表位的单克隆抗体用于建立双单克隆抗体夹心法ＥＬＩＳＡ检测ＩＬ－８，其中以４Ｄ７为包被抗体，４Ｃ３为酶标抗体的组合可获得最高的敏感性，对单核细胞源性ｒｈＩＬ－８（ＭＩＬ－８）及内皮细胞源性ｒｈＩＬ－８（ＥＩＬ－８）检测的敏感性可分别达到１５６ｐｇ／ｍｌ及３１２ｐｇ／ｍｌ。     

A comparison of two dissimilar monoclonal antibodies that are directed to a common epitope in the reduced and carboxymethylated chicken riboflavin carrier protein

Two monoclonal antibodies, D2A1 and D5G3 were elicited by immunization with a preparation of chicken egg riboflavin carrier protein which had been reduced and carboxymethylated. Epitopes recognised by the monoclonal antibodies were mapped using the Pepscan method. Epitopic determinants for D2A1 as well as D5G3 were identified within a region spanning 13 amino acids (residues 170–182) in the primary sequence of riboflavin carrier protein. Interestingly, these monoclonal antibodies, despite sharing a common epitope exhibited a marked difference in their binding to native (folded) riboflavin carrier protein versus reduced carboxymethylated (unfolded) riboflavin carrier protein. Both monoclonal antibodies bound reduced carboxymethylated riboflavin carrier protein to comparable extents in solid phase (ELISA and immunoblots) and liquid phase (radioimmunoassay) assays. However, while D5G3 could bind native riboflavin carrier protein in solid and liquid phase assays, D2A1 showed negligible binding to the native structure. Alanine substituted peptide analogs of the epitope in question defined the crucial amino acids of the epitope needed for binding to the two antibodies.     

A comparison of two dissimilar monoclonal antibodies that are directed to a common epitope in the reduced and carboxymethylated chicken riboflavin carrier protein

Two monoclonal antibodies, D2A1 and D5G3 were elicited by immunization with a preparation of chicken egg riboflavin carrier protein which had been reduced and carboxymethylated. Epitopes recognised by the monoclonal antibodies were mapped using the Pepscan method. Epitopic determinants for D2A1 as well as D5G3 were identified within a region spanning 13 amino acids (residues 170-182) in the primary sequence of riboflavin carrier protein. Interestingly, these monoclonal antibodies, despite sharing a common epitope exhibited a marked difference in their binding to native (folded) riboflavin carrier protein versus reduced carboxymethylated (unfolded) riboflavin carrier protein. Both monoclonal antibodies bound reduced carboxymethylated riboflavin carrier protein to comparable extents in solid phase (ELISA and immunoblots) and liquid phase (radioimmunoassay) assays. However, while D5G3 could bind native riboflavin carrier protein in solid and liquid phase assays, D2A1 showed negligible binding to the native structure. Alanine substituted peptide analogs of the epitope in question defined the crucial amino acids of the epitope needed for binding to the two antibodies.     

Murine monoclonal antibodies to PorA of Neisseria meningitidis show reduced protective activity in vivo against B:15:P1.7,16 subtype variants in an infant rat infection model

The major outer membrane protein PorA of Neisseria meningitidis is the target for bactericidal serosubtyping antibodies and is currently considered as a potential vaccine candidate against group B meningococcal disease. Although the minor antigenic variability of the PorA has been increasingly recognized and described, its implication for vaccine design remains unclear. In this study, the protective activity of murine monoclonal PorA specific antibodies against four isogenic meningococcal P1.7,16 target strains, the prototype P1.7,16a and three loop 4 point mutation variants (designated P1.7,16b to d) constructed from reference strain H44/76 (B:15:P1.7,16a), was evaluated in the infant rat infection model. All monoclonal antibodies had been obtained by immunization of mice with outer membrane protein preparations from meningococcal serosubtype P1.7,16 reference strain H44/76. A challenge dose of 10(5)cfu/pup was given i.p. 1-2 h after the i.p. injection of 1:100 diluted antibodies, and the development of bacteremia was assessed by culturing blood samples taken 6 h after challenge. MN14C11.6, a reference monoclonal antibody for serosubtype P1.7 epitope located in predicted loop 1 (VR1) identical in all the variants, was equally protective against all loop 4 variants. The three P1.16 specific monoclonal antibodies tested (MN5C11G, MN12H2 and 62D12-8) all completely protected animals against the prototype P1.7,16a, variably against the P1.7,16b and P1.7,16c, but not against the P1.7,16d variant. Our findings therefore suggest that certain subtype variants may escape protection in vivo conferred by PorA specific antibodies.     

The immunochemistry of sandwich ELISAs--I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates

Radiolabelled bovine IgG1, IgG2, SIgA and IgM and heavy-chain specific polyclonal and monoclonal antibodies to these isotypes were employed as models to investigate immunochemical aspects of sandwich enzyme immunoassays (ELISAs). The titration plots obtained by measuring enzyme activity paralleled those obtained when the binding of radiolabelled immunoglobulins to solid-phase capture antibodies was quantitated. As predicted from the Mass Law, the percentage of labelled immunoglobulin which was bound remained constant over the range in which the sandwich ELISA titration was linear on a log-log plot. Also as predicted from the Mass Law, increasing the solid-phase concn of polyclonal antibodies by affinity purification increased the linear region of the log-log ELISA plot and the corresponding region over which a constant percentage of immunoglobulin binding was observed. When used as capture antibodies adsorbed on plastic at equal concns, the best monoclonal antibodies were 1/8- less than 1/16 as effective as their polyclonal counterparts in binding iodinated bovine immunoglobulins; these differences can be directly interpreted to result from an 8 and greater than 16-fold higher functional, relative affinity of the polyclonal reagents. Steric hindrance was shown to occur when symmetrical sandwich ELISAs, i.e. capture and detection antibody are both heavy-chain specific, are used to measure monomeric but not IgM immunoglobulins. The use of an asymmetrical configuration, i.e. anti-Fab antibody-enzyme conjugates, avoids this problem. Symmetrical conjugates based on the avidin-biotin system, horseradish peroxidase or alkaline phosphatase, were less effective than their asymmetrical (anti-Fab) counterparts. Evidence that the lower activity of symmetrical conjugates was due to steric hindrance was illustrated using horseradish peroxidase-antibody conjugates of different sizes. Sandwich assays using affinity-purified, polyclonal solid-phase antibodies and an asymmetrical conjugate were judged to be immunochemically and economically optimal. Using an asymmetrical configuration, the non-linear nature of sandwich ELISA titration plots is the predictable result of changing antibody to antigen ratios in an antibody-limiting system, and not the result of steric hindrance of the detection system.     

Cytological evidence for 5-azacytidine-induced demethylation of the heterochromatic regions of human chromosomes

This work was aimed at studying the effects of the demethylating agent 5-azacytidine (5-azaC) on the constitutive heterochromatin of human chromosomes at the cytological level. Metaphase preparations from peripheral blood lymphocyte and lymphoblastoid cultures obtained by standard methods were treated with the agent. Labelling of the heterochromatic regions was achieved by the indirect immunostaining method using anti-5-methylcytosine (5MeC) monoclonal antibodies and peroxidase-tagged second antibodies. 4-Chloro-1-naphthol (4C1N) was used as the substrate. The rate of methylation of individual chromosomes or chromosome groups was measured as the frequency of binding of anti-5MeC antibodies to specific chromosome regions. The following results were obtained: (i) in control cultures high intra- and interindividual variability in the binding frequencies of anti-5MeC antibodies to the short arm region of the acrocentrics was observed; (ii) 5-azaC consistently affects the methylation status of the constitutive heterochromatin; (iii) preferential demethylation occurs in the heterochromatic regions of specific chromosomes; (iv) under our experimental conditions the demethylating effect of 5-azaC appears not to be related to the well-known uncoiling effect of this drug.accepted for publication by M. Schmid     

The use of monoclonal antibodies to define levels of cystatin C in normal human serum

We established four hybridoma cell lines which secreted monoclonal antibodies against human cystatin C. These monoclonal antibodies were of IgA(kappa), IgG2a(lambda), IgG1(kappa), and IgG1(lambda) isotypes, respectively. The association constants (Ka) of the three IgG monoclonal antibodies ranged from 3.6 x 10(9) M-1 to 7.3 x 10(10) M-1. An ELISA technique for the measurement of cystatin C was established by using one of these monoclonal antibodies. The assay procedure was highly specific, sensitive, and reproducible. The lower detectable limit of cystatin C by this procedure was 1.9 ng/ml. The level of cystatin C in normal human serum was 1.16 +/- 0.91 micrograms/ml (mean +/- S.D., n = 274).     

Determination of affinity constants and the number of binding sites of monoclonal antibodies specific for southern bean mosaic virus

Values of the equilibrium dissociation constant and the number of attachment sites for the binding of radiolabeled F(AB) fragments prepared from four monoclonal antibodies to southern bean mosaic virus have been determined. Monoclonal antibodies B6 and B10 produced against the bean type strain of southern bean mosaic virus (SBMV-B) and 1C4 and 1D6 produced against the cowpea type strain (SBMV-C) were more reactive with the untreated virus than with EDTA-swollen particles. B6 and B10 reacted with 180 and 90 sites, respectively, on the SBMV-B particle and were not reactive with SBMV-C. Antibody 1D6 reacted with 180 sites on both SBMV-B and SBMV-C, whereas 1C4 bound to 180 sites on SBMV-C and 60 sites on SBMV-B. The equilibrium dissociation constants for B10, 1C4, and 1D6 were similar but antibody B6 bound with an 8- to 10-fold lower affinity. Solid phase competitive antibody inhibition analysis showed that 1C4 and 1D6 were mutually inhibitory and that these antibodies also inhibited the binding of B6 and B10. Antibody B6 partially inhibited the binding of 1D6 but did not affect 1C4 or B10 binding. No inhibition of B6, 1C4, or 1D6 binding was observed when B10 was the competing antibody. Possible sites of reaction of these monoclonal antibodies on the SBMV particle are discussed.     

Production, binding characteristics, and immunogenicity of heterologous anti-idiotypic antibody to herpes simplex virus glycoprotein C

A monoclonal antibody specific for glycoprotein C (gC) of herpes simplex virus type 1 (HSV-1) was used to prepare a heterologous anti-idiotypic antibody in rabbits. After absorption with normal mouse immunoglobulin (NMS) the anti-idiotypic (anti-id) antibody retained binding activity for MoAb D4.1, the immunogen. The anti-id (anti-id C) also demonstrated a cross-reactive binding activity, as shown by ELISA, for MoAb D4.2 and MoAb D4.8 which was specific for glycoprotein D (gD) and glycoprotein B (gB) of HSV-1, respectively. Also, anti-id C bound to and eluted from MoAb D4.2 and MoAb D4.8 affinity columns retained the ability to bind all three monoclonal antibodies. This cross-reactive anti-id could inhibit the binding of each of the three monoclonal antibodies to their respective proteins, suggesting an antigen combining site specificity. Subsequently, the idiotope on MoAb D4.8 was shown to be outside the antigen combining site, since anti-id C recognized MoAb D4.8 complexed with gB. The anti-id, however, did not bind MoAb D4.1 or MoAb D4.2, if these monoclonals were bound to gC or gD, respectively, suggesting the cross-reactive determinant was paratopic on those two monoclonals. Immunization of mice with anti-id C could prime splenocytes in vivo to proliferate in response to HSV antigen stimulation in vitro. Thus, spleen cells involved in the HSV immune response in vitro recognized the anti-idiotypic antibody in vivo.     

Monoclonal antibodies to cyanogen bromide fragments of glycophorin A

Eleven mouse monoclonal antibodies directed against epitopes on CNBr peptides of the major sialoglycoconjugate of the human red blood cell, glycophorin A, have been produced by hybridomas derived from P3-X63-Ag8.653 myeloma cells and spleen cells from BALB/c mice immunized with purified glycophorin. The monoclonal antibodies could be divided into four groups according to their reactivities with CNBr peptides in a direct ELISA assay: one antibody (6B5) that binds solely to the aminoterminal octapeptide (CNBr3); two antibodies (8F10 and 9C3) that bind to CNBrl (residues 9-81); two antibodies (3D2 and 4C6) that are reactive with CNBr2, The C-terminal portion of the molecule (residues 82-131); six antibodies (1B4, 4C3, 4E7, 7B10, 7C11 and 9D6) which are cross-reactive with an epitope on both CNBr1 and CNBR3 glycopeptides. This cross-reactive epitope(s) appears to involve both carbohydrate and protein residues.     

Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin

The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin.     

A convenient method for epitope competition analysis of two monoclonal antibodies for their antigen binding

Choosing optimum pair of capturing antibody and detecting antibody when developing monoclonal antibody (MAb)-based, sandwich enzyme-linked immunosorbent assays is a time-consuming process requiring the coupling of individual antibodies to an enzyme like horseradish peroxidase or alkaline phosphatase. The MAbs required for the two-site sandwich ELISA should bind to distinct epitopes of the antigen, and their binding should not be mutually exclusive. To determine if two monoclonal antibodies would occupy distinct sites of their antigen in binding, and enzyme-linked immunosorbent assay was devised, which is easy-to-use and does not require any coupling of monoclonal antibodies to enzymes. Microplate wells are coated with rabbit polyclonal antibodies raised against the same antigen of MAbs. After blocking, a limited amount of the antigen is added for incubation with the rabbit antibodies. Mouse monoclonal antibody 1 (Mab 1) is added to saturation. A serial dilution of MAb 2 (for analysis) or MAb 1 (for control) is added subsequently. An enzyme-labeled, goat anti-mouse secondary antibody and its substrates are added for color development. Thus, the epitope competition of two MAbs for their antigen binding is easily determined by the measurement and comparison of color development between the two MAb additions.     

Inadequacy of traditional ELISA for screening hybridoma supernatants for murine monoclonal antibodies

Hybridomas producing anti-creatine kinase (CK) and anti-lactate dehydrogenase (LDH) antibodies were screened by enzyme linked immunosorbant assay (ELISA) with antigen coated onto plastic wells. Out of seven antibodies positive for each isoenzyme of CK, four antibodies failed to bind to the radiolabeled antigen in solution phase radioimmunoassay (RIA) or native antigen in competitive ELISA. Moreover, out of nine antibodies shown reactive with LDH-1 and seven antibodies binding to LDH-5 by ELISA, not a single antibody bound to either radiolabeled or native antigen in solution. Our results along with other recent studies strongly suggest that antigen conformation on solid phase may frequently be different from in solution and that screening by ELISA in which antigen is attached to solid phase is often inappropriate for determining the presence of useful monoclonal antibodies.