Enzyme immunoassay of immune complexes formed in vitro via interactions of serum antibodies with diphtheria toxin

The interaction of diphtheria toxin with serum antitoxin antibodies has been studied by enzyme immunoassay at variable ratios of the original amounts of the antigen and antibodies in the reaction mixture. Under the conditions of excess of the antibodies, the free toxin was not detected, and free antibodies accounted for 68 to 98% of the original amount of the antibodies. Under the conditions of excess of the toxin, free antibodies account for 2 to 7% of the original amount and free toxin, for 80–100% of its original level. Under the conditions where the toxin is taken in excess, and the amounts of the toxin and the antibodies are equivalent, formed immune complexes are regularly detected in the reaction mixtures. In these complexes, part of the epitopes of the toxin remains free from antibodies. The data obtained are interpreted from the viewpoint of epitope heterogeneity, bivalence of serum antibodies, and monovalence of the toxin epitopes. A new model of the toxin-antibody interaction is proposed.Translated from Prikladnaya Biokhimiya i Mikrobiologiya, Vol. 41, No. 2, 2005, pp. 235–242.Original Russian Text Copyright © 2005 by Titova, Sviridov.     

Application of magnetite nanoparticles for the development of highly sensitive immunochromatographic test systems for mycotoxin detection

Highly sensitive immunochromatographic test systems were developed for the detection of zearalenone (ZEA) and T-2 toxin (T2T) using magnetite nanoparticles (MNPs) for the labeling. In order to detect an analyte with high sensitivity, the competitive reaction was performed with free specific antibodies, while immune complexes were detected by the reaction with label-conjugated anti-species antibodies. The conditions for the synthesis of magnetite nanoparticles conjugated to anti-species antibodies were optimized. The concentrations of specific reagents that provided the highly sensitive detection of T-2 toxin and zearalenone were found. The instrumental detection limit for the determination of T-2 toxin and zearalenone in baby food samples (oat flakes) was 0.1 and 0.05 ng/mL (2.0 and 1.0 ng/g), respectively. The assay time was 15 min. The results of the present study confirm the possibility of the practical use of magnetite nanoparticles for immunochromatographic assay labeling.     

Submicrodeterminations of thiols, disulphides and thiol esters in serum by using o-hydroxymercuribenzoic acid and dithiofluorescein

1. Methods are described for selective estimation of thiols, disulphides and thiol esters in standard solutions and in serum. The methods are based on the reaction with the excess of o-hydroxymercuribenzoic acid (HMB) in alkaline solution with subsequent addition of dithiofluorescein in excess and determination of the extinction at 588mmu. The sensitivity of the methods amounts to 1.5x10(-9)g.equiv. in 5ml. of final solution. Of results obtained on standard solutions 80% have the errors within the range +/-4%. 2. It has been found that serum contains an unidentified substance (substance X) producing green complexes with dithiofluorescein which undergo decomposition on addition of formaldehyde. The correction for substance X must be estimated in a separate sample and taken into account. The concentration of substance X can be calculated from extinctions measured at 588mmu and 635mmu in the presence of dithiofluorescein in excess. 3. The selective determination of thiols and disulphides is based on different reaction rates with formaldehyde. The complexes between HMB and cysteine can be selectively decomposed by formaldehyde, and free glutathione can be selectively removed by formaldehyde in the presence of protein thiols. 4. Thiols are determined in the presence of triethylamine, thiols plus disulphides in the presence of triethylamine and sulphite, and thiols plus thiol esters in the presence of dimethylamine, with subsequent addition of ammonium sulphate.     

Interaction of antibodies specific towards the 2,4-dinitrophenyl group and of their fragments with hapten

The interaction of hapten (ε-DNP lys) with native and S-sulfonated antibodies specific towards the 2,4-dinitrophenyl group, as well as the interaction with isolated chains and a complex obtained by mixing light, (L) and heavy (H) chains of these antibodies, were followed both by polarography and by equilibrium dialysis. With the S-sulfonated antibodies and with the mixture of H and L chains the binding heterogeneity observed in the original antibodies was much lowered or entirely removed. At the same time, the amount of active proteins in the sample decreased approximately by half. The association constants of modified antibodies were of the same order as the average association constants of the original antibodies. A slow increase of the amounts of hapten bound with proteins was observed on mixing the H and L chains and adding hapten. This slow reactivation was not obtained with the original or S-sulfonated antibodies and with isolated chains. It was shown that the reaction determining the kinetics of this reactivation (the slowest reaction) was not the association of H and L chains but the interaction of complexes of the H and L chains with hapten. It was reported previously that H chains were nonspecifically reactivated by binding L chains. The amount of hapten bound by the complex of H and L chains increased with increasing excess of L chains following a curve resembling the Langmuir isotherm. The limiting value of the amount of hapten bound when using antibody L chains was higher than in the case of nonspecific L chains.     

Studies on the influence of Trasylol on the partition of trypsin between the human plasma protease inhibitors in vitro.

The distribution of trypsin between the protease inhibitors of human serum with and without Trasylol was studied in vitro. 1) Trypsin was preferentially bound by alpha2-macroglobulin on addition of small amounts of the enzyme to normal serum in both the presence and absence of Trasylol in a molar concentration equal to that of alpha2-macroglobulin. 2) On saturation of alpha2-macroglobulin, a considerable amount of trypsin was bound by Trasylol even when most of the serum alpha1-antitrypsin was in a free form. 3) In reaction mixtures containing small amounts of trypsin, Trasylol was identified in a free form as well as in complex with trypsin-alpha2-macroglobulin complex and to a limited extent with trypsin. 4) With larger amounts of trypsin, sufficient to saturate alpha2-macroglobulin, increasing amounts of Trasylol were bound to trypsin. The relative amount of Trasylol bound to trypsin-alpha2-macroglobulin complexes was now smaller. This was explained by a higher affinity (or binding rate) of Trasylol for trypsin than for trypsin-alpha2-macroglobulin complexes. 5) Trypsin-Trasylol complexes showed no signs of dissociation after 5 h incubation at 37 degrees C in serum.     

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: determination of affinities and specificities of monoclonal antibodies by using biotin-labeled antibodies and avidin as precipitating agent in a solution phase immunoassay

A general method is described for the determination of affinity constants and antigen cross-reactivities of monoclonal antibodies. The method employs biotin-labeled antibody, radiolabeled antigen, and avidin as a precipitating agent in a homogeneous phase, competitive radioimmunoassay. This method eliminates incomplete or variable precipitation of antigen-antibody complexes often encountered in immunoassays in which monoclonal antibodies are employed. Using this assay system, we were able to rapidly determine the affinity constants for a number of monoclonal antibodies elicited to carcinoembryonic antigen (CEA). In the preceding paper it was shown that five of the monoclonal antibodies recognized distinct epitopes on CEA. In antigen-binding experiments with these five monoclonal antibodies, the percent of radiolabeled CEA bound in antibody excess ranged from 30 to 92%. The CEA cross-reacting antigens, normal cross-reacting antigen (NCA), and tumor-extracted, CEA-related antigen (TEX) were significantly bound by one, and to a lesser degree, by two of the five antibodies. Two antibodies did not bind significant amounts of NCA or TEX. In inhibition studies, the amount of unlabeled CEA leading to 50% inhibition of 125I-labeled CEA-binding was in the range of 3.7 to 760 ng per tube. The amount of TEX showing the same degree of inhibition was 23-fold greater than the amount of CEA for two antibodies and 351-fold greater than the amount of CEA for a third antibody. The affinity constants for CEA were in the range of 1.0 x 10(8) to 5.1 x 10(10) M-1. The affinity constants for NCA and TEX, determined for one of the antibodies, were three orders of magnitude lower in comparison to CEA. The heterogeneity of radiolabeled CEA as indicated by the low fraction bound by one of the monoclonal antibodies is shown to be most probably an artifact resulting from radioiodination damage. The application of the approach described in this report should eliminate the problems most commonly encountered in the determination of affinity constants for monoclonal antibodies or the use of monoclonal antibodies in competitive, homogeneous-phase immunoassays.     

Kinetic study of the HIV-1 DNA 3'-end processing

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min(-1)), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.     

Microchip fluorescence-enhanced immunoaasay for simultaneous quantification of multiple tumor markers

A microchip fluorescence-enhanced immunoassay method was developed for simultaneous detection of carcinoma antigen 125 (CA125) and carbohydrate antigen 15-3 (CA15-3). In this method, CA125 and CA15-3 react with excess amount of fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (Ab(*)) of CA125 and CA15-3 to form CA125-Ab(125)(*) and CA15-3-Ab(15-3)(*) complexes. Microchip electrophoresis (MCE) separation of free Ab(125)(*), Ab(15-3)(*), and CA125-Ab(125)(*), CA15-3-Ab(15-3)(*) complexes were then performed. The separated species were sensitively detected by laser-induced fluorescence detection (LIF). CA125 and CA15-3 were quantified simultaneously by measuring the fluorescence intensity of CA125-Ab(125)(*) and CA15-3-Ab(15-3)(*) complexes, respectively. Under the optimum conditions, the limits of detection were 0.23 U/mL for CA125 and 0.09 U/mL for CA15-3. The present MCE-LIF method was applied to the determination of CA125 and CA15-3 in serum from healthy subjects and cancer patients. The levels of CA125 and CA15-3 in these sera samples were found to be in the ranges of 15.6-36.1 U/mL and 13.8-28.4 U/mL for healthy subjects, and 192.5-368.3 U/mL and 63.3-198.4 U/mL for cancer patients.     

Two desired epitopes of cTnI benefit for preparation of standardized monoclonal antibodies

Acute myocardial infarction (AMI) is one of the most severe cardiovascular diseases in humans, often resulting in unexpected death. Early detection is critical for patient survival. Sandwich ELISA is a common method for the detection of AMI. However, ELISA kits from different manufacturers can give different results, in part because of the lack of standardized epitopes. Therefore, the purpose of this study was to find two standardized epitopes. We predicted two antigen epitopes and respectively immunize mice to manufacture standardized monoclonal antibodies. Eight monoclonal antibodies were prepared. Monoclonal antibodies 7D2 and 2C3 were selected with high affinity, and their characteristics were explored. The results show that monoclonal antibodies 7D2 and 2C3 can both bind to various modified forms and complexes of cardiac troponin I (cTnI), were not cross‐reaction with related antigens of normal human serum and can be paired. Therefore, we deem epitopes 30 to 42 and 77 to 89 standardized epitopes.     

Epitopes of human interferon-alpha defined by the reaction of monoclonal antibodies with alpha interferons and interferon analogues

Five monoclonal antibodies reactive with human interferon (HuIFN)-alpha 2, but not with HuIFN-alpha 1, have been analyzed for their reaction with a series of IFN analogues and hybrid IFN molecules. Using analogues containing alpha 1 or gamma substitutions, it was shown that amino acids in the 107 to 113 region are implicated in the epitopes recognized by four of the five antibodies tested. Surprisingly, two of the antibodies that did not react with [alpha 1(113 to 114)112 to 113]alpha 2 also did not react with a truncated IFN-alpha 2(4 to 155). The presence of an epitope determined by amino acids at 112 and 113 and by the amino and carboxyl ends of the molecule supports a model for IFN where the carboxyl- and amino-terminals are adjacent to the proposed reverse turn around amino acid 110 to 115. The fifth alpha 2 specific antibody whose reaction with HuIFN-alpha 2 is not affected by the above substitutions and truncations recognizes IFN or IFN hybrids that, like alpha 2, have arginine at position 120, but does not react with IFN that, like alpha 1, have lysine at position 120. Amino acids 107 to 113 and 120 lie in regions of the molecule that have high hydrophilicity and are probably structurally involved in the epitopes recognized by the antibodies. Under conditions of antibody excess, the antibodies described here inhibit binding of HuIFN-alpha 2 to both human and bovine cells.     

Failure of the usual anti-Ig antibody method for quantitative measurement of low affinity antibodies

The usual anti-Ig antibody method, consisting of the precipitation of soluble antigen-antibody complexes by heterologous anti-Ig antibody, was applied for quantitative estimation of guinea pig IgG2 anti-ovalbumin and anti-2,4-dinitrophenyl (DNP) antibodies by measuring the maximum amounts of antibody-bound antigens. However, the amounts of antibodies estimated were less than those obtained by other methods: the precipitin reaction, the precipitation of antigen-antibody complexes with 50% saturated ammonium sulfate, and equilibrium dialysis. In particular, the anti-Ig antibody method greatly underestimated the amount of anti-DNP antibody with low affinity for epsilon-DNP-L-lysine. Thus, it was concluded that partial dissociation of the antigen-antibody complexes occurring upon precipitation with anti-Ig antibody made the anti-Ig antibody method unsuitable for quantitative determination of antibodies.     

The recognition domain for the low density lipoprotein cellular receptor is expressed once on each lipoprotein particle

The stoichiometry of binding of monoclonal antibodies and Fab fragments to LDL was assessed. Increasing amounts of two [125I]-labelled antibodies which define epitopes at or near the LDL-receptor recognition domains of apoB were incubated with fixed amounts of LDL and antibody-LDL complexes were separated from free antibodies by heparin-MnCl2 precipitation. Saturation kinetics were obtained and data were analyzed according to Scatchard. One antibody or Fab fragment was bound per LDL particle. Homogeneity of binding was indicated by straight Scatchard lines and by the binding of virtually all LDL particles by an antibody affinity chromatographic column.     

Relationship between poliovirus neutralization and aggregation.

The interaction of mono- and polyclonal neutralizing antibodies with poliovirus was studied. In all cases, neutralization was due to antibody-mediated virus aggregation, and the unpolymerized virions accounted for the residual infectivity. The effect of papain on previously neutralized virus was to deaggregate the virus to fully infective single virions. With some antibodies, the amount of aggregated virus regressed in the region of greatest antibody excess, even though the virus remained fully neutralized. Under these conditions, noninfective, unaggregated immune complexes were formed. A mutant resistant to one of the monoclonal antibodies was selected. The mutant virions were still bound but no longer aggregated or neutralized by the selecting antibodies.     

An immunoradiometric assay for 1,25-dihydroxyvitamin D3 receptor

A ligand-independent, quantitative assay has been developed for the measurement of 1,25-dihydroxyvitamin D receptor utilizing purified receptor from pig intestine as a standard and two high affinity monoclonal antibodies directed to two different epitopes on the receptor. In this assay a fixed amount of 125I-labeled antibody is incubated with a fixed amount of a second antireceptor antibody linked to biotin and increasing amounts of purified receptor protein or sample. Antibody-receptor complexes can then be immunoprecipitated with avidin-Sepharose beads and counted. This method is highly reproducible and can detect 150 pg of 1,25-dihydroxyvitamin D3 receptor in crude extracts with intra- and interassay coefficients of variation of 8.6 and 18.2%. The monoclonal antibodies used recognize both native and denatured receptors from several different species, including human. This immunoradiometric assay should prove useful for studies of receptor regulation, occupancy, distribution, and turnover.     

The role of free amino groups of peptides and proteins in the Folin-Lowry and biuret methods

On an equal weight basis polymyxin B and EM 49 which do not contain tyrosine or tryptophan yielded the same colour intensity as proteins in the Folin-Lowry and biuret methods. But, in the absence of reagent C (alkaline copper reagent) polymyxin B and EM 49 yielded no colour in the Folin-Lowry method. Mono-, di- and tri-formyl polymyxins B formed identical amounts of coloured complexes as polymyxin B in the two methods. However, the tetra- and penta-formyl polymyxins B yielded only one-fifth and one-sixth, respectively, of the expected colour in the Folin-Lowry method. Similarly, 40% and 30%, respectively, of the anticipated amount of colour is formed in the biuret method. Formylated and methylated lysozyme and bovine serum albumins form only 70–75% of the expected colour in the Folin-Lowry method. Since formation of colour by reduction of Folin reagent, in the Folin-Lowry method, is at least partly due to complexes of copper, it was inferred that polymyxin B as well as its mono-, di- and tri-formyl derivatives on the one hand and the tetra- and penta-formyl derivatives on the other differ in their ability to complex Cu(II) The former group of compounds was indeed found to complex as many as three Cu(II) ions whereas the tetra- and penta-formyl polymyxins B complexed only one equivalent, under conditions of excess Cu(II). Under conditions of low Cu(II), polymyxin B and all its derivatives complexed only one Cu(II). In proteins, sites other than amino groups which complex Cu(II) probably play a major role in the reduction of the Folin reagent, since methylated lysozyme and bovine serum albumin yield 70–75% of the colour formed by the unmodified proteins in the Folin-Lowry reaction.     

Polypeptides belonging to each of the three major chlorophyll a + b protein complexes are present in a chlorophyll-b-less barley mutant

Polypeptides of the three major chlorophyll a + b protein complexes were detected in a chlorophyll-b-less barley mutant (chlorina f2) using immunological techniques. Antibodies to CP Ia, a photosystem I complex containing both the reaction center (CP I) and the chlorophyll a + b antenna (LHCI), detected substantial amounts of LHCI polypeptides in mutant thylakoids. Some polypeptides of the two photosystem-II-associated chlorophyll a + b complexes, CP 29 and LHCII, were also detected using antibodies raised against these complexes. The CP 29 apoprotein and the minor 25-kDa polypeptide of LHCII were present in amounts that could be seen by Coomassie blue staining. In contrast, the two major polypeptides of LHCII were greatly diminished in amount, and one of them may be completely absent. These data suggest that the absence of chlorophyll b may have differing effects on the synthesis, processing or turnover of the various chlorophyll a + b binding polypeptides. They also show that these polypeptides can be inserted into thylakoids in the absence of Chl b, and that significant amounts of some of them are accumulated in the mutant thylakoids.     

Antibody-mediated cell cytotoxicity in a defined system: regulation by antigen, antibody, and complement.

The use of a serum-free environment and target cells carrying defined amounts of radiolabeled antigen allowed a quantitative study of the role of antigen, antibody, and complement on antibody-mediated cell cytotoxicity (AbMC). For lysis to occure, a minimum number of antigen molecules must be present on the target cell. 51Cr release from target cells with lower antigen density requires larger concentration of effector cells and antibodies. Target cell-bound complement, itself unable to mediate cytotoxicity, reduces the number of IgG molecules required for lysis. The antibody and complement, however, have to be bound to the same target cell. Bystander complement-coated erythrocytes, present in the same reaction mixture with IgG-coated targets, are not lysed. Blocking of AbMC is effected only by antigen, either soluble or in immune complexes prepared in antigen excess. Antigen competes at the level of the target cell. Blocking at the level of the effector cell, by use of immune complexes prepared at equivalence or in antibody excess, is difficult to achieve. The large number of cells with Fc receptors contained in mouse spleens may explain this finding. Arming of effector cells by passive binding of immune complexes is poorly effective as a means of obtaining lysis of the target cells. In all situations, the outcome of the reaction is determined by the presence of free antibody-combining sites, alone, or in immune complexes, that are able to combine with the target cell membrane antigen. The requirements for lysis are rather stringent.     

Comparison of solution structures of dihydrofolate reductases and enzyme-ligand complexes using cross-reacting antibodies

Polyclonal antibodies against dihydrofolate reductase (DHFR) from the human lymphoblastoid cell line WIL-2/M4 were used as probes to compare the antigenic structures in solution of native DHFRs obtained from a broad range of species and their complexes with substrate, cofactor, and folate antagonist inhibitors. All these antibodies could bind to the denatured human DHFR, indicating that they were specific for the primary structure of this enzyme. Denatured chicken liver and L1210 murine leukemic DHFRs competed for all of the antibodies that bound to the human enzyme, although less effectively than the denatured human enzyme, showing the presence of similar epitopes among the vertebrate enzymes. However, both direct binding and competition experiments showed low antibody cross-reactivities with native chicken liver (8%) and murine (10%) DHFRs, suggesting differences in the disposition of similar epitopes in these enzymes. The lactobacillus casei DHFR showed a low amount (less than 2%) of cross-reactivity with the antibodies while the same antibodies did not cross-react with the Escherichia coli enzyme. DHFR from soybean seedlings competed for a large proportion (70%) of the anti-human DHFR antibodies, indicating a close similarity in the antigenic structures of plant and animal DHFRs. Binary complexes of the L. casei, avian, murine, and human DHFRs with dihydrofolate, methotrexate (MTX), trimethoprim (TMP), NADPH, and NADP+ all showed significantly lower antibody binding capacity as compared with the corresponding free enzymes. Further, these ligands inhibited antibody binding to the enzyme to varying degrees.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solid-Phase Radioimmunoassays for Quantitative Antibody Determination of Clostridium perfringens Type D ε Toxin

Two reversed solid-phase radioimmunoassays were developed for quantitative determination of antibodies against Clostridium perfringens type D epsilon toxin. 125I-labeled prototoxin was used in the bromoacetylcellulose-bound antibody method and in the antibody-coated tube method. The antibody values which can be detected by the assays are in the range of 0.004 IU/ml of investigated serum. The methods allow the screening investigation of large groups of vaccinated sheep in a rapid and inexpensive way, and are very suitable for measuring small amounts of C. perfringens D epsilon antibodies with a small experimental error.     

Immune complexes with cationic antibodies deposit in glomeruli more effectively than cationic antibodies alone

In previously published studies, highly cationized antibodies alone and in immune complexes bound to glomeruli by charge-charge interaction, but only immune complexes persisted in glomeruli. Because normal IgG does not deposit in glomeruli, studies were conducted to determine whether cationized antibodies can be prepared which deposit in glomeruli when bound to antigen but not when free in circulation. A series of cationized rabbit antiHSA was prepared with the number of added amino groups ranging from 13.3 to 60.2 per antibody molecule. Antibodies alone or in preformed soluble immune complexes, prepared at fivefold or 50-fold antigen excess, were administered to mice. With the injection of a fixed dose of 100 micrograms per mouse, antibodies alone did not deposit in glomeruli with less than 29.6 added amino groups by immunofluorescence microscopy. In contrast, 100 micrograms of antibodies with 23.5 added amino groups in immune complexes, made at fivefold antigen excess, formed immune deposits in glomeruli. With selected preparations of cationized, radiolabeled antibodies, deposition in glomeruli was quantified by isolation of mouse glomeruli. These quantitative data were in good agreement with the results of immunofluorescence microscopy. Immune complexes made at 50-fold antigen excess, containing only small-latticed immune complexes with no more than two antibody molecules per complex, deposited in glomeruli similar to antibodies alone. Selected cationized antibodies alone or in immune complexes were administered to mice in varying doses. In these experiments, glomerular deposition of immune complexes, made at fivefold antigen excess, was detected with five- to 10-fold smaller doses than the deposition of the same antibodies alone. These studies demonstrate that antibody molecules in immune complexes are more likely to deposit in glomeruli by charge-charge interactions than antibodies alone.