Non-precipitating Antibodies Research Articles

Immunochemical characterization of cobrotoxin modified with glutaraldehyde.

Modification of cobrotoxin with glutaraldehyde, a bifunctional reagent, resulted in the formation of cross-inked derivatives.The intra-molecular cross-linked monomeric derlvatives as well as dimers, trilllers, and higher molecular weigllt derivatives arising from intermolecular reactions were separated by gel filtration. Polymeric dsrivatives were completely detoxified and the others lost almost completely their lethality. However, all the modified derlvatives retained relatively high antigenic activities towards the antibody against native toxin . Rabbits immunized with these modified derivatives (except for trimeric derivatives) produced considsrable amounts of non-precipitating antibody but much less precipitating antibody to cobrotoxin. The neutralizing capacity of anti-onomeric derivatives sera had 1.4 times more potent than that of anti-cobrotoxin sera. Antisera prepared by immunization with the unfractionated reaction products of cobrotoxin or with dimeric derivatives had the same or almost the same capacity as that of anti-cobrotoxin sera for neutralizing the lethality of cobrotoxin. These antisera were obtained in only one-half period required for anti-cobrotoxin sera. However, polymeric cobrotoxin derivatives were poor linmunogens for the production of neutralizing antibody against native toxin.

Responses of european green lizards Lacerta viridis following administration of Leishmania agamae promastigotes

European green lizards ( Lacerta viridis) were injected intraperitoneally, subcutaneously or orally with viable Leishmania agamae promastigotes. Neither promastigotes nor amastigotes were later found in blood and tissue impression smears, or in blood and selected organ cultures. However, by the use of an immunoperoxidase technique, parasite antigens were detected in the liver, stomach, small intestine, kidney, gonad, heart, lung and skin but not in the bone marrow, brain or spleen. Non-precipitating antibodies with β 2-electrophoretic mobility were induced against L. agamae. They were detected in the sera by enzyme-linked immunosorbent assay 3–7 days post-infection. The titres increased significantly above background levels ( P < 0.001) and reached maxima after 6–7 weeks, with 27 out of 29 lizards producing antibodies. The mean serum protein concentration significantly increased after infection ( P < 0.005) with no significant differences in mean values between male and female animals. Lizard sera separated into 7 components on cellulose acetate membranes with migration rates comparable to albumin, α- and β-globulins of human serum; γ-globulins were absent. Significant decreases occurred ( P < 0.05) in the albumin fraction, with significant increases in the β-globulin region of anti- L. agamae sera. C-reactive protein was not detected in either normal or immune lizard sera.

A procedure for study of immunochemical properties of megagametophyte tissue of PinusStrobus L.

Seeds from six trees of Pinusstrobus L. were germinated. Excised megagametophyte tissue preparation from each was injected into female New Zealand white rabbits. After 7 weeks, sodium dodecyl sulfate (SDS) – polyacrylamide slab gel electrophoretic separation of antigens was performed. Electrophoretic transfer of antigens from gels to nitrocellulose blots was accomplished, followed by treatment of blots with antibody, 125I-labelled protein A, and autoradiography. Each tree could be uniquely identified based on comparisons among the other trees. This procedure is highly sensitive and avoids lack of detection of nonprecipitating antibodies in agar preparations.

Grouping of haemolytic streptococci by monoclonal antibodies: Determinant specificity, cross-reactivity and affinity

Streptococcal group polysaccharide (CHO), A-, A-variant-, B-, C-, D- and G-specific monoclonal antibodies were prepared by the hybridoma technique employing spleen cells of several inbred mouse strains which are either high or low responders to the group A-CHO. The isotypes of these reagents were restricted to the class mu and IgG subclasses gamma 3 and--in small numbers--gamma 1. Two distinct categories of antibodies were identified for all but group D specificity: those which agglutinate suspended bacteria but do not precipitate purified soluble antigen, and those which show both agglutinating and precipitating properties. The group D antibodies described here were only of the latter category. The reactions were inhibitable by haptens in as far as these were known. Cross-reactions were observed in group-A-specific antibodies with E and L polysaccharides. Most G-CHO-specific antibodies cross-reacted with B-CHO. Association constants determined by fluorescence quenching measurements were for binding of complete A and C polysaccharides in the range of 10(6) to greater than 10(8) M-1, and for hapten binding by A-, Av- and C-CHO-specific antibodies in the range of 10(3) to 10(4) M-1. These results support a model of steric arrangements of antigenic determinants on A-variant bacteria and solubilized antigen [42] and allow its extension to streptococcal groups A, B, C and G. This model explains the observed functional differences by postulating single, terminal determinants which interact with the prevailing non-precipitating antibodies and internal repeating determinants which react with precipitins, respectively. No significant differences were found in the reactivity patterns to these streptococcal group antigens between strains of mice in terms of their ability to respond with high or low serum antibody titres to group A-CHO. On the other hand, within high and low responder strains, different kinetics of the optimal timing of fusion after initiation of the secondary immune reaction by boosting was observed. Low responders were most efficiently used for fusion 1.5 days later than high-responder spleen cells. This feature is interpreted to indicate an earlier proliferation of B lymphocytes in high responders, due to either an improved responsiveness to T-lymphocyte help or a reduced reactivity with T suppressor cells in comparison to low-responder B lymphocytes.

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Lacerta viridis produced relatively heat-stable, dithiothreitol-sensitive, non-precipitating antibodies with β 2-electrophoretic mobility following exposure to Leishmania agamae promastigotes but no histopathological or clinical signs of infection were seen. Significant four-and two-fold increases in the mean lysozyme and protein levels respectively were found in immune sera. Immunoenzyme techniques proved the most sensitive for detection of parasite antigens in selected body organs and immunoglobulins in sera.

Production of antibody for α1-acid glycoprotein using carcinoembryonic antigen and normal fecal antigen

The structural characteristics of carcinoembryonic antigen (CEA) and CEA-like antigen in feces and meconium were examined using antibody raised against 2 antigens. When antibody to CEA was made, anti- α 1-acid glycoprotein (AG) (non-precipitating antibody) besides the precipitating antibody for CEA was made. When antibodies against each CEA-like antigen (mol. wt. 180,000) purified from feces and meconium were tested, each antibody showed a fused precipitin line for 2 antigens. Some antibodies showed the non-precipitating antibody for AG. CEA and CEA-like antigen in feces and meconium may be composed of 2 portions (AG portion and non-AG portion). Antibody specific to these antigens may be the antibody directed to the non-AG portion.

Application of denatured, electrophoretically separated, and immobilized lysates of herpes simplex virus-infected cells for detection of monoclonal antibodies and for studies of the properties of viral proteins.

We report the use of herpes simplex virus type 1 (HSV-1)- and HSV-2-infected cell polypeptides (ICPs) separated by electrophoresis in polyacrylamide gels and transferred to nitrocellulose to (i) detect monoclonal antibodies to viral polypeptides and to (ii) study the properties of the proteins with the monoclonal antibodies. Our results were as follows. (i) When the antigens were electrophoretically separated in denaturing gels and then immobilized on nitrocellulose strips, we detected a greater diversity of monoclonal antibodies to viral proteins than when we used the technique of immune precipitation of soluble, nondenatured viral antigens. The primary advantage of the technique is in the detection of nonprecipitating antibody and of antibody to poorly soluble antigens not available for reaction in preparations cleared by high-speed centrifugation before immune reaction. (ii) Studies of the viral polypeptides reactive with three monoclonal antibodies indicated that the technique can be used to investigate several properties of the antigens. Specifically, monoclonal antibody to ICP 4 confirmed the accumulation of viral protein in the nucleus and the mapping of the gene in the S component. The results showed, however, that HSV-1 and HSV-2 ICP 4 do have common antigenic determinants. The reaction of a nonprecipitating monoclonal antibody with electrophoretically separated, immobilized polypeptides contained in cytoplasmic and nuclear fractions, those chemically deglycosylated, or those specified by specific HSV-1 x HSV-2 intertypic recombinants identified the antigens reactive with the second monoclonal antibody as various forms of glycoprotein gC. Of particular interest was a set of four antigens, 39,000 to 46,500 in apparent molecular weight, reactive with each of several monoclonal antibodies. These studies showed that two polypeptides partition in the cytoplasm and two in the nucleus and that all comap with the previously mapped ICPs 35 and 37 in the region of the genome defined by the viral thymidine kinase gene on the left and the glycoprotein gA/B gene on the right. Unlike ICP 4 and gC, the four polypeptides are linked by intermolecular bisulfide bonds, inasmuch as the polypeptides were not at the expected locations upon denaturation and electrophoresis in the absence of reducing agents.

The primary humoral immune response of European green lizards (Lacerta viridis) to Leishmania agamae.

European green lizards, Lacerta viridis, produced relatively thermostable, dithiothreitol-sensitive, non-precipitating, agglutinins and complement-fixing antibodies (CFA) to Leishmania agamae administered subcutaneously (SC), intraperitoneally (IP) or orally (OR). Antibodies were also detected by the immobilization test (IMM) and by enzyme-linked immunosorbent assay (ELISA). The most sensitive method for the detection of stimulated immunoglobulins was ELISA. Antibodies were detected as early as 3 days post-infection with ELISA and between 5 and 7 for CFA, direct agglutination (DA) and indirect haemagglutination (IHA). In the case of IMM, the times of first detection varied from 14 to 28 days. Maximum CFA (2(-8)), DA (2(-8)), IHA (2(-11)) and ELISA (2(-16)) titres were reached from 42 to 49 days with significantly higher values occurring in the OR and IP groups. With IMM, maxima occurred after 5 or 6 weeks. Following exposure, two- to five-fold significant increases in serum lysozyme levels were demonstrated but the concentrations in sera following SC, IP or OR routes of antigen administration were not significantly different when the groups were compared with each other. The highest lysozyme values (approximately 12.3 - 12.5 micrograms ml(-1)) were found in the SC and OR groups when compared to the IP (7.40 micrograms ml(-1)).

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Nonprecipitating antibodies directed toward human-F VIII R: Ag were found in the plasma of a multitransfused patient with severe von Willebrand's disease (VIII: C 2%, VIII R: Ag 30min.) during replacement therapy with VIII concentrate for intracranial hemorrhage.Such antibodies were detected with newly developed semiquantitative assays of anti-VIII R: Ag antibodies by using ELISA. Partially purified AHF made from commercially available F VIII concentrate by Gel filtration was coated as solid phase antigen on the well of plastic microplate. The reacted patient's antibodies with VIII: Ag on the surface of the wells were detected and titrated by using enzyme conjugated anti-human IgG heavy chain, k or λ Light chain. These studies showed that the types of the anti-VIII R: Ag antibodies were IgG heavy chain, mainly k light chain and partially λ light chain. The anti-VIII R: Ag IgG antibody in the patient's plasma diluted (1:10, 000) with PBS was detected 3 weeks after initation of the repalacement therapy by this method. These antibodies were absorbed with normal plasma but not with severe von Willebrand's plasma.The anti-VIII R: Ag antibodies in patient's plasma blocked platelet retention by glass beads column measured by Bowie's method. But they did not block ristocetin-induced platelet aggulutination at all, and they weakly inhibited procoagulant activity of AHF in non-specific manner.These data suggest that the site or sites on the AHF complex molecule that are associated with ristocetin-induced platelet agglutination differ qualitatively from those associated with enhancement of platelet retention by glass beads.

Methylazoxymethanol: Effect on the Immune Response of Rats to Human Serum Albumin&lt;xref ref-type="fn" rid="fn2"&gt;2&lt;/xref&gt;&lt;xref ref-type="fn" rid="fn3"&gt;3&lt;/xref&gt;

Inbred male F344 rats treated with 20 mg methylazoxymethanol (MAM)/kg body weight showed minimal damage to DNA in bone marrow, spleen, and thymus and no suppression of the humoral immune response to human serum albumin (HSA). At high doses of MAM (40, 60, and 80 mg/kg), damage to DNA of bone marrow, spleen, and thymus was noted. The effect on the humoral immune responses was nonuniform. Some rats responded normally by eliciting precipitating antibodies, whereas others responded by synthesizing nonprecipitating antibodies at low levels. The production of nonprecipitating antibodies may indicate a restriction of the recognition of HSA antigenic determinants in rats given injections of MAM prior to immunization. The restriction may be a consequence of suppression or elimination of certain clones of lymphocytes responsible for recognition of certain antigenic determinants. This interference in some clones of lymphocytes would result in the synthesis of antibodies against one or two antigenic sites on the HSA and the concomitant inability of the produced antibodies to form complete lattice structure with antigen.

Complement-mediated fragmentation of soluble and insoluble immune complexes containing porcine anti-DNP antibodies

Complement-mediated release of soluble immune complexes and immune precipitates containing DNP-PSA and precipitating or non-precipitating porcine anti-DNP antibodies was studied. A decrease in the average size of soluble immune complexes indicating their fragmentation was observed during incubation in excess human serum, the extent of the complex release was found to be in direct proportion to the time of incubation. The effect was complement-dependent. In the second part of the study, complement-dependent solubilization of the immune precipitates of the precipitating antibody preparation was compared to the solubilization of the precipitates of the non-precipitating antibody formed in the presence of PEG. Although, both types of precipitates activated complement in about the same extent, complexes of non-precipitating antibody were solubilized much slower than those of the precipitating one. As avidity of both antibody preparations to the antigen was high, the observed differences in the rate of the complex solubilization probably reflected differences in the structure of the two types of complexes.

Effect of polyethylene glycol on the rate of immune complex formation by non-precipitating antibody

A kinetic study formation of large size complexes of non-precipitating pig-Dnp anti-Dnp antibody and multivalent dinitrophenylated serum albumin was performed using light scattering and absorption spectroscopy of the Dnp-group. A very rapid phase of the process resulted in the formation of complexes having molecular weight of about 2 × 10 6. Further increase of the complex size was much slower. Addition of PEG affected positively the rate of complex growth even in concentrations below 1%. The spectroscopic kinetic curves also showed a rapid and a slow phase, sensitive to the presence of PEG. The character of the kinetic data does not support the simple view that polymers enhance precipitate-formation by the steric exclusion of complexes from the polymer domains. It can be assumed that the interaction of the polymer with the antigen-antibody system consists of a subtle temporary attachment of the polymer to the antibody molecule resulting in a change of the shape and/or flexibility of the antibody molecule, favouring its cross-linking capacity.

Enzymatic and immunochemical properties of lysozyme—Restriction of recognition of lysozyme antigenic determinants in pigs

Pigs immunized with lysozyme responded by producing only nonprecipitating antibody throughout the immunization period. Fig antilysozyme antibodies were found to be resistant to papain fragmentation, only 33% of the antibodies were fragmented with papain. From the binding of fluorescein labeled or 14C-labeled lysozyme to antilysozyme antibodies it was concluded that the antibodies elicited in pigs recognized only two antigenic determinants of lysozyme. These results were confirmed from the binding of Fab fragments to 14C-lysozyme. Fab fragments prepared from precipitating rabbit antilysozyme antibody bound 14C-lysozyme at a molar ratio of Fab/lysozyme = 3. Therefore nonprecipitating antibodies are the outcome of recognition of only two antigenic determinants on lysozyme and inability to form a lattice structure when antibody and antigen interact. This work emphasizes the limitations of using antibodies as a biological reagent for delineating the antigenic determinants on proteins.

Evaluation of polyethylene glycol's suitability for quantitative determination of non-precipitating antibody

Polyethylene glycol (PEG) has been used extensively in the quantitative precipitin reaction and for the determination of soluble immune complexes. It has now been found that the optimum concentration of PEG for precipitation of non-precipitating antibodies in the quantitative precipitin reaction varies with the dilution of antisera. Immune precipitates obtained with PEG were examined for their protein content by polyacrylamide gel electrophoresis to detect non-specific precipitation of serum proteins. Determination of non-precipitating antibodies in sera of goats and pigs immunized with chicken egg-white lysozyme by the quantitative precipitin reaction in the presence of PEG, yielded lower values than obtained by immunoabsorbents. Care should be exercised in interpreting results when PEG is used to enhance precipitation.

General shape and hapten-induced conformational changes of pig antibody against dinitrophenyl. A small-angle scattering study.

Pig antibodies against dinitrophenyl were studied by neutron small-angle scattering and X-ray small-angle scattering with particular attention to the analysis of cross-section plots and determination of the radii of gyration of cross-section. The experimentally determined molecular parameters Rg (radius of gyration), Rq1 and Rq2 (two different radii of gyration of cross-section characterizing every antibody sample) show that the shapes of the two antibody types, precipitating and non-precipitating, are similar. The non-precipitating antibody is slightly more compact. The parameters Rg, Rq1 and Rq2 of complexes of antibodies with the hapten, 8-dinitrophenyl-5,8-aza-4-oxo-octanoic acid, are smaller than those of the free antibody. This indicates that a conformational change is induced by the binding of the hapten. The character of the change of parameters is consistent with a view that the observed contraction of the molecule proceeds via similarity transformation. In order to design a model of a pig antibody molecule, isolated building blocks of the molecule, the Fab and Fc fragments, were first studied. A comparison of the scattering curves with various models of fragments showed, however, that the isolated fragments acquire in solution elongated rod-like shapes. Over 300 tentative models of the intact antibody molecule, built of small identical spheres, were constructed before a good fit with the experimental data was achieved. The most probable models have a cavity in the Fc part and the Fab parts are either fully extended or slightly bent downwards to the Fc part.

Toward a system for detecting somatic-cell mutations. V. Preparation of fluorescent antibodies to hemoglobin Hasharon, a human α-chain variant

Antibodies against the abnormal human hemoglobin, Hb Hasharon ( α 47Asp→His), were raised in horse and purified by absorption against Sepharose 4B to which normal hemoglobins or Hb Hasharon were bound. The purified, non-precipitating antibodies were tested for specificity against normal hemoglobins and Hb Hasharon by immunodiffusion in the presence of anti-horse IgG, and by exposing mixtures of normal and Hb Hasharon-containing red cells to the antibodies after conjugation of the latter with fluorescein isothiocyanate. The ease with which antibodies specific for different variant hemoglobins have been prepared, and their potential for identifying individual erythrocytes that contain these hemoglobins by virtue of somatic mutation, underscore their value as aids to detection and analysis of mutational events in human subjects.

22] Antigen quantitation by a reverse hemolytic assay

This chapter describes the antigen quantitation by a reverse hemolytic assay (RHA). Erythrocytes, chemically coated with purified antibody, can lyse through a reaction that is mediated specifically by the complementary antigen. This antigen-mediated hemolysis, wherein the relative position of antigen and antibody on the erythrocytes is reversed, can be exploited for detecting the antigen. The RHA provides a sensitive, specific, and precise assay for determining the antigen levels in fluids. The antigen can be quantitated in absolute weight units when the standard antigen is available in purified and homogeneous form. The assay involves the reaction of the antigen with its specific antibody in a gel. The antibody is anchored to erythrocytes that are immobilized in the gel. The RHA may be more accurate and more precise than radial immunodiffusion (RID), because the hemolytic edges are sharp at any antigen concentration, thus facilitating measurement. RHA may make use of flocculating and nonprecipitating antibody, unlike RID. The RHA dishes can be read immediately after complement incubation or at later times, if treated with 2% formaldehyde, to fix the indicator cells.

A hierarchy of responsiveness and the production of nonprecipitating antibodies to F antigen

The immune response to F antigen by a variety of inbred strains and F1 hybrids has been studied. All of the mice responding to appropriate preparations of F antigen share ak allele atH-2K orI-A. In F1 hybrids, however, this permissive gene is sometimes expressed as dominant responsiveness, while in other combinations as dominant nonresponsiveness. There appears to be a hierarchy of responsiveness among the responder strains tested. Finally, some strains produce nonprecipitating antibodies against F antigen which may represent a genetically controlled restriction of the response to this antigen.

Nonprecipitating pig anti-dinitrophenyl antibody: factors influencing the conversion into precipitating antibody.

AbstractA high affinity nonprecipitating anti‐2,4‐dinitrophenyl (DNP) IgG antibody constitutes a major fraction of circulating anti‐DNP antibodies at the decline of the anti‐ DNP antibody response in pigs. The purified nonprecipitating antibody forms exclusively soluble complexes with DNP‐PSA (pig serum albumin) or DNP‐PGG (pig IgG). The binding sites are accessible to large ligands; one antibody molecule can combine with two molecules of DNP‐PSA simultaneoulsy.The formation of complexes with DNP‐PSA was studied in various media. No precipitate was formed in saline, in 0.9 M or 0.05 M sodium chloride, in 0.5 M potassium thiocyanate and in several nonionic detergent solutions (0.5% and 1%). Normal pig or guinea pig sera were found to contain a factor, removable by an antigen‐antibody precipitate that caused a partial precipitation of the complexes. A mild alteration of the integrity of the antibody molecule by cleavage of interchain disulfide bonds did not affect the nonprecipitating character of the antibody. In media containing hydrophilic polymers, i.e. various dextrans, polyethylene glycols or a soluble polyamide (poly‐α,β5‐[N(2‐hydroxyethyl)‐DL‐aspartamide]), a functional conversion occurred; the antibody behaved like a precipitin. The precipitate with DNP‐PSA could not be resolubilized upon removal of the polymer by washing. The magnitude of the functional conversion depended on the concentration of the polymer and on its molecular weight, larger molecules being more effective. The effect of the hydrophilic polymers could be potentiated by replacing water in the solvent partly by deuterium oxide.The findings are compatible with a notion, that in polymer‐containing media the nonprecipitating antibody acquires a conformation functionally equivalent to the conformation of a precipitating antibody and that the altered conformation is stabilized by the lattice of the precipitate with DNP‐PSA.

Synthetic antigens—V: Properties of rabbit antibodies against a synthetic antigen: Interpolymer of styrene and maleic acid

Properties of rabbit antibodies against a new synthetic antigen: interpolymer of styrene and maleic acid (PSM)‡ are described. Rabbits immunized with PSM produced precipitating and nonprecipitating antibodies of IgG class. Interactions of anti-PSM antibodies with PSM are mainly of an electrostatic character and were inhibited by various low molecular weight compounds resembling the structural unit of PSM. Anti-PSM antibodies belong to low-affinity antibodies and show multispecificity. The apparent affinity constant for PSM was of the order of 10 5 M −1. Anti-PSM antibodies showed no restriction of heterogeneity in isoeleetric focusing. The results of spectral measurements showed that tyrosine residues may be involved in the interaction of antibodies with PSM. No major change of conformation of anti-PSM antibodies occurred during the interaction with PSM or hapten-like compounds.