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.     

Antigen mimicry by anti-idiotypic antibodies: study of interactions between complementary surfaces in macromolecules.

Anti-idiotypic antibodies were obtained from New Zealand White rabbits injected with affinity-purified rabbit anti-TrpR antibodies. In gel mobility shift studies, such immunoglobulin preparations were shown to contain one or more species able to form specific complexes with DNA molecules bearing a trp operator. In competitive ELISA assays, the binding of anti-idiotypic antibodies to operator-bearing DNA was reversed by TrpR. The demonstration that the immune repertoire contains information for operator-specific DNA-binding proteins may be relevant to the etiology of certain autoimmune diseases.     

Expression of anti-idiotypic clones against auto-anti-DNA antibodies in normal individuals

A serum pool from 280 blood donors and individual samples from blood donors were assayed for anti-idiotypic activity to auto-anti-DNA antibodies by competitive radioimmunoassays. We found that serum from several normal blood donors inhibited the binding activity of anti-DNA antibodies affinity purified from the sera of patients with systemic lupus erythematosus. This inhibition was due to immunoglobulin molecules but was not due to rheumatoid factor activity. Antiallotypic antibodies were not responsible for the anti-anti-DNA activity detected. Because this inhibition was blocked by DNA molecules, the observed reactivity was probably caused by idiotype-anti-idiotype interactions. These results provide evidence that anti-idiotype antibodies against anti-DNA autoantibodies are present in certain normal human sera. Anti-anti-DNA antibodies could play a role in the regulation of autoimmunity to DNA.     

Idiotypic determinants of monoclonal antibodies that bind to 3-fucosyllactosamine

Many monoclonal antibodies that react with the lacto-N-fucopentaose III (LNF III) antigenic determinant, Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc, have been described recently. The terminal trisaccharide of this determinant, fucosyllactosamine, is present on glycolipids and glycoproteins and on the surface of granulocytes, monocytes, and other cells. To study the structural and genetic diversity of these antibodies, syngeneic anti-idiotypic monoclonal antibodies were produced in BALB/c mice against PMN 6, a monoclonal antibody directed against this sequence. Anti-idiotypic antibodies 6B1 and 6C4 reacted with 50% of a panel of 20 anti-LNF III monoclonal antibodies, whereas 6A3 reacted strongly only with PMN 6. This indicates that the determinants recognized by 6C4 and 6B1 represent major cross-reactive idiotopes of this family of antibodies. The binding of idiotypic antibodies to a glycolipid bearing this antigenic determinant was completely inhibited by the three anti-idiotypic antibodies, 6A3, 6B1, and 6C4. The idiotopes could be demonstrated on the heavy chain of the monoclonal antibodies by an antibody transfer technique when mild reducing conditions were employed, but a high concentration of reducing agent destroyed the idiotypic determinants. This suggests that the anti-idiotypic antibodies recognize conformational structures expressed on the heavy chain molecules. The binding of 18 monoclonal antibodies to two glycolipid antigens and to a fucosyllactosamine-bovine serum albumin conjugate was compared. Antibodies that possessed the 6C4 cross-reactive idiotope bound to fucosyllactosamine-bovine serum albumin more weakly than idiotype-negative antibodies (p = 0.001). This suggests that the 6C4-positive antibodies might represent germline structures.     

SDR grafting--a new approach to antibody humanization

A major impediment to the clinical utility of the murine monoclonal antibodies is their potential to elicit human anti-murine antibody (HAMA) response in patients. To circumvent this problem, murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts. To that end, murine antibodies have been humanized by grafting their complementarity determining regions (CDRs) onto the variable light (V(L)) and variable heavy (V(H)) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site. However, the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients. To minimize the anti-Id response, a procedure to humanize xenogeneic antibodies has been described that is based on grafting, onto the human frameworks, only the specificity determining residues (SDRs), the CDR residues that are most crucial in the antibody-ligand interaction. The SDRs are identified through the help of the database of the three-dimensional structures of the antigen-antibody complexes of known structures or by mutational analysis of the antibody-combining site. An alternative approach to humanization, which involves retention of more CDR residues, is based on grafting of the 'abbreviated' CDRs, the stretches of CDR residues that include all the SDRs. A procedure to assess the reactivity of the humanized antibody to sera from patients who had been administered the murine antibody has also been described.     

Anti-angiotensin II anti-idiotypic antibodies bind to angiotensin II receptor

An anti-idiotypic serum from a rabbit immunized with one anti-angiotensin II (AII) monoclonal antibody (A25) was shown to identify a cross-reactive idiotope (CRI) shared by six anti-AII monoclonal antibodies, in addition to a binding site-associated private idiotope. This anti-idiotypic reagent bound to rat liver membranes bearing AII receptors; binding was abolished after pretreatment of the membranes with AII. In immunoblotting experiments with rat liver membranes, as well as with rat pituitary homogenates, a 63,000 +/- 2,000 dalton protein was revealed that co-migrated with the AII receptor. After purification by affinity chromatography on an immobilized CRI+-antibody (A41), anti-CRI antibodies could immunoprecipitate the hormone binding activity from detergent-treated rat liver membranes and still recognize the 63,000 dalton protein. In contrast, anti-idiotypic antibodies specific for the private idiotope failed to interact with the AII receptor. Similar results were obtained with a second anti-idiotypic serum produced by immunization with another CRI+ anti-AII monoclonal antibody (A22). The sharing of the CRI determinant between the AII receptor and anti-AII antibodies might account for the reactivity of anti-idiotypic antibodies towards the AII receptor.     

Epitopic and paratopically directed anti-idiotypic factors in the regulation of resistance to murine schistosomiasis mansoni

In this study we investigated aspects of targets and regulatory mechanisms of immunologically mediated resistance to schistosomiasis. The interactions of antigen, monoclonal antibodies (MAb), and anti-idiotypic antibodies were studied by using competitive inhibition ELISA, radioimmunoprecipitation, and direct-binding ELISA techniques. MAb, either protective or nonprotective against challenge with Schistosoma mansoni, recognize either discrete or shared epitopes. MAb that recognize the same specific epitope may or may not express the ability to adoptively transfer resistance to syngeneic recipients. These results suggest that the functional as well as the epitopic specificity must be considered in an evaluation of protective mechanisms. The antibodies also can be characterized by both unique and cross-reacting idiotypic determinants. In addition, a relationship between antigen and anti-idiotypic antibody activity has been demonstrated. The immunologic analogy between antigenic epitopes and anti-idiotypic antibodies has been demonstrated by the ability of these two moieties to reciprocally inhibit the recognition of paratope-associated idiotypes, expressed by the protective MAb. This anti-idiotypic activity can be demonstrated in serum of infected animals. In this study we have identified two specific epitopes related to protection, and we illustrate here the steric relationship between antigen and anti-idiotypic antibody. The presence of idiotypically directed regulatory pathways within actively infected animals suggests that the immune response can be differentially regulated at the clonal level.     

Circulating monoclonal IgM proteins in B cell chronic lymphocytic leukemia: their identification, characterization and relationship to membrane IgM

Accumulated evidence indicates that there is a circulating monoclonal Ig protein related to the leukemic cell-associated Ig in the majority of patients with B cell chronic lymphocytic leukemia (CLL) despite the failure to demonstrate such a protein by conventional serum electrophoresis. Methodology has been developed to reveal these hidden monoclonal bands and to show that they are related to the leukemia-associated membrane Ig (mIg). Of nine CLL cases with stainable mIgM and without discernable plasma Ig bands, marked hypogammaglobulinemia was evident in six. In the other three, a significant amount of protein was present in the gamma region. IgM was isolated from the plasma of these patients by affinity chromatography with Sepharose-4B, conjugated with affinity purified anti-human IgM antibodies. One to 3 mg were isolated from 20 to 40 ml of plasma. Agarose electrophoresis revealed a monoclonal Ig band in the isolated IgM in all cases. Eight of these IgM proteins were analyzed by high-pressure liquid chromatography. Five were found to be pentameric IgM. In the remaining three, various amounts of monomeric IgM were detected. Attempts to make anti-idiotypic antibodies to the isolated proteins have been successful. Thus far, a rabbit anti-idiotypic antiserum was obtained in one case and two mouse monoclonal anti-idiotypic antibodies in two additional cases. Immunofluorescence analysis revealed that plasma IgM and mIgM shared similar idiotypic determinants. One other monoclonal antibody was shown to be specific for a V region marker of a minor Ig population. These findings indicate that B leukemic lymphocytes do secrete a small amount of IgM and lend further support to the thesis that the maturation defect in CLL is incomplete. It is also feasible to isolate the secreted IgM and to produce anti-idiotypic antibodies to them. In view of the potential therapeutic effect of anti-idiotypic antibodies, this may offer an alternative and efficient approach to generate a large panel of anti-idiotypic antibodies for clinical trials. The possibility also exists that this approach is applicable to other B cell proliferative disorders such as the non-Hodgkin B cell lymphomas.     

A syngeneic monoclonal anti-idiotypic antibody with internal image properties restricted to a single aldosterone-binding system

Immunization with a murine anti-aldosterone mAb (AAC) resulted in the isolation of a syngeneic monoclonal anti-idiotypic antibody, LH9G4. LH9G4 bound to Fab fragments of AAC and was affinity-purified on an AAC column. LH9G4 inhibited the binding of aldosterone to AAC in a dose-dependent manner with an apparent dissociation constant of 0.5 nM as determined by competitive inhibition assays in ELISA and RIA. LH9G4 and aldosterone have similar relative affinities for AAC. Kinetic studies and Scatchard plot analysis support a reversible and reciprocal competitive inhibition mechanism between LH9G4 and aldosterone for the paratope of AAC. The possibility of a steric hindrance mechanism was eliminated. No cross-reactivity was seen with six other murine anti-aldosterone mAb, with a rabbit polyclonal antibody or with aldosterone receptor. The anti-idiotypic antibody, defined as a "restricted" internal image of aldosterone, is apparently directed at a private idiotope present in the paratope of AAC but not in binding sites of other aldosterone-binding proteins. Biophysical considerations involving characteristics of nonbonded attractive forces can explain these findings. An advantage of the one-step auto-anti-idiotypic procedure for the generation of Ab2-beta or internal image antibodies is discussed.     

Interaction between immobilized antibodies and the antigen-enzyme complex]

The interaction between immobilized antibodies against human immunoglobulin G (IgG) and the immunoenzyme complex IgG-peroxidase (IgG-P) was studied. The complex was obtained by covalent binding of IgG to peroxidase modified by sodium periodate. Study of the IgG-P binding kinetics and dissociation of the antibody-(IgG-P) complex showed that the antibodies immobilized on Sepharose reversibly interacted with IgG-P, similar to the antigen-antibody reaction in solution. The efficient values of the binding constants for the antibodies binding to Sepharose covalently and through the antigen-antibody bond are (2,2+/-0,5) 10(8) M-1 and (4,2+/-0,2) 10(8) M-1, respectively. The nature of a carrier and the immobilization method used do not significantly affect the rate of the complex binding to the antibodies. The activation energy of the reaction of IgG-P binding to the antibodies immobilized on Sepharose covalently and through the antigen-antibody bond is 7,3 and 4,1 kcal/mole, respectively. A procedure of titration of immobilized antibodies active sites with the antigen-enzyme complex is discussed.     

Kinetic Studies of a Doubly Bound Red Cell Antigen-Antibody System

The Polybrene method for detection of red cell antibodies which utilizes continuous flow equipment was modified so that kinetic studies could be performed on red cell antibodies doubly bound between adjacent red cells. In the anti-Rh_o-Rh_o erythrocyte system, deaggregation by temperature was studied over an antibody concentration range of from approximately 1 to 500 antibody molecules per erythrocyte, a residence time range of approximately eightfold, and a temperature range of from 10 to 55°C. The rate of dissociation of antigen-antibody complex, as determined from deaggregation of antibody-dependent red cell aggregates, was found to be of apparent zero order. The apparent activation energy for the antigen-antibody reaction under the experimental conditions was determined and found to be higher than would be expected for singly bound antigen-antibody systems. Possible explanations are considered for these findings in terms of an antigen-antibody bond-breaking model.     

Identification of Serotonin Receptors Recognized by Anti-Idiotypic Antibodies

Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.     

Interferon-like activity in an anti-interferon anti-idiotypic hybridoma antibody

We describe here an anti-idiotypic hybridoma antibody directed against affinity-purified rabbit idiotypic antibodies (Rb-Id) to a homogeneous protein, the recombinant human leukocyte A interferon (rIFN-alpha A). The supernatant of the hybridoma, designated 3-1B, was able to inhibit the neutralization of rIFN-alpha A activity by the idiotypic antibodies. An in vivo passage of uncloned 3-1B cells yielded hybridoma cells (presumably a subclone), designated 3B1, the supernatant of which exhibited interferon-like antiviral activity with both bovine kidney (MDBK) cells and human amnion (WISH) cells. This activity could be absorbed by polymer-bound goat anti-mouse immunoglobulin serum and by Rb-Id coupled to Affi-Gel 10, and could be partially eluted from the latter at pH 2.5. The anti-idiotypic hybridoma antibody was able to compete with 125I-rIFN-alpha A for binding to the Rb-Id and also to interferon receptor-bearing MDBK cells. The clinical significance of an interferon-like anti-idiotypic antibody is discussed.     

Quantification and specific detection of collagenous proteins using an enzyme-linked immunosorbent assay and an immunoblotting for cyanogen bromide peptides

A method for the detection of collagenous proteins within cyanogen bromide digests of tissues has been devised. The peptides produced by digestion with cyanogen bromide were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter. They were stained on the filter by incubation first with antibodies to collagen and then with a second antibody covalently linked to horseradish peroxidase, 4-chloro-1-naphthol was added, and the bound enzyme was assayed. This procedure is useful for the identification and characterization of collagens of types I, III, IV, and V in tissues. In addition, we have developed a sensitive and specific competitive enzyme-linked immunosorbent assay (ELISA) which is convenient for quantifying collagens (types I, III, and IV) in tissues. In this kind of assay, soluble cyanogen bromide peptides compete with cyanogen bromide peptides adsorbed onto a solid-phase support for rabbit anti-collagen antibodies. We determined the amount of bound antibody by using goat anti-rabbit immunoglobulin G covalently conjugated to horseradish peroxidase and then provided a substrate for the enzymatic reaction. The sensitivity range of the ELISA is 0.09 micrograms/ml in the region of 90 to 10% binding.     

Spontaneous development of anti-hepatitis B virus envelope (anti-idiotypic) antibodies in animals immunized with human liver endonexin II or with the F(ab')2 fragment of anti-human liver endonexin II immunoglobulin G: evidence for a receptor-ligand-like relationship between small hepatitis B surface antigen and endonexin II.

In a previous study, we have identified endonexin II (E-II) on human liver plasma membranes as a specific, Ca(2+)-dependent, small hepatitis B surface antigen (HBsAg)-binding protein. In this article, we describe the spontaneous development of anti-HBs antibodies in rabbits immunized with native or recombinant human liver E-II and in chickens immunized with the F(ab')2 fragment of rabbit anti-human liver E-II immunoglobulin G. Anti-HBs activity was not observed in rabbits immunized with rat liver E-II. Cross-reactivity of anti-E-II antibodies to HBsAg epitopes was excluded, since anti-HBs and anti-E-II activities can be separated by E-II affinity chromatography. The existence of an anti-idiotypic antibody is further demonstrated by competitive binding of human liver E-II and this antibody (Ab2) to small HBsAg, suggesting that Ab2 mimics a specific E-II epitope that interacts with small HBsAg. In addition, it was demonstrated that anti-HBs antibodies developed in rabbits after immunization with intact human liver E-II or in chickens after immunization with F(ab')2 fragments of rabbit anti-human liver E-II immunoglobulin G recognize the same epitopes on small HBsAg. These findings strongly indicate that human liver E-II is a very specific small HBsAg-binding protein and support the assumption that human liver E-II is the hepatitis B virus receptor protein.     

Idiotypes of Lewis rat antibodies to encephalitogenic peptides of guinea pig myelin basic protein: in vitro and in vivo studies

Lewis rat antibodies raised by immunization with encephalitogenic peptide 68-88 guinea pig myelin basic protein were purified by affinity chromatography and used to immunize rabbits. After exhaustive absorption of the rabbit antisera to remove anti-rat immunoglobulin activity, the antisera retained activity against the immunogen, shown by the ability to block reaction of radioiodinated peptide with the active site of the rat anti-peptide antibodies. Intrastrain idiotypic cross-reactivity was assessed by testing the rabbit antisera against a panel of Lewis anti-peptide antibodies. Each anti-idiotypic antiserum displayed a unique pattern of reactivity with the panel. Similar tests in which a panel of anti-peptide antibodies raised in F344 rats was used demonstrated the presence of interstrain cross-reactive idiotopes. When seven rabbit anti-idiotypic antisera were tested by pretreatment of rats before challenge with encephalitogen for effect in vivo, five were without effect. Of the remaining two, one caused a slight suppression of disease; the other enhanced disease compared to control animals.     

Antigen-antibody interaction. The immunodominant region of EDP208 pili

The EDP208 pilus contains a major antigenic determinant in the N-terminal dodecapeptide, as shown by E. A. Worobec, A. K. Taneja, R. S. Hodges, and W. Paranchych ((1983) J. Bacteriol. 153, 955-961). This peptide was chemically synthesized, coupled to bovine serum albumin with N-hydroxysuccinimidyl p-azido-benzoate, and used in immunoblot and enzyme-linked immunosorbent assays to show it was capable of reacting with anti-EDP208 pilus antibodies. Antibodies raised against the synthetic peptide conjugate were also capable of reacting with whole pili in these assays. To further examine the specific residues responsible for the antigenicity of this site, several peptide analogs were chemically synthesized. The relative affinity of these peptides for anti-EDP208 pilus antibodies was determined by a competitive enzyme-linked immunosorbent assay using the Fab fragment of anti-EDP208 pilus immunoglobulin G. From these results we established that the antigenic region of this peptide was the N-terminal pentapeptide, N-acetyl-Thr-Asp-Leu-Leu-Ala, and the key residues responsible for the antibody-antigen interaction are the N-acetyl-Thr1, Leu3, and Leu4. Hydrophobic interactions involving the methyl of the acetyl group and the leucine side chains make the largest contributions to the antigen-antibody interaction, while a lesser contribution is made by the Thr1 hydroxyl. The side chains of Asp2 and Ala5 contribute only weakly to the stabilization of the antigen-antibody complex.     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.     

Idiotypy of human anti-Candida albicans antibodies: recurrence, presence of a cross-reactive autoanti-idiotypic-like activity, and role in the induction of specific in vitro antibody response

Rabbit anti-idiotypic antibodies (L12) were raised against human anti-mannan of Candida albicans (CA) antibodies isolated from the serum of a normal donor. The absorbed anti-idiotypic antiserum bound to donor anti-CA mannan antibodies but not to control immunoglobulins. Binding was inhibited by CA mannan but not by other polysaccharide antigens. L12 was shown to cross-react with anti-CA mannan-isolated antibodies or with anti-CA antibody-containing sera from individuals unrelated to the donor. IgG fraction isolated from the donor serum was repeatedly absorbed on CA mannan Sepharose to remove anti-mannan antibodies. This IgG fraction (named autoanti-idiotypic fraction) blocked, in a dose-dependent fashion, the binding of rabbit anti-idiotype to donor anti-CA mannan antibodies. Moreover, this CP-depleted IgG fraction cross-reacted with public idiotypic determinants of unrelated anti-CA mannan antibodies. Finally, L12 induced sensitized lymphocytes to produce anti-CA mannan antibodies in vitro in the absence of antigen.     

Immunogold for detection of antigen on nitrocellulose paper

The application of immunogold for the detection of antigen-antibody complexes on nitrocellulose paper is described. Tobacco mosaic virus (TMV) protein, either purified or in leaf extract, was bound to the nitrocellulose paper and then exposed to rabbit anti-TMV serum. The antigen-antibody complexes were detected by gold-labeled goat anti-rabbit immunoglobulin G. The gold-labeled antibody is directly visible because of its pink color. This method can detect 1-5 pg of TMV protein, either in purified form or in the unpurified plant extract, with high specificity.