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At least 19 recordsLinked to original sources
PubMed1980

Antibodies to synthetic antibody-combining sites. Antibodies against a surface-simulation peptide with antibody-combining activity toward lysozyme antigenic site 3 react with lysozyme antibodies.

Recently, we reported the synthesis and immunochemistry of two peptides designed, by complementarity and surface-simulation synthesis, to mimic antibody-combining sites against two antigenic sites of lysozyme. In the present work antibodies were raised against one of these peptides, which is complementary to antigenic site 3 of lysozyme, to determine whether these antibodies will react with anti-lysozyme antibodies. Radioiodinated antipeptide antibodies were bound by immunoadsorbents of the immune IgG from two goats anti-lysozyme antisera but not by adsorbents of myoglobin, non-immune goat IgG or immune IgG of antisera against cytochrome c. The binding of anti-peptide antibodies to adsorbents of anti-lysozyme antibodies was fully inhibited by free lysozyme but not by bovine serum albumin, human hemoglobin A, horse cytochrome c or bovine ribonuclease A. Thus, antisera against an antibody-combining site can be raised by immunizing with a peptide which probably does not exist in the antibody but is designed by surface-simulation synthesis to mimic an antibody-combining site.

Animals↗
PubMed1982

Experimental antigen-antibody complex disease in mice. The role of antibody levels, antibody affinity and circulating antigen-antibody complexes.

Chronic antigen-antibody complex disease (A-ACD) was induced in mice by daily injections of HSA. The disease was characterized by death, intense glomerular basement membrane deposition of complexes in the kidneys, proteinuria and an impairment of the glomerular filtration rate. In mice that had been selectively bred to produce antibody of low affinity to protein antigens injected in saline, the incidence of chronic A-ACD was 61% compared to 21% in mice selectively bred to produce antibody of high affinity. Low affinity female mice produced approximately ten times as much free antibody to HSA as did low affinity males and high affinity males and females, but this did not result in an increase incidence of chronic A-ACD in low affinity female mice. Low affinity female mice also had the highest levels of circulating antigen-antibody complexes detected by conglutinin and Clq binding assays, but there was no correlation between the presence of high levels of complexes in serum and tissue damage. In both lines of mice the mean affinity of free antibody to HSA started low and increased with time and the number of injections of antigen. However, development of chronic A-ACD in individual low affinity mice appeared to be associated with a failure to produce this time-associated increase in antibody affinity. These results are discussed with reference to the relevance of the measurement of the amount of affinity of free antibody in serum and the levels of circulating antigen-antibody complexes to the production of tissue damage in this model of chronic A-ACD.

Animals↗
PubMed1969

Idiotypy of rabbit antibodies. I. Comparison of idiotypy of antibodies against Salmonella typhi with that of antibodies against other bacteria in the same rabbits, or of antibodies against Salmonella typhi in various rabbits.

Sera of rabbits immunized against Salmonella typhi have been studied for the idiotypy of certain of their components, i.e., the property of these components to possess an antigenic specificity which is different in individual rabbits, and which varies with the antigens against which these rabbits have been immunized. The reagent used (precipitating anti-idiotypic sera) have been prepared by injecting rabbits with bacteria agglutinated by anti-S. typhi sera (immunizing sera) as was done in the first observations by the authors of the phenomenon in the rabbit. These first observations have been confirmed and extended. In contrast to allotypy, the anti-idiotypic sera precipitate the corresponding immunizing sera, but not the sera taken in the immunizing rabbits prior to their immunization against S. typhi, nor the immunizing sera absorbed with the somatic antigen of S. typhi, demonstrating that idiotypes are antibodies. The idiotypic specificities of the antibodies of one rabbit against S. typhi are not detected in the antibodies of the same rabbit against another noncross-reacting Salmonella (S. tranoroa) and vice versa; nor are they detected in the anti-pneumococcal antibodies of the same rabbit. Each anti-idiotypic serum fails to precipitate anti-S. typhi sera of rabbits other than the immunizing one except for certain extremely faint reactions, the significance of which has not been established. The idiotypic specificities of anti-S. typhi antibodies of three rabbits were not found in anti-S. typhi antibodies of their parents. This lack of a sign of hereditary transmission of idiotypic specificities contrasts with allotypy. The apparent role of random chance in the determinism of the idiotypic patterns or of the idiotypic determinants has been discussed. Unless it were admitted that antibodies with similar functions do not exist in different individuals, idiotypy apparently adds an order of magnitude to the antibody variability which had been previously envisaged. In one given individual, the heterogeneity of the idiotypic specificities seems to be less extended than that of the antibody functions. The possible relationships between these two levels of molecular variability and between the corresponding levels of cellular variability have been discussed.

Animals↗
PubMed1991

Relationship between IgG1 and IgG4 antibodies to foods and the development of IgE antibodies to inhalant allergens. II. Increased levels of IgG antibodies to foods in children who subsequently develop IgE antibodies to inhalant allergens.

In the present investigation we have tested the hypothesis that children with a high IgG antibody response to foods have an increased risk of developing IgE antibodies to inhalant allergens. Sera from 106 children with an increased risk of developing IgE-mediated allergy were analysed. During the follow-up, in 54 of these children IgE antibodies to inhalant allergens appeared. A positive/negative IgG1 and IgG4 anti-food score was determined as described previously: sera from age-clustered unselected children were tested for the levels of IgG1 and IgG4 antibodies to common foods. For each IgG RAST and each age group, the 75-percentile was chosen as cut-off value. Each antibody level was thus converted into a positive (higher than the 75-percentile of the age group) or negative value. The number of positive tests was used as the score. High-risk children with a high IgG1 anti-food score more often developed inhalant-specific IgE antibodies than high-risk children with low IgG1 titres: 50% of the children with a high IgG1 anti-food score developed IgE antibodies to grass pollen. Fifty per cent of the children with a high and 14% of the children with a low IgG1 anti-food score developed IgE antibodies to cat dander. For the prediction of the development of IgE anti-mite (house dust mite), the IgG4 anti-food scores appeared less useful than the IgG1 anti-food scores; 46% of the IgG4 high responders versus 22% of the IgG4 low responders acquired IgE anti-mite, whereas for IgG1 these percentages were 73 and 19, respectively.

Allergens↗
PubMed1990

Analysis of the role of antibody-dependent cellular cytotoxic antibody activity in murine neonatal herpes simplex virus infection with antibodies to synthetic peptides of glycoprotein D and monoclonal antibodies to glycoprotein B.

The role of antibody in neonatal herpes simplex virus (HSV) infection remains controversial. A battery of well-characterized monoclonal antibodies to HSV glycoprotein B (gB), and polyclonal antibodies against synthetic peptides of predicted epitopes of HSV glycoprotein D (gD) were used to determine in vitro functional activity and association with protection against lethal infection in a murine model of neonatal HSV disease. Antiviral neutralization activity of HSV was not associated with antibody-dependent cellular cytotoxicity (ADCC) activity to HSV-infected cells in vitro. In a model of high dose challenge (10(4) PFU), protection was not afforded by any antibody alone, but was by antibody plus human mononuclear cells, and highly associated with ADCC functional activity (P less than 0.001). In a low dose challenge model, neutralizing activity of antibody alone was associated with protection in vivo (P less than 0.001). Of the nine neutralizing epitopes of gD in vitro, eight were predicted surface regions. Four of the five epitopic sites of gD (2-21, 267-276, 288-297, and 303-312) that were determined to be important targets of ADCC and in vivo protection were also predicted to be surface regions. The only exception was the antiserum to region 52-61 which was predicted to be buried and also showed these activities. ADCC as well as neutralizing antibody activity are important in protection against neonatal HSV infection.

Animals↗
PubMed1993

A multinuclear NMR study of the affinity maturation of anti-NP mouse monoclonal antibodies: comparison of antibody combining sites of primary response antibody N1G9 and secondary response antibody 3B62.

On the basis of multinuclear NMR data, the structures of the antibody combining sites of anti-4-hydroxy-3-nitrophenylacetyl (NP) antibodies were compared for N1G9, which is one of the primary response antibodies with low affinity for NP, and 3B62, which is one of the secondary response antibodies with high affinity for NP. It has been concluded, on the basis of the results of antibody engineering, that in most secondary response antibodies a Trp-->Leu exchange at position 33 of the heavy chain is primarily responsible for the increased affinity for NP [Allen, D., Simon, T., Sablitzky, F., Rajewsky, K., & Cumano, A. (1988) EMBO J. 7, 1995-2001]. Although 3B62 exhibits one of the highest affinities for NP, it lacks the Trp-->Leu exchange at position 33 of the heavy chain. A variety of stable isotope-labeled Fab analogues of N1G9 and 3B62 have been prepared. Chain-specific resonance assignments were made by recombination of the heavy chains and light chains of the Fab fragments. Binding experiments of a spin-labeled hapten and NOESY experiments have demonstrated that, compared with the environment of the antibody combining site of N1G9, the combination of mutations (including one codon deletion) and the particular D-JH rearrangement in the heavy chain of 3B62 affords a more hydrophobic environment, which is formed by one Tyr residue originating from the light chain and two Tyr residues originating from the heavy chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2003

Iodine-131 Tositumomab: (131)I-anti-B1 antibody, (131)I-tositumomab, anti-CD20 murine monoclonal antibody-I-131, B1, Bexxar, (131)I-anti-B1 antibody, iodine-131 tositumomab, iodine-131 anti-B1 antibody, tositumomab.

Iodine-131 tositumomab [B1, Bexxar , iodine-131 anti-B1 antibody] is a murine antibody conjugated to iodine 131 that recognises and binds to the B1 (CD20) antigen which is found specifically on B lymphocytes. Iodine-131 tositumomab has a dual mechanism of action. It is capable of initiating a host immune response to those B cells to which it is attached, and it also triggers apoptosis in a significant proportion of the cells to which it binds. The product was first discovered by Coulter Corporation, Miami, in collaboration with the Dana-Farber Cancer Institute and the University of Michigan. The spin-off company Coulter Pharmaceutical, upon its formation, obtained worldwide rights to iodine-131 tositumomab. (Coulter Corporation was acquired subsequently by Beckman Instruments in October 1997. The union of the two companies produced Beckman Coulter.) In December 2000, Coulter Pharmaceutical was acquired by, and merged into, Corixa Corporation.Iodine-131 tositumomab is available for licencing in Japan. Corixa Corporation and GlaxoSmithKline signed an agreement to jointly develop and commercialise iodine-131 tositumomab. The total agreement has a potential value of up to $US132 million, plus shared profits and royalties. The two companies will jointly market the antibody in the US following regulatory approval. Corixa Corporation has announced it expects iodine-131 tositumomab to be approved in the US for non-Hodgkin's lymphoma (NHL). Under the terms of the original agreement, GlaxoSmithKline will receive exclusive marketing rights outside the US, excluding Japan. However, an amended agreement between the two companies will allow Corixa Corporation to also market the product outside the US. In February 2003, the European Commission granted iodine-131 tositumomab orphan-drug designation. Corixa Corporation and GlaxoSmithKline also intend to jointly investigate the use of the product in other indications. GlaxoSmithKline may also receive access to second generation anti-CD20 compounds under its agreement with Corixa Corporation. In May 2003, Corixa Corporation entered into an agreement that will see GlaxoSmithKline market Bexxar in Canada. Under the terms of the agreement, Corixa Corporation will manufacture and supply the product to GlaxoSmithKline, who will register, market and sell it in Canada. In October 2001, Amersham PLC, a supplier of medical equipment, announced that its Amersham Health unit had signed a marketing agreement with Corixa Corporation. The agreement allows Amersham Health to market iodine-131 tositumomab in Europe. Corixa Corporation formed agreements with Boehringer Ingelheim Pharma KG and Lonza Biologics to produce the B1 antibody and radiolabelling of the antibody has been contracted out to MDS Nordion.Iodine-131 tositumomab has received orphan drug and fast track designation for the treatment of NHL. Corixa Corporation submitted a Biologics Licence Application (BLA) to the US FDA in 1999, seeking permission to market Bexxar in the US for the treatment of relapsed or refractory, low grade or transformed low grade B-cell NHL. Following a priority review, the US FDA requested that Corixa Corporation reformat certain sections and perform additional analyses of existing data in its BLA. Corixa Corporation and GlaxoSmithKline resubmitted their BLA to the US FDA in September 2000. The BLA was subsequently accepted by the US FDA in November 2000. However, in March 2001, the US FDA requested additional information in its complete review letter to Corixa Corporation and marketing partner GlaxoSmithKline. The two companies submitted data pertaining to the chemistry, manufacturing and controls section of the BLA, and to the majority of the questions regarding the clinical section of the BLA in August 2001. Corixa Corporation and GlaxoSmithKline submitted the remainder of the response to the US FDA in September 2001, following an independent review of clinical trial data. In March 2002, Corixa Corporation received another complete review letter from the US FDA, which stated that additional clinical trials would have to be conducted in order to provide adequate evidence of the safety and clinical benefit of Bexxa. The US FDA also denied Corixa Corporation's request for accelerated approval, stating that the data provided was inadequate to show that Bexxar filled an unmet medical need. Corixa Corporation has met formally with the US FDA but the two were unable to resolve their differences. Corixa Corporation will now file a formal request for dispute resolution under the Food and Drug Administration Modernisation Act. Corixa Corporation also requested a presentation of Bexxa data to the US FDA's scientific advisors. In June 2002, the US FDA granted the company's appeal for additional regulatory review. In December 2002, the US FDA's Oncologic Drugs Advisory Committee agreed that Bexxar has clinical benefit for patients with NHL. In May 2003, Corixa Corporation and GlaxoSmithKline announced that they had fulfilled many of the steps required for US FDA approval, however the US FDA has extended its review of the application for another 3 months. This extension will allow for further refinement of post marketing commitments and package insert language, and to ensure they are consistent with an updated safety database requested by the US FDA and submitted by Corixa Corporation in early April. GlaxoSmithKline was waiting for the outcome of the situation before deciding on marketing plans for Bexxar. Corixa Corporation and GlaxoSmithKline will conduct a head-to-head study of Bexxar and Idec's Zevalin, planned for mid-2003. The trial will likely be one of three phase IV studies that the US FDA requires for accelerated approval of Bexxar. Corixa Corporation initiated its Expanded Access Program for Bexxar in response to requests from physicians and patients for continued access to Bexxar during the period prior to potential US FDA marketing approval.A phase II multicentre trial of Bexxar in combination with CHOP chemotherapy is underway in the US as first-line therapy in patients with intermediate-grade NHL. Corixa Corporation has initiated a phase II trial of iodine-131 tositumomab in combination with cyclophosphamide, vincristine and prednisone for the treatment of previously untreated low-grade NHL. The trial was initiated while the company was preparing its BLA for Bexxar for use as a single agent for relapsed or refractory NHL. Corixa Corporation intends to pursue additional trials to expand the potential use of iodine-131 tositumomab to other indications, including chronic lymphocytic leukaemia. The agent is also in a clinical trial for preparation in autologous bone marrow transplant patients. The trial is designed to test the combination of iodine-131 tositumomab and chemotherapy. The trial began in 1995 and has so far enrolled 40 patients. In addition, a phase II dose-escalation trial has begun at the University of Nebraska for the combined use of iodine-131 tositumomab and chemotherapy as preparation for autologous bone marrow transplant. Corixa Corporation has received an issued US patent covering methods for administering and dosing radioimmunotherapy for the treatment of B-cell lymphomas. The patent covers iodine-131 tositumomab and other anti-CD20 antibodies used to aid in selective tumour targeting. Corixa Corporation has exclusive rights to the patent.A February 2000 media release from GlaxoSmithKline and Corixa Corporation stated that they had been issued a composition patent relating to radiolabelled monoclonal antibodies (including Bexxar) for the treatment of B-cell lymphomas. On 11 September 2001, IDEC announced that it had filed two separate lawsuits. The first lawsuit is against Corixa Corporation and the University of Michigan on six patents pertaining to products and processes related to radioimmunotherapy. They seek a declaration that Zevalin does not infringe Corixa Corporation's issued US patents. The second lawsuit involves two patents relating to cell culture media, and is against GlaxoSmithKline. IDEC's lawsuit in this case, seeks a declaration that its manufacture of Zevalin does not infringe GlaxoSmithKline's issued US patents. Corixa Corporation and GlaxoSmithKline have also filed a complaint for patent infringement against IDEC. These actions however, should have no effect on the regulatory process that Zevalin is completing, or prevent IDEC from launching the drug before iodine-131 tositumomab.A year earlier, in March 2001, the Financial Times reported that Bexxar could reach peak sales of $US120 million. In 1998, Coulter Pharmaceutical received a licencee fee payment of $US34 million from SmithKline Beecham (now GSK) in the fourth quarter of the year, as part of the joint development and commercialisation agreement for Bexxar.

Adjuvants, Immunologic↗
PubMed1981

Comparison of fluorescent-antibody, neutralizing-antibody, and complement-enhanced neutralizing-antibody assays for detection of serum antibody to respiratory syncytial virus.

A comparison of three assays for the detection of serum antibody to respiratory syncytial virus (RSV) was carried out on 47 serum samples obtained sequentially from infants and young children with RSV infection. Neutralizing-antibody (NA) activity was determined by a semimicromethod of plaque reduction. Complement-enhanced NA activity was determined by the addition of guinea pig complement to NA assays. RSV antibody responses in immunoglobulin G, immunoglobulin M, and immunoglobulin A classes were determined by using indirect immunofluorescence techniques for fluorescent-antibody (FAb) assay. Antibody to RSV was detectable by all three techniques as early as 4 days after the onset of illness. At all phases of illness, titers obtained by complement-enhanced NA assays were significantly greater than those obtained by NA or FAb assays (P less than 0.01). RSV-FAb titers determined in the immunoglobulin G class correlated well with those determined by complement-enhanced NA or NA assays. The data suggest that the FAb assay for detection of RSV antibody in serum is somewhat less sensitive but also less laborious and more rapid than NA assays.

Animals↗
PubMed1983

The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody.

The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-epsilon-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.

Animals↗
PubMed2005

Herpesvirus interference with virus-specific antibodies: bridging antibodies, internalizing antibodies, and hiding from antibodies.

Herpesviruses have developed different tools to thwart efficient antibody-dependent neutralisation and lysis of virions and elimination of infected cells. This overview will briefly summarize different of these tools, including (i) viral Fc receptors and the resulting process of antibody bridging, (ii) internalization of individual viral proteins and clustered antibody-antigen complexes from the plasma membrane of infected cells, and (iii) directed egress of virus particles to sites of intimate cell-cell contact that are difficult to access for antibodies.

Animals↗
PubMed2005

Fucose removal from complex-type oligosaccharide enhances the antibody-dependent cellular cytotoxicity of single-gene-encoded antibody comprising a single-chain antibody linked the antibody constant region.

Fucose removal from complex-type oligosaccharide of human IgG1-type antibody results in a great enhancement of antibody-dependent cellular cytotoxicity (ADCC). The aim of this study was to clarify the effect of fucose removal on effector functions of a single-gene-encoded antibody with an scFv used as the binding domain. We generated both a fucose-negative anti-tumor associated glycoprotein (TAG)-72 scFv-Fc using alpha-1,6-fucosyltransferase knock-out CHO cells and a highly fucosylated scFv-Fc from parental CHO cells. Expression, assembly and antigen binding activity of the scFv-Fcs were not influenced by fucose removal. The scFv-Fc lacking fucose exhibited significantly more potent FcgammaRIIIa binding and ADCC compared to highly fucosylated scFv-Fc. These results prove that ADCC enhancement by fucose-removal is effective in not only whole IgG1, but also scFv-Fc, and thus increases the potential of Fc-fusion proteins as therapeutic candidates.

Animals↗
PubMed2006

Fucose removal from complex-type oligosaccharide enhances the antibody-dependent cellular cytotoxicity of single-gene-encoded bispecific antibody comprising of two single-chain antibodies linked to the antibody constant region.

Bispecific antibodies (bsAbs) have the potential to extend binding selectivity, increase avidity and exert potent cytotoxicity due to the combination of dual specificities. scFv2-Fc type of single-gene-encoded bispecific antibody, composed of two different single-chain Fvs and an Fc, has been reported to be capable of binding to different antigens. The aim of this study was to determine the effect of fucose removal on effector functions of scFv2-Fc since fucose depletion from oligosaccharide of human IgG1 and scFv-Fc results in significant enhancement of ADCC. We generated novel single-gene-encoded bsAb with dual specificity against tumor associated glycoprotein (TAG)-72 and MUC1 mucin as fucose-negative scFv2-Fc from alpha-1,6-fucosyltransferase knock-out CHO cells and a highly fucosylated scFv2-Fc comparator from parental CHO cells. Expression, assembly and the antigen-binding activity of the scFv2-Fc were not influenced by removal of fucose. The fucose negative scFv2-Fc bound with higher avidity to FcgammaRIIIa and enhanced ADCC compared to the highly fucosylated scFv2-Fc. These results demonstrate that ADCC-enhancement by removal of fucose is effective in not only whole IgG1 and scFv-Fc, but also scFv2-Fc targeting two different antigens, and thus increases the potential of fucose-negative scFv2-Fcs as novel therapeutic candidates.

Animals↗
PubMed1988

Neutralizing antibodies in relation to antibody-dependent cellular cytotoxicity-inducing antibodies against human immunodeficiency virus type I.

The presence of neutralizing antibodies against human immunodeficiency virus type 1 (HIV-1) was investigated in sera from 73 HIV-1 seropositive subjects at different clinical stages. Virus neutralization was measured as survival of MT-4 cells in a 6-7 day microassay. Sixty-three sera were also tested for antibodies inducing virus-specific antibody-dependent cellular cytotoxicity (ADCC). Neutralizing antibodies were found in 59% of sera tested, the positivity rate being 50% (9/17) in asymptomatic subjects, 67% (12/18) in patients with persistent generalized lymphadenopathy (PGL) and 54% (14/26) in AIDS patients (not significant differences). ADCC antibodies were present in 43% of the sera. Neutralizing antibodies and ADCC-inducing antibodies were found simultaneously in 35% (22/63) of the sera. Neutralizing antibodies alone were found in 22% (14/63) and ADCC antibodies alone in 6% (4/63) of the sera tested. Thirty-seven per cent (23/63) of the sera were negative for both types of antibodies, 62% of the sera with neutralizing antibodies also had ADCC inducing antibodies and 85% of the sera with ADCC antibodies had neutralizing antibodies. The titres of ADCC antibodies were higher than those of neutralizing antibodies. Thus, the presence of ADCC antibodies was related to the presence of neutralizing antibodies, but no correlation was found between the titres of these antibodies in sera positive for both activities.

AIDS-Related Complex↗
PubMed1983

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.

Animals↗
PubMed1965

ANTIBODY SYNTHESIS AT THE CELLULAR LEVEL. ANTIBODY-INDUCED SUPPRESSION OF 19S AND 7S ANTIBODY RESPONSE.

The suppressing activity of passively transferred antibodies on antibody synthesis against sheep red cells was investigated at the cellular level by the agar-plaque technique developed by Jerne. Humoral antibodies injected prior to the antigen suppressed the appearance of plaque-forming spleen cells producing 19S antibodies completely. Antibodies given during the first 4 days after antigen injection also showed such action, but only after a latency period of 40 hours. The inhibiting efficiency of 7S antibodies was about 100 to 200 times greater than that of 19S antibodies. The results support the conclusion that humoral antibodies inhibit the immune response by removing the stimulus for the proliferation of the antibody producing cells and not by directly depressing antibody synthesis in already committed cells. Passively transferred antibodies inhibited the 7S response if given prior to, or 24 hours after the antigen injection, in analogy with previous results concerning 19S response. In contrast to these previous results on 19S synthesis, antibody transfer had no detectable effect during the early exponential phase of 7S production (5 to 7 days after antigen injection). Only limited inhibition was observed 3 days after the antigen. One possible explanation of this difference is that 7S-producing cells do not divide, or divide at a slow rate. Antigen injection would stimulate the proliferation of 19S-producing cells. Subsequently these would switch to the synthesis of 7S antibodies. These would inhibit the initiation of new 19S-producing cells by combining with the antigen. They would thus suppress the recruitment of their own precursors. A steady state of 7S antibody production by cells with a long lifetime would be the result. This hypothesis ascribes an important regulatory function to 7S antibodies. They would be parts of a feed-back system preventing excessive cell multiplication in response to a single antigen.

Animals↗
PubMed1987

T cell activation by cross-linking anti-CD3 antibodies with second anti-T cell antibodies: dual antibody cross-linking mimics physical monocyte interaction.

The anti-CD3 antibody BMA030 (IgG2a isotype) induces T cell activation and proliferation if an interaction with monocytes is provided. In contrast to other anti-CD3 antibodies, it is unable to induce interleukin (IL)2 responsiveness through cross-linking by plastic-bound goat anti mouse Ig antibodies (panning). Cross-linking BMA030 with a second anti-T cell antibody is, however, able to induce IL 2 responsiveness in monocyte-depleted T cell cultures. In this report we show that a large number of different antibodies are suitable for this dual antibody stimulation, and that the extent of proliferation corresponds to the percentage of T cells expressing the respective T cell antigen. Proliferation induced by low concentrations (0.1-1 ng/ml) of other anti-CD3 antibodies requires also cross-linking with second anti-T cell antibodies. The proliferative response of monocyte-depleted T cells to two cross-linked anti-T cell antibodies plus added IL 2 is of the same magnitude as the one induced by anti-CD3 antibodies plus monocytes. On the other hand, if monocytes are present, soluble anti-CD2, -CD4, -CD8, -LFA-1 antibodies (IgG1 or F(ab')2 fragments) can inhibit OKT3 or BMA030-induced T cell activation. Anti-CD6 antibodies do not interfere with this monocyte-dependent T cell stimulation. We conclude that dual antibody stimulation mimics the physical contact of T cells with monocyte membranes, where the T cell receptor CD3 complex is cross-linked with neighboring structures (mainly so-called adhesion molecules) through the interaction with respective counter-structures on monocyte membranes. Dual antibody cross-linking bypasses this interaction and can be used to stimulate IL 2 responsiveness of antibody-defined T cells.

Adult↗
PubMed1994

Combined analysis of islet cell antibodies that cross-react with mouse pancreas, antibodies to the M(r) 64,000 islet protein, and antibodies to glutamate decarboxylase in type I diabetic patients.

OBJECTIVE: A combined analysis of whether islet cell autoantibodies (ICAs) are cross-reactive with mouse pancreas, with glutamate decarboxylase (GAD) antibodies, and with 64K antibodies was performed in a large sample of recently diagnosed type I diabetic patients. The disappearance rates of these different autoantibodies were compared in some patients after onset of the disease. The aims were to determine patterns in GAD/64K antibodies with regard to cross-species reaction of ICA and to assess whether GAD could contribute to ICA positivity in mouse and human pancreases and whether the simultaneous search for all the antibody specificities enhances the detection of autoimmune stigma. RESEARCH DESIGN AND METHODS: ICA detected by immunofluorescence in human and mouse pancreases, antibodies immunoprecipitating the 64K rat islet antigen, and antibodies immunotrapping brain GAD activity were quantified at diagnosis of diabetes in 95 patients and in sequential samples during 1 year after diagnosis in 13 patients. The contribution of GAD to ICA positivity in mouse and human pancreases was evaluated by the analysis of correlations between tests and by the ability of brain homogenate to block ICA reactivity in pancreases from both species. RESULTS: ICAs were detected in human pancreases in sera from 63 (66%) patients, among which 61% bound also to a mouse pancreas. GAD and 64K antibodies were strongly correlated (P < 0.0001) and were detected in 69 and 73% of the patients, respectively. All but two patients with ICA in human pancreas also displayed either ICA in mouse pancreas or GAD/64K antibodies. Among 32 patients without ICA in human pancreas, 54% displayed either GAD/64K antibodies or ICA in mouse pancreas. Only 16% of the patients displayed neither ICA nor GAD/64K antibodies. A correlation (P < 0.005) was found between ICA in human and mouse pancreases. GAD or 64K antibodies were strongly correlated with ICA in human pancreas (P < 0.0001), but not with ICA in mouse pancreas. After preincubation of six sera with GAD-containing brain homogenate, ICA titers were unaffected in mouse pancreas but reduced in human pancreas. ICA titers in mouse pancreas were decreased after 3 months (P < 0.01) in diabetic patients, contrasting with the stability of ICA in human pancreas and GAD antibodies by 1 year after diagnosis. CONCLUSIONS: According to cross-species reaction, we confirm the heterogeneity of ICA in a large series of type I diabetic patients, ICAs that cross-reacted with mouse pancreas being more frequent than ICAs without cross-species reactivity. GAD and 64K antibodies were also present in a majority of patients. The simultaneous search for all the antibody specificities enhances the detection of autoimmune stigma so that only a few patients did not display any autoantibody at diagnosis. GAD is not the target of ICAs in mouse pancreas, whereas GAD accounts for ICA positivity in human pancreas. The conclusion that ICAs in mouse pancreas are not GAD-reactive is reinforced by the fact that they are more transient after onset of diabetes than are GAD antibodies or the complex mixture of ICAs in human pancreas.

Adolescent↗