Antigen-binding Capacity Research Articles

Mass sensitivity of acoustic plate mode liquid sensors on ZX–LiNbO3

Acoustic plate mode (APM) devices have been used as detectors in liquid environments. One of the primary detection mechanisms in such devices is mass loading. In the present work, the potential of APMs as mass sensitive biosensors is evaluated using five APM devices on ZX–LiNbO3 with frequencies of operation ranging from 13 to 42 MHz. Mass sensitivity is measured as a function of frequency and is compared to data predicted by perturbation theory. Discussions are given relative to typical antigen binding capacities of derivatized crystal surfaces.

Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli

The ability of immunoglobulin Fab and single chain (ScFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates as drug delivery systems and imaging tools. This study investigates the use of single chain antibody fragment bacterial expression vectors as a possible strategy for the production of these molecules. We have modified the pSW1-VHD1.3-VKD1.3-TAG1 vector [Ward et al. (1989) Nature 341, 544–546] which originally, when expressed in E. coli, produced an Fab fragment. In an effort to improve the affinty of the parent vector product a novel single chain antibody construct which encodes a protein with anti-P. aeruginosa activity was generated using a 14 amino acid linker [Chaudhary et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1066–1070]. In addition to the heavy and light chain variable domain genes, our construct also contained the light chain kappa constant domain gene to aid purification of the fragments. To underline this difference from the conventional ScFv fragment we have described this protein as a ScAb. The ScAb generated had an antigen binding capacity similar to the parent anti- P. aeruginosa antibody but was superior to the recombinant anti- P. aeruginosa Fab fragment. On HPLC and non-denaturing gel electrophoresis analysis, the ScAb was found to exist in multimeric forms while the Fab fragment existed only as a single unit. Dimeric ScAb had a similar antigen binding profile to the parent antibody.

The stabilization of a human IgM monoclonal antibody with poly(vinylpyrrolidone).

An IgM anti-group B Streptococcus monoclonal antibody (4B9) was found to undergo irreversible heat-induced aggregation at 50 degrees C. A variety of excipients was tested for their ability to inhibit antibody aggregation. The amount of 4B9 aggregation, which was determined by analysis on a size-exclusion HPLC, was significantly reduced in the presence of low concentrations [between 0.1 and 1.0% (w/v)] of poly(vinylpyrrolidone) (PVP) molecules ranging in molecular weight from 10 to 40 kDa. When the PVP concentration was greater than 1.0%, antibody aggregation was enhanced, and with the highest molecular weight PVP, antibody precipitation occurred. HPLC was used to show that more PVP was associated with the 4B9 at 50 degrees C than at 25 degrees C. Differential scanning calorimetry revealed that PVP concentrations greater than 2.0% decreased the antibody thermal transition temperature. Enzyme-linked immunosorbent assays were used to assess the effects of PVP on the antigen binding capacity of 4B9 and on 4B9 quantitation. At 4 degrees C, PVP solutions of up to 5.0% had no effect on either 4B9 quantitation or antigen binding. At 50 degrees C, however, less 4B9 was detected in the 5.0% PVP solution. The heat stabilization of the 4B9 antibody by low concentrations of PVP can be explained by a weak binding of PVP to the native protein. The PVP may sterically interfere with protein-protein interactions, thus reducing aggregation. Higher concentrations of PVP lead to protein aggregation and precipitation, probably by a volume-exclusion mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Assembly of monoclonal antibodies with IgG1 and IgA heavy chain domains in transgenic tobacco plants

The genes encoding the heavy and light chains of a murine monoclonal antibody (mAb Guy's 13) have been cloned and expressed in Nicotiana tabacum. Transgenic plants have been regenerated that secrete full-length Guy's 13 antibody. By manipulation of the heavy chain gene sequence, constant region domains from an immunoglobulin alpha heavy chain have been introduced, and plants secreting Guy's 13 mAb with chimeric gamma/alpha heavy chains have also been produced. For each plant antibody, light and heavy chains have been detected by Western blot analysis and the fidelity of assembly confirmed by demonstrating that the antibody is fully functional, by antigen binding studies. Furthermore, the plant antibodies retained the ability to aggregate streptococci, which confirms that the bivalent antigen-binding capacity of the full length antibodies is intact. The results demonstrate that IgA as well as IgG class antibodies can be assembled correctly in tobacco plants and suggest that transgenic plants may be suitable for high-level expression of more complex genetically engineered immunoglobulin molecules. Since mAb Guy's 13 prevents streptococcal colonization in humans, transgenic plant technology may have therapeutic applications.

Dextran modified gold surfaces for surface plasmon resonance sensors: immunoreactivity of immobilized antibodies and antibody-surface interaction studies

Recently, the modification of gold surfaces with covalently bound carboxylated dextran was described, for use in biospecific interaction studies in an analytical system based on surface plasmon resonance (SPR). In this study, the antigen binding immunoreactivity of antibodies immobilized on the modified surfaces was investigated with the SPR-based system. Four different monoclonal antibodies specific against the antigens β 2-microglobulin, luteinizing hormone, human transferrin, and immunoglobulin E, were covalently attached by amine coupling to the dextran layer and the antigen binding capacities were evaluated for different amounts of bound antibody. High immunoreactivities were obtained with molar activities ranging from 0.6 to 1.5 over a wide range of surface concentrations. Interaction studies between carboxymethylated dextran surfaces and antibodies showed that, in comparison with unmodified gold surfaces, the modified surface had low nonspecific adsorption and high capacity for antibody immobilization.

Immunological properties of a radioiodinated monoclonal antibody and its F(ab')2mu fragments directed against the polymorphic epithelial mucin of human breast cancer.

The purification of the IgM monoclonal antibody 436 against a breast tumor antigen from mouse ascitic fluid is reported. The purified immunoglobulin was radioiodinated and the resulting product assessed for its binding capacity and binding specificity. Purified IgM-436 served for F(ab')2 mu preparation which was tested for its antigen binding capacity. Radioiodinated IgM-436 and its F(ab')2 mu retained their immunological activity which was never lower than those of the corresponding cold products.

Transient suppression of a secondary humoral response in rats is evoked by lithium-pilocarpine-induced limbic seizures

Several experiments were designed to evaluate a secondary humoral response following limbic seizures. After baseline antigen binding capacity (ABC) had been determined for the primary response, a second subcutaneous injection of the antigen (human serum albumin) was accompanied by an injection of either lithium (3 mEq/kg)-pilocarpine (30 mg/kg) or one of two comparator treatments: metrazol (30 mg/kg) or cyclophosphamide (50 mg/kg); other rats served as drug controls. Only the groups that received the lithium-pilocarpine (status epilepticus) or cyclophosphamide (no seizure) displayed significant immunosuppression after 5 but not 10 days. The results support the hypothesis that seizure activity within the amygdaloid-hippocampal complex modulates immunocompetence through corticotropin mechanisms.

Characterization of monoclonal antibodies physically adsorbed onto polystyrene latex particles

The physicochemical and immunochemical properties of monoclonal antibodies (MAbs), adsorbed onto polystyrene latex particles, have been investigated. Both native and pH 2 pretreated MAbs were compared before and after immobilization. It was found that the antigen binding capacity of the immobilized, acidic pretreated MAbs was significantly higher than for the immobilized, native IgG molecules. This enhanced antigen binding capacity appeared to be due to an improved molecular orientation following adsorption of the monomeric, pH 2 treated IgG fraction. Additionally, experiments using F(ab′) 2 fragments demonstrated that the Fc portion of the MAb molecule is of major importance for achieving the enhanced binding capacity. Binding studies showed that the (apparent) affinity of native and pH 2 pretreated MAbs were similar; the K a values of the immobilized MAbs were higher than those of MAbs in solution.

Purification of MPB70 and production of specific monoclonal antibodies.

MPB70 is a protein secreted into the culture filtrate of Mycobacterium bovis BCG (substrain Tokyo 172), which is able to induce a delayed-type hypersensitivity (DTH) skin reaction in guinea pigs immunized with BCG-Tokyo. By high-pressure chromatofocusing and size-exclusion high performance liquid chromatography, a further purified MPB70 protein was obtained, which was visualized as a single band with a molecular mass of 22 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis. A series of hybridoma cell lines that produced monoclonal antibodies (MAbs) against the purified MPB70 protein was prepared, and three MAbs, Bov-1, Bov-2, and Bov-3, with strong antigen-binding capacities were established. Bov-1 was the most potent MAb among them and binds to only a 22 kDa protein band in culture filtrates of M. bovis, but not to bands in those of M. tuberculosis by Western immunoblotting analysis, suggesting that Bov-1 recognize different epitope of MPB70 from MAbs that have been shown previously to recognize several species of molecules in culture filtrates of M. bovis. The purified MPB70 protein elicited a strong DTH skin reaction in guinea pigs sensitized with BCG-Tokyo vaccine. Bov-1 had no inhibitory effect on generation of the DTH skin reaction, showing that MAb bound to an epitope distinct from that inducing the skin reaction. All of the three MAbs were specific to MPB70 and each recognized a different epitope on MPB70. MPB70 was not detected in the culture filtrate of M. tuberculosis H37Rv. Thus, these MAbs may be useful for detecting MPB70 in studies on discriminating infection with M. bovis in domestic animals or in distinguishing vaccination with BCG-Tokyo from other mycobacterial infections in humans.

Infantile stimulation produces mild enhancement in a primary humoral response of adult albino rats.

Rat pups from 12 litters were handled daily, once every three days, or never touched between postnatal Days 5 and 20. At 100 days of age, 20 days after inoculation with human serum albumin, trunk blood was obtained to measure the antigen binding capacity (ABC). Rats handled as infants displayed relative immunofacilitation compared to those that had not been handled; the effect explained 7% of the variance in ABC values. In comparison, sex differences explained 14% of the variance. The results support the hypothesis that early disruptions in mother-infant bonding increase the likelihood of developing allergic responses following exposure to novel antigens during adulthood.

Characterization of a recombinant chimeric plasminogen activator composed of a fibrin fragment-D-dimer-specific humanized monoclonal antibody and a truncated single-chain urokinase.

A recombinant chimeric plasminogen activator, MA-15C5Hu/scu-PA-32k, composed of a humanized fibrin fragment-D-dimer-specific monoclonal antibody (MA-15C5Hu) and a recombinant low-molecular-mass single-chain urokinase-type plasminogen activator, comprising amino acids Leu144-Leu411 (scu-PA-32k), was produced by cotransfecting Chinese hamster ovary (CHO) cells with the cDNA encoding the MA-15C5Hu light-chain sequence and the cDNA encoding the MA-15C5Hu heavy-chain sequence fused with the cDNA encoding scu-PA-32k. Purified MA-15C5Hu/scu-PA-32k migrated as a 215-kDa band on non-reducing SDS/PAGE, which is consistent with a molecule composed of one antibody and two scu-PA-32k moieties. However, the chimera was obtained as a mixture of single-chain u-PA-32k (37%) and amidolytically inactive (50%) and active (13%) two-chain u-PA-32k, the latter of which was removed by immunoadsorption on a monoclonal antibody specific for two-chain urokinase. The fragment-D-dimer affinity and enzymatic properties of MA-15CHu/scu-PA-32k were similar to those of MA-15C5Hu or of scu-PA-32k. In an in vitro system composed of a 125I-fibrin-labeled human plasma clot submerged in citrated human plasma, MA-15C5Hu/scu-PA-32k had a 12-fold higher fibrinolytic potency than scu-PA-32k: 50% lysis in 2 h required 0.43 +/- 0.12 micrograms u-PA-32k equivalent of the chimera/ml versus 5.4 +/- 0.3 micrograms/ml of scu-PA-32k (mean +/- SEM, n = 4). Addition of purified fibrin fragment-D dimer reduced the fibrinolytic potency of MA-15C5Hu/scu-PA-32k in a concentration-dependent way, indicating that the increased potency is the result of antibody targeting. Thus, a recombinant humanized antifibrin antibody/u-PA chimera has been obtained in which only the variable domains of the antibody moiety are of non-human origin. The chimera has intact antigen-binding capacity, u-PA enzymatic activity and a significantly increased fibrinolytic potency in a plasma medium in vitro.

Immobilization of monoclonal antibodies for affinity chromatography using a chelating peptide

A procedure was developed for oriented immobilization of monoclonal antibodies on a solid support. The technique involves the specific oligosaccharide-directed covalent modification of the monoclonal antibody (mAb) with the chelating peptide, Lys-Gly-(His) 6, in conjunction with immobilized metal ion affinity chromatography. Chelating peptide-mAb conjugates with a molar ratio of 2.2 retained full antigen binding activity. On immobilization of the modified antiboides on a nickel affinity resin, The molar antigen binding ratio was 1.4. The high antigen binding capacity is indicative of oriented immobilization providing maximum access for the antigen. The described method can be used for the preparation of high-capacity immunosorbents for affinity chromatography and it is applicable for all immunoglobulin classes.

Coupling of antibody-binding fragments to solid-phase supports: site-directed binding of F(ab') 2 fragments

A method to covalently bind antibody fragments, via their carboxyl termini to solid supports, is presented. The strategy involves: (1) reversibly blocking all the accessible carboxyl groups on the antibody molecule with phenylhydrazine, (2) exposing the carboxyl termini of the fragment by enzymatic digestion with pepsin and (3) subsequently coupling the fragment to an appropriate support. Experiments with an anti-bovine serum albumin monoclonal antibody and C-14 phenylhydrazine revealed that the blocking step was nearly completely reversible with a dilute solution of FeCl 3. Radioiodinated blocked F(ab') 2 fragments were then coupled to an amino-functionalized Sepharose 4B column, and characterized as to their coupling capacity (mass of protein coupled/ml of bead), and antigen-binding activity. The coupling cf the blocked fragments was found to be 12%, half the coupling efficiency of unmodified radioiodinated F(ab') 2. The antigen-binding capacity (mol antigen bound per mol antibody coupled) for the blocked F(ab') 2, on the other hand, was found to be 1.9, which was approx. 3.5-times greater than for the unmodified F(ab') 2. Comparisons with other conventional coupling techniques were also made. These preliminary studies suggest that this technique can provide one with the means to obtain more uniform and active populations of immobilized antibody fragments.

The influence of sucrose, dextran, and hydroxypropyl-beta-cyclodextrin as lyoprotectants for a freeze-dried mouse IgG2a monoclonal antibody (MN12).

The influence of lyophilization on the stability of a monoclonal antibody (MN12) was investigated. MN12 was freeze-dried in different formulations [without lyoprotectant or in the presence of sucrose, dextran, or hydroxypropyl-beta-cyclodextrin (HP beta CD)] and under varying conditions (with or without secondary drying). Subsequently, the monoclonal antibody was stored for 18 or 32 days at various temperatures (4, 37, or 56 degrees C). For comparison, solutions of MN12 were stored under the same conditions. Regardless of the lyoprotectant used, precipitation and a concomitant reduction of the antigen-binding capacity by about 10% were observed upon reconstitution of lyophilized MN12. HP beta CD proved to be the most effective stabilizer to prevent degradation of lyophilized MN12 during storage. Compared with MN12 solutions, HP beta CD-containing lyophilized MN12 cakes were more resistant to heat-induced charge alterations and loss of antigen-binding capacity.

Immunoassay in the presence of water-soluble polymers

To overcome the adverse consequences of antibody immobilization (impairment of the antigen-binding parameters) the interaction of a number of antigens with both soluble and covalently immobilized antibodies in the presence of water-soluble polymers has been investigated. Incorporation of some water-soluble polymers (Ficoll, Dextran) into the assay mixture was shown to substantially increase the antigen-binding capacity of both soluble and immobilized antibodies. The use of dextran T70 enhanced the sensitivity of competitive radioimmunoassays for CEA and β 2-microglobulin 3–4-fold. This procedure may be also applied to shorten the incubation period when performing an assay.

The effects of incubation temperature and coating procedure on the measurement of antibodies to cardiolipin

Many laboratories have established ELISAs for the routine detection of anti-cardiolipin antibodies (ACA). Earlier studies had indicated that assay incubation at 37°C may interfere with the antigen binding capacity of these antibodies. We have reexamined this phenomenon by comparing ACA titers obtained when incubations are performed at either 37°C or at room temperature (RT). In addition, the effect of coating antigen in aqueous or organic solution was compared. The sera tested included a set of recognized ACA standards and samples from 19 patients with SLE, two with primary anti-phospholipid syndrome, 71 patients with a variety of autoimmune and non-autoimmune disorders and 210 blood bank controls. The results show that while some sera do perform better under either incubation temperature there was no correlation between ACA titers and incubation temperature on a population basis either for IgG or IgM isotypes. This was seen both for positive standards and patient sera. For IgG ACA a similar phenomenon was seen if the microplates were coated with cardiolipin either in sodium carbonate or ethanol. For IgM ACA there was a significant increase in ACA titers at RT when cardiolipin was coated in ethanol. The data suggest that for most sera neither the antigen coating medium nor the assay incubation temperature are important variables in the determination of IgG ACA. Factors contributing to the influence of either variable in individual sera could not be identified.

Autoimmunity in non-human primates: the role of major histocompatibility complex and T cells, and implications for therapy

Two autoimmune disease models were studied in rhesus monkeys: type II collagen-induced arthritis (CIA) and experimental allergic encephalomyelitis (EAE). Unrelated outbred animals were used in these studies. In both models disease resistant and susceptible individuals could be identified. Susceptibility correlated with in vitro cellular responsiveness to antigen in the CIA model. In both models resistant as well as susceptible individuals developed a humoral response to the inducing antigen. However, there is an indication that IgM antibodies play a crucial role in the induction of CIA. No clear association between major histocompatibility complex (MHC) type and disease incidence was found although a higher frequency of a certain DR type was observed in EAE susceptible monkeys. It is likely that both the antigen binding capacity of the MHC class II molecules and the T-cell repertoire play an important role in determining whether disease will develop or not.

Effect of antigen size on optimal ligand density of immobilized antibodies for a high-performance liquid chromatographic support

The antigen binding capacities for purified polyclonal antiboides immobilized onto a silica-based high-perforamnce liquid chromatographic (HPLC) affinity support are described for three serum proteins over a range of antibody ligand densities. The rate of decline in the specific activity of the immobilized antibodies with respect to increasing liquid density was found to increase with the molecular weights of the antigens. The antibodies used were purified from whole antiserum using high-performance affinity chromatography and were examined using HPLC on an SCX stationary phase. Conditions are also described for efficient coupling of the ligand to the support.

Fixation of cryo-sections under HIV-1 inactivating conditions: integrity of antigen binding sites and cell surface antigens

Cryostat-sections of biopsies from HIV-infected patients or HIV/SIV-infected experimental animals pose a biohazard risk to laboratory workers. The objective of this study was to select a procedure that appropriately fixes cryo-sections and reduces the risk of HIV-1 infectivity. This inactivation procedure should preserve antigen binding capacity of host-produced antibodies and the antigenic structure of epitopes present in these tissues, while retaining sufficient morphologic detail. We tested the effect of seven different established fixation-inactivation procedures for HIV-1 on the detection of specific antibodies and membrane markers, compared to acetone fixation as a reference. Frozen sections of spleens from mice immunized with trinitrophenyl (TNP)-Ficoll were incubated with TNP-alkaline phosphatase to detect specific antibody-forming cells and follicular immune complexes containing TNP-specific antibodies. In addition, sections were stained with monoclonal antibodies directed against IgM (187-1), T-cells (anti Thy-1), and marginal metallophilic macrophages (MOMA-1). Five procedures proved useful as they gave results similar to regular acetone fixation. In contrast, two procedures with a methanol-containing fixative obscured both antigen binding sites and membrane antigens. Subsequently, these five selected procedures were tested on glass slide preparations of HIV-1 infected cell lines, expressing HIV-1 determinants defined by monoclonal antibodies. Finally, the procedures were tested on sections of an HIV-1 infected human lymph node, for detection of HIV-specific B-cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex differences in primary humoral responses of albino rats to human serum albumin

Primary humoral responses to human serum albumin, with or without concomitant Freund's adjuvant, were determined for over 300 male and female Wistar albino rats. Groups of female rats always displayed significantly more antibody production than males. Without adjuvant only 70% of the males responded compared to 99% of the females. When the antigen was accompanied by adjuvant, more than 95% of both sexes responded. Whereas without adjuvant the female response was only twice that of the males, adjuvant facilitated the response to a factor of 10 greater than the males. Both the gender and adjuvant effect each accounted for about 16% of the variance in the measurements of antigen binding capacity. Post-weaning castration did not stimulate the gender differences; there were no group differences in ABC at the extremes of estrus-related ambulatory cycles.