Enhanced production of antibody with specific antigen.

On inoculation of nonspecific stimulator of immunity (NSI), prepared from Mycobacterium phlei (M. phlei), simultaneously along with sheep pox virus (SPV) in sheep, the recipient has exhibited appreciable level of SPV specific antibody as early as on 10th day which reached at peak level on 20th day and remained unaltered on 30th day of postimmunisation as evinced by serum neutralisation test (SNT), enzyme linked immunosorbant assay (ELISA) indirect, fluorescent antibody technique (FAT) indirect, counter immunoelectrophoresis (CIEP) and finally by virulent SPV challenge. On the contrary, sheep, when immunised with SPV only could not produce appreciable level of antibody on 10th day but did so on 20th day of inoculation. SPV and NSI immunised sheep produced enhanced protection against virulent SPV challenge in comparison with sheep immunised with SPV only. Healthy control sheep, however, could not resist challenge.     

Effects of early antigen exposure through lactation on later specific antibody responses in mice

This study characterized totally the effects of early Ag exposure by the suckling route on later specific antibody responses. When mother mice of BALB/c or C57BL/6 strains were injected with deaggregated human gamma-globulin (HGG) immediately after delivery, total amounts of HGG in sera of offspring increased until 2 wk of age. The catabolism of transferred HGG was extremely slow and the half-life was about 3 wk in both strains. Hence, small amounts of Ag in mothers, 0.5 micrograms in C57BL/6 and 50 micrograms in BALB/c, could tolerize their offspring effectively. As these were minimum tolerogenic doses, the strain difference in ease of tolerance induction is apparent already during suckling. The study on timing dependent effects of HGG-specific antiserum on tolerance induction by mothers given 50 micrograms HGG demonstrated that the tolerance is achieved within the 1st wk of lactation in C57BL/6 offspring, but not in BALB/c offspring, and the restoration from the tolerance needs more than 6 wk under circumstances, supposedly, without free Ag. Whereas the tolerance was induced in a dose-dependent manner in each class of antibody, the dissociation of tolerant states between IgM, IgG, and IgE antibody classes was found in C57BL/6 offspring. It is interesting that C57BL/6 offspring were sensitized weakly, but significantly, by mothers given subtolerogenic doses. However, this was not apparent in BALB/c. Thus, the Ag dose and the animal strain are related closely to the consequences of this Ag exposure. The aging of suckling mice within the first 2 wk of life or immunomodulators administered early in life did not seriously affect the consequences. Studies on a cellular basis showed that the tolerance is caused by the selective defect in helper T cell function and the suppressor cell activity is not associated with the mechanisms. This contrasts with other models of oral tolerance.     

Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.     

Linear PADRE T helper epitope and carbohydrate B cell epitope conjugates induce specific high titer IgG antibody responses

Linear carbohydrate-peptide constructs based on the 13 amino acid nonnatural pan DR epitope (PADRE) and carbohydrate B cell epitopes are demonstrated to be potent immunogens. These data support our belief that PADRE should be considered as an alternative to more complex carriers for use in prophylaxis and therapeutic vaccines. Two model carbohydrate-PADRE glycoconjugates were used to demonstrate that PADRE could effectively provide T cell help for carbohydrate-specific Ab responses. Conjugates of PADRE covalently linked to the human milk oligosaccharide, lacto-N-fucopentose II or a dodecasaccharide derived from Salmonella typhimurium O-Ag induced high titer IgG Ab responses in mice, which were comparable to glycoconjugates employing human serum albumin (HSA) as the carrier protein. Different adjuvants, in combination with PADRE conjugates, allowed for the modulation of the isotype profile with alum supporting an IgG1 profile; QS-21 an IgG2a, 2b profile, while an alum/QS-21 mixture generated a balanced IgG1/IgG2b isotype profile. As defined by binding to synthetic glycoconjugates, dodecasaccharide-specific Abs exhibited fine specificity similar to protective polyclonal Ab responses previously reported for dodecasaccharide-protein conjugates. The same Abs bound to intact S. typhimurium cells, suggesting that biologically relevant specificities were produced. The affinity of the dodecasaccharide-specific Abs was further shown to be comparable to that of a well-characterized, high affinity monoclonal anti-carbohydrate Ab recognizing the same epitope.     

Investigating specific antigen/antibody binding with the atomic force microscope

The aim of this work is to detect immune complexes without any kind of labelling of each of the immunological species, with a view to create a very sensitive biosensor. This is achieved by using the atomic force microscopy. We have proceeded by imaging the antibody (anti-rabbit IgG) or anti-rabbit IgG moieties adsorbed onto mica surface, before and after incubation of two kinds of antigens: a specific (rabbit IgG) and a non-specific one (sheep IgG). The analysis using the height histograms reveals many interesting features. We propose a general framework for interpreting these analysis, which enables the discrimination between specific and non-specific complexes.     

Desensitization of delayed-type hypersensitivity by antigen and specific antibody.

The role of antibody in the desensitization of delayed-type hypersnsitivity (DTH) to dinitrophenylated bovine gammaglobulin (DNP-BGG) was studied in rats. Rats sensitized by a subcutaneous injection of DNP₃₂-BGG in Freund's complete adjuvant (FCA) were desensitized 14 days later with various doses of DNP₃₂-BGG injected intravenously. It was found that only certain doses (100–500 μg) of DNP-BGG effectively desensitized, antigen doses outside this optimum range being ineffective in suppressing DTH. In adoptive cell transfer experiments, it was shown that sensitized peritoneal cells incubated with optimum doses of the antigen in the presence of specific antiserum in vitro failed to transfer the delayed response to normal recipients, whereas the treatment of the sensitized cells with the antigen or with the antiserum separately did not impair the ability of these cells to transfer DTH. The effect of desensitization is specific and is not permanent. The DTH reappears 3–4 wk after desensitizing injection.     

Biochemical properties of conjugates of urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin

Equimolar mixtures of recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody (MA-15C5) directed against fragment-D dimer of human cross-linked fibrin were conjugated, using the cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (PySSProSu). The conjugate (rscu-PA/MA-15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment-D dimer with a yield of 42 +/- 15% (mean +/- SD, n = 3), contained an average of 1.2 +/- 0.3 IgG molecules/rscu-PA molecule. On non-reduced SDS/PAGE it migrated as a main band with apparent Mr of 200,000. Specific amidolytic activities expressed/mass of u-PA were less than 250 IU/mg for rscu-PA/MA-15C5 and rscu-PA, 140,000 +/- 13,000 IU/mg and 100,000 +/- 17,000 IU/mg for their plasmin-generated two chain derivatives rtcu-PA/MA-15C5 and rtcu-PA respectively. Specific activities on fibrin plates were 100,000 +/- 24,000 IU/mg and 130,000 +/- 49,000 IU/mg for rscu-PA/MA-15C5 and rtcu-PA/MA-15C5 respectively, as compared to 180,000 +/- 15,000 IU/mg for both rscu-PA and rtcu-PA. Activation of plasminogen with rscu-PA/MA-15C5 (Km = 0.37 +/- 0.16 microM, k2 = 0.0063 +/- 0.0030 s-1 or rtcu-PA/MA-15C5 (Km = 19 +/- 3.0 microM, k2 = 2.0 +/- 0.10 s-1) in purified systems followed Michaelis-Menten kinetics with Km and k2 values comparable to those of rscu-PA and rtcu-PA. In an in vitro system composed of a 125I-fibrin-labeled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained; 50% lysis in 2 h required 1.4 microgram/ml of rscu-PA or 0.33 microgram/ml of rtcu-PA, but only 0.22 microgram u-PA/ml of rscu-PA/MA-15C5 or 0.15 microgram u-PA/ml of rtcu-PA/MA-15C5. Addition of purified fragment-D dimer reversed the increased fibrinolytic potency of rscu-PA/MA-15C5 in a concentration-dependent way (50% inhibition at 7.2 micrograms fragment-D dimer/ml). Thus, conjugation of u-PA moieties with the fibrin-specific antibody MA-15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4-fold for rscu-PA and 2.2-fold for rtcu-PA.     

A specific DNA unwinding activity associated with SV40 large T antigen

The incubation of highly purified large T antigen with relaxed, circular SV40 DNA in the presence of topoisomerase I (nicking closing enzyme) resulted in the introduction of negative superhelical turns in the DNA. ATP was not required for this reaction. A similar introduction of superhelical turns could also be obtained when a recombinant plasmid DNA (Y182), which contains sequences from both SV40 DNA and pBR322, was used. However, no effect was observed when relaxed pBR322 DNA, which does not contain SV40 DNA sequences, was incubated with T antigen in the presence of topoisomerase. These results are consistent with the hypothesis that large T antigen can recognize and unwind specific sequences on SV40 DNA.     

Monoclonal anti-A antibody removal by synthetic A antigen immobilized on specific antibody filters

Removal of blood group anti-A and anti-B antibodies can prevent hyperacute organ rejection in ABO-incompatible transplantation. We are developing an extracorporeal-specific antibody filter (SAF) as an immunoadsorption device for direct removal of ABO blood group antibodies from whole blood, without the need for plasma separation and plasma exchange. A hollow fiber-based small scale SAF (mini-SAF) device was fabricated and synthetic A antigen, Atrisaccharide (Atri) conjugated to activated polyacrylic acid, was immobilized on the fiber lumen surface. Monoclonal antibody anti-A IgM were specifically removed up to 70% of initial antibodies using mini-SAF device. The monoclonal anti-A capture experiments on mini-SAF indicated that antibody removal relative to the initial concentration is independent of inlet concentration in the beginning; however, as the surface starts saturating with bound antibodies, removal becomes dependent on inlet concentration. No significant effect of flow rate on removal rate was observed. The radial diffusion and axial convection-based mathematical model developed for unsteady state antibody removal was in good agreement with the experimental data and showed that the antibody removal rate can be maximized by increasing the antibody-binding capacity of the SAF.     

Rapid and efficient production of radiolabeled antibody conjugates using vacuum diafiltration guided by mathematical modeling

Increasing interest in the use of radiolabeled antibodies for cancer imaging and therapy drives the need for more efficient production of the antibody conjugates. Here, we illustrate a method for rapid and efficient production of radiolabeled antibody conjugates using vacuum diafiltration guided by mathematical modeling. We apply this technique to the production of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated antibodies at the milligram and gram production scale and achieve radiolabeling efficiencies >95% using In-111. Using vacuum diafiltration, antibody-chelate conjugation and purification can be accomplished within the same vessel, and the entire process can be completed in <24 h. Vacuum diafiltration also offers safer and gentler processing conditions by eliminating the need to keep the retentate vessel under positive pressure through applied gas pressure or shear-inducing restriction points in the retentate flow path. Experimental data and mathematical model calculations suggest there exists a weak binding affinity (approximately 10(4)M(-1)) between the charged chelate molecules (e.g., DOTA) and the antibodies that slows the removal of excess chelate during purification. By analyzing the radiolabeling efficiency as a function of the number of diavolumes, we demonstrate the importance of balancing the removal of free chelate with the introduction of metal contaminants from the diafiltration buffer and also illustrate how to optimize radiolabeling of antibody conjugates under a variety of operating conditions. This methodology is applicable to the production of antibody conjugates in general.     

Medullary substrate and differential cardiovascular responses during stimulation of specific acupoints

Electroacupuncture (EA) at P5-P6 acupoints overlying the median nerve reduces premotor sympathetic cardiovascular neuronal activity in the rostral ventral lateral medulla (rVLM) and visceral reflex pressor responses. In previous studies, we have noted different durations of influence of EA comparing P5-P6 and S36-S37 acupoints, suggesting that point specificity may exist. The purpose of this study was to evaluate the influence of stimulating P5-P6 (overlying the median nerve), LI4-L7 (overlying branches of the median nerve and the superficial radial nerve), LI6-LI7 (overlying the superficial radial nerve), LI10-LI11 (overlying the deep radial nerves), S36-S37 (overlying the deep peroneal nerves), or K1-B67 (overlying terminal branches of the tibial nerves) specific acupoints, overlying deep and superficial somatic nerves, on the excitatory cardiovascular reflex and rVLM responses evoked by stimulation of chemosensitive receptors in the cat's gallbladder with bradykinin (BK) or direct splanchnic nerve (SN) stimulation. We observed point-specific differences in magnitude and duration of EA inhibition between P5-P6 or LI10-LI11 and LI4-L7 or S36-S37 in responses to 30-min stimulation with low-frequency, low-current EA. EA at LI6-LI7 and K1-B67 acupoints as well as direct stimulation of the superficial radial nerve did not cause any cardiovascular or rVLM neuronal effects. Cardiovascular neurons in the rVLM, a subset of which were classified as premotor sympathetic cells, responded to brief (30 s) stimulation of the SN as well as acupoints P5-P6, LI10-LI11, LI4-L7, S36-S37, LI6-LI7, or K1-B67, or underlying somatic pathways in a fashion similar to the reflex responses. In fact, we observed a significant linear relationship (r(2) = 0.71) between the evoked rVLM response and reflex change in mean arterial blood pressure. In addition, EA stimulation at P5-P6 and LI4-L7 decreased rVLM neuronal activity by 41 and 12%, respectively, for >1 h, demonstrating that prolonged input into the medulla during stimulation of somatic nerves, depending on the degree of convergence, leads to more or less inhibition of activity of these cardiovascular neurons. Thus EA at acupoints overlying deep and superficial somatic nerves leads to point-specific effects on cardiovascular reflex responses. In a similar manner, sympathetic cardiovascular rVLM neurons that respond to both visceral (reflex) and somatic (EA) nerve stimulation manifest graded responses during stimulation of specific acupoints, suggesting that this medullary region plays a role in site-specific inhibition of cardiovascular reflex responses by acupuncture.     

Monoclonal anti-idiotypic antibodies induce humoral immune responses specific for simian virus 40 large tumor antigen in mice.

Mouse monoclonal anti-Id antibodies were generated against a mouse mAb (Ab-1) preparation specific for SV40 large tumor Ag (T-Ag). Four monoclonal anti-Id preparations each inhibited the binding of the monoclonal anti-SV40 T-Ag Ab-1 preparation to SV40 T-Ag. These anti-Id preparations appeared to recognize similar idiotopes on the monoclonal anti-SV40 T-Ag Ab-1 based on competitive cross-inhibition studies. One of these anti-Id preparations, designated 57B, was examined further for its in vivo modulatory capacity in mice. This anti-Id induced an Ab-3 response in BALB/c mice that recognized SV40 T-Ag (Ag+) and expressed an Id that was shared by the monoclonal anti-SV40 T-Ag Ab-1 preparation (Id+). The Id expressed on the Ab-3 differed from the Id induced in BALB/c mice immunized with the nominal SV40 T-Ag. Furthermore, characterization of the humoral immune response induced by anti-Id immunization indicated that the Ab-3 also recognized different epitopes on SV40 T-Ag when compared to the anti-SV40 T-Ag Ab-1 preparation used to generate the anti-Id. These studies indicate that monoclonal anti-Id can be used to induce humoral immune responses to a viral encoded tumor-associated Ag in vivo with 1) and Id specificity that differs from that expressed on antibodies produced by immunization with the nominal Ag and 2) an epitope specificity distinct from the Ab-1 preparation used for the production of the anti-Id.     

Idiotype and antigen-specific T cell responses in mice on immunization with antigen, antibody, and anti-idiotypic antibody.

Idiotypic determinants of immunoglobulin molecules can evoke both CD4(+) and CD8(+) T responses and exist not only as the integral components of a bona fide antigen binding receptor but also as distinct molecular entities in the processed forms on the cell surface of B lymphocytes. The present work provides experimental evidence for the concept that regulation of memory B cell populations can be achieved through the presentation of idiotypic and anti-idiotypic determinants to helper and cytotoxic cell. The potential of B cells to present antigens to helper and cytotoxic T cells through class II and class I MHC suggests a mechanism by which both B and T cell homeostasis can be maintained. We provide evidence for the generation of idiotype- and antigen-specific Th and Tc cells upon immunization of syngenic mice with antigen or idiotypic antibody (Ab1) or anti-idiotypic antibody (Ab2). The selective activation and proliferation of the antigen-specific Th and Tc cells mediated by idiotypic stimulation observed in these experiments suggests a B-cell-driven mechanism for the maintenance of antigen-specific T cell memory in the absence of antigenic stimulation, under certain conditions.     

Bacterial single-chain antibody fragments, specific for carcinoembryonic antigen.

We have produced single-chain antibody (scFv) fragments in bacteria specific for carcinoembryonic antigen (CEA). Polymerase chain reaction (PCR) was used for the cloning and modification of the heavy and light variable regions (VH and VL) of the mouse monoclonal antibody (MAb) CB-CEA.1. A 14-amino acid linker was used in the synthesis of the scFv gene. The VH and VL regions were amplified from cDNA by PCR using 5' end FR1 and 3' end constant region primers, and then sequenced. VH was then amplified by PCR using an exact 5' end FR1 primer, and a phosphorylated (PP) 3' end primer for J2 that also encoded the first 7 amino acids of the linker. VL was amplified with a PP 5' end primer for FR1, also encoding the remaining 7 amino acids of the linker, and a 3' end primer for J5, plus a stop codon and a BglII restriction site. The fragments were ligated and reamplified with the PP VH 5' and VL 3' end primers. The VH-linker-VL structure was blunt-cloned into expression vectors bearing the tryptophan promoter and pelB or ompA signal peptide sequences. Culture supernatant, bacteria pellet and periplasm preparations were assayed in Western blot and a protein of about 27 kDa was identified with rabbit antibodies specific for the Fab of CB-CEA.1. Bacterial supernatant and periplasm preparations also inhibited the recognition of CEA by HRP-labeled CB-CEA.1 in enzyme-linked immunosorbent assay (ELISA). Periplasm preparations were purified by affinity chromatography with specific anti-idiotypic MAbs. The Western blot of the eluates identified a protein of approximately 27 kDa that blocked the recognition of CEA by HRP-labeled CB-CEA.1 in ELISA. The VH-linker-VL structure was cloned into a vector bearing the lacZ promoter and the pelB signal peptide. The recombinant bacterial clones also expressed about 27 kDa scFv, specific for CEA.