Preparation of antibody against agatharesinol, a norlignan, using a hapten-carrier conjugate.

In order to immunolabel heartwood extractives in Japanese cedar (Sugi, Cryptomeria japonica), we attempted to prepare antibodies against agatharesinol, a major norlignan of these heartwood extractives. Agatharesinol by itself is not antigenic due to its low-M(r), and thus was covalently bound to bovine serum albumin in order to synthesize an antigenic hapten-carrier conjugate (artificial antigen). The number of agatharesinol molecules per artificial antigen molecule was estimated as 27-28 by quantifying Lys in an acid hydrolysate of the artificial antigen by HPLC. Reaction between the artificial antigen and serum obtained from a rabbit immunized with the artificial antigen was competitively inhibited by agatharesinol, indicating the successful production of anti-agatharesinol antibodies. Inhibition by sequirin C, another major norlignan in Sugi, was weaker than that by agatharesinol. Furthermore, an EtOAc soluble fraction, which contains mainly norlignans, inhibited the reaction more strongly than any of the other fractions of Sugi heartwood extractives. Thus, the antiserum we have produced reacts most strongly with agatharesinol and recognizes norlignans almost selectively among Sugi heartwood extractives.     

Interaction between F1-ATPase and its naturally occurring inhibitor protein. Studies using a specific anti-inhibitor antibody

An antibody was raised to cross-linked ox-heart mitochondrial inhibitor protein, which cross-reacts with the free inhibitor but with no other mitochondrial membrane protein. This antibody yields an immunoprecipitate with the cross-linked inhibitor protein, but a soluble antibody-antigen complex with free inhibitor. The antibody binds well to inhibitor protein whether the latter is complexed with F1-ATPase or not. Antibody binding has no effect on the ability of the inhibitor protein to inhibit the ATPase activity of F1. These findings suggest that the antibody does not block the site of interaction between the inhibitor and F1. The inhibitor protein content of submitochondrial membrane preparations was determined by radioimmunoassay, activity measurements and an immunochemical 'back titration' technique. The inhibitor content of the membranes is shown to decrease after energisation, suggesting a loss of inhibitor from the membranes into solution. Binding antibody to the inhibitor protein on submitochondrial particles has no effect on the steady-state rate of phosphorylation, but it increases the lag phase preceding phosphorylation from 30 to 54 s. The rate constant for the approach to the steady state drops from 0.078 to 0.052 s-1. This effect confirms that the lag phase is due to inhibition of phosphorylation by the inhibitor protein. The increase in ATPase activity following energisation takes place by a fast phase (80% maximal activity reached within 90 s) and a slower phase (lasting about 10 min.). The rate constant of the rapid phase (0.017 s-1) is of the same order as that for the activation of phosphorylation. It is concluded that the rapid phase of ATPase induction is fast enough for this process to occur simultaneously with the activation of phosphorylation.     

Distances of tyrosine residues from a spin-label hapten in the combining site of a specific monoclonal antibody

The nuclear magnetic resonance spectra of an Fab fragment of a monoclonal antibody specifically directed against a nitroxide spin-label hapten have been recorded at different concentrations of the hapten. The hybridoma producing this antibody was grown on deuterated phenylalanine, tryptophan, and 3,5-dideuteriotyrosine or 2,6-dideuteriotyrosine. Difference spectra--without hapten minus with hapten--were calculated for each concentration of hapten. The difference spectra reveal five well-resolved singlet proton resonance signals from tyrosine deuterated in the 3,5-positions (H 2,6 Tyr) and nine from tyrosine deuterated in the 2,6-positions (H 3,5 Tyr). The measured intensities of these signals as a function of combining site occupation have been interpreted in terms of a theory involving intrinsic line widths (T2), the hapten off-rate (k), and distances to the paramagnetic center. Good agreement with theory is found for all of the isolated proton signals. The best estimate of k is 350 s-1; distances in the range 13 to less than 9 A are calculated. Extension of this analysis to other amino acids is discussed.     

Functional properties of a rat monoclonal IgE antibody specific for Schistosoma mansoni

A rat monoclonal antibody of IgE isotype (B48-14) raised against Schistosoma mansoni has been generated by the fusion of mesenteric lymph node cells from LOU/M rats immunized with a preparation of adult schistosome worms and IR973F nonsecreting rat myeloma cells. Investigation of the in vitro effector functions of this IgE antibody showed a high level of cytotoxicity against S. mansoni schistosomula in the presence of eosinophils, macrophages, and platelets. A significant level of protection (40 to 60%) against a challenge infection with S. mansoni cercariae was achieved by passive transfer experiment of B48-14 IgE to naive recipient rats. By immunoprecipitation, B48-14 IgE antibodies were shown to react with an antigen of 26 kDa present in excretion-secretion products of schistosomula, previously described as a potential immunogen eliciting a protective IgE response against schistosomiasis.     

IgE antibody against hepatitis B surface antigen in mice

Hepatitis B surface antigen (HBs antigen) was examined for elicitation of IgE production by injection into mice. The Prausnitz-Küstner (PK)-type skin test in the rat was employed for detection of IgE antibody to HBs antigen, because no sufficient purified HBs antigen was available as the challenging antigen for the passive cutaneous anaphylaxis (PCA) test in rats. The positive PK test was considered to be due to IgE antibody, since the active principle was inactivated by heating the sera at 56 C for 30 min, did not bind to protein A and was eliminated by anti-mouse IgE antisera. These data indicate that the PK-type test in rats can be used for detection of mouse IgE antibody when the amount of a test sample is not sufficient for the PCA test in rats.     

Detection of Ki-ras proto-oncogene protein by a specific monoclonal antibody.

We have used a commercially available mouse monoclonal antibody and shown it to bind specifically to cellular Ki-ras p21 proteins and not to cellular N- and Ha-ras p21 proteins. In conjunction with electrophoresis and Western blotting, this antibody can be used, with further detailing, to assess levels of the cellular Ki-ras p21 against a background of total p21s.     

Interaction of antibodies specific towards the 2,4-dinitrophenyl group and of their fragments with hapten

The interaction of hapten (ε-DNP lys) with native and S-sulfonated antibodies specific towards the 2,4-dinitrophenyl group, as well as the interaction with isolated chains and a complex obtained by mixing light, (L) and heavy (H) chains of these antibodies, were followed both by polarography and by equilibrium dialysis. With the S-sulfonated antibodies and with the mixture of H and L chains the binding heterogeneity observed in the original antibodies was much lowered or entirely removed. At the same time, the amount of active proteins in the sample decreased approximately by half. The association constants of modified antibodies were of the same order as the average association constants of the original antibodies. A slow increase of the amounts of hapten bound with proteins was observed on mixing the H and L chains and adding hapten. This slow reactivation was not obtained with the original or S-sulfonated antibodies and with isolated chains. It was shown that the reaction determining the kinetics of this reactivation (the slowest reaction) was not the association of H and L chains but the interaction of complexes of the H and L chains with hapten. It was reported previously that H chains were nonspecifically reactivated by binding L chains. The amount of hapten bound by the complex of H and L chains increased with increasing excess of L chains following a curve resembling the Langmuir isotherm. The limiting value of the amount of hapten bound when using antibody L chains was higher than in the case of nonspecific L chains.     

IgE antibody responses against Japanese cedar pollen in the mouse

IgE antibody responses against Japanese cedar pollen in the mouse were investigated to develop a mouse model of human allergy for combinations of factors including pollen administration routes, elicitation antigens and inbred mouse strains. Daily short term inhalation of native pollen or intratracheal administration of pollen suspended in saline induced IgE antibody responses in DBA/2, BDF1 and Balb/c mice, but failed to induce any detectable responses in C57BL/6 and C57BL/10 mice. Intraperitoneal injection of pollen suspension also induced IgE antibody responses in DBA/2, BDF1 and Balb/c mice but not in C57BL/6 mice. IgE antibody responses against pollen described above were detected by passive cutaneous anaphylaxis (PCA) reactions using crude extract of pollen as an elicitation antigen. On the other hand, IgE antibodies specific for antigen Sugi basic protein (AgSBP), which is a major allergen of pollen in humans (Yasueda, H., Yui, Shimizu, T., and Shida, T., 1983. Isolation and partial characterization of the major allergen from Japanese cedar (Cryptomeria japonica) pollen. J. Allergy Clin. Immunol. 71: 77-86), were also detected by PCA reactions using AgSBP in the sera from mice which received secondary or the tertiary stimulation by pollen. These results suggest that IgE antibody responses against Japanese cedar pollen in the mouse can be induced by airway sensitization and that the responses are genetically controlled by H-2-linked immune response genes. The results also suggest that not only IgE antibody responses specific for components other than AgSBP but also responses specific for AgSBP can be induced in the mouse by repeating appropriate sensitization by pollen.     

Isolation and characterization of a specific antibody population against human fibrinogen

A specific antibody population against human fibrinogen was isolated from a rabbit antiserum by affinity chromatography on fibrinogen-bound Sepharose gel. Using a sensitive competitive radioimmunoassay, the antibody population was found to recognize epitopes on native fibrinogen but crossreacted minimally with fibrinogen fragment D and an early plasmin-degraded fibrinogen A alpha-chain product, but not at all with fragment E or fibrinopeptides A and B. Fibrin monomers shared part of these epitopes. The antibody population crossreacted to a small extent with bovine, horse and baboon fibrinogens and not at all with fibrinogens from sheep, rat, pig, goat, guinea pig, dog and rabbit.     

Use of a hapten specific anti-dansyl antibody for the localization of ribosomal proteins by immuno electron microscopy

The fluorescent reagent dansyl chloride has been used as an immunological marker for the electron microscopic localization of ribosomal proteins on the surface of 50S ribosomal subunits. The proteins BstL1 from Bacillus stearothermophilus and EcoL1 from Escherichia coli were dansylated to various degrees and reconstituted into the L1-deficient E. coli 50S subunits from mutant MV17-10. Using antibodies specific to dansyl chloride, both proteins were mapped at the lateral protuberance near the peptidyl transferase center.     

Comparative fluorescent antibody staining of histoplasma capsulatum and histoplasma duboisii with a specific anti-yeast phase H. Capsulatum conjugate

Summary The specific anti-yeast phaseHistoplasma capsulatum conjugate has been tested against 13 yeast phase strains ofH. capsulatum and 9 ofH. duboisii. The conjugate was specific forH. capsulatum, no yeast phase form ofH. duboisii obtained in vitro or in vivo reacted with it. The taxonomic implications of these results are discussed.     

A monoclonal antibody against a 135-K Golgi membrane protein.

A monoclonal antibody ( 53FC3 ) has been produced against a Golgi membrane protein with a mol. wt. of 135 000 which was originally identified using a polyclonal antiserum. Treatment of isolated, intact Golgi vesicles with protease caused a decrease in mol. wt. of 5000-10 000, whereas in the presence of Triton X-100, the protein was completely degraded. This shows that the protein spans the bilayer and that most of its mass is on the luminal side of Golgi membranes. Using two immunoelectron microscopic techniques, the protein was found in one or two cisternae on one side of the Golgi stack which, in normal rat kidney cells, had 4-6 cisternae. As an illustration of the use to which this monoclonal antibody can be put we present a light microscopic study of the disassembly and reassembly of the Golgi complex during mitosis.     

Protective effects of anti-antiidiotypic IgE antibodies obtained from an IgE monoclonal antibody specific for a 26-kilodalton Schistosoma mansoni antigen

A rat IgE mAb specific for larval Ag (26 kDa, 56 kDa) has been shown to protect rats against Schistosoma mansoni infection. Immunizations of Lou/M rats performed with this IgE (Ab1) induced the production of antiidiotypic antibodies (Ab2). Moreover, after this Ab2 production, anti-antiidiotypic antibodies (Ab3) were revealed. The screening of Ab3 isotypes showed the presence of IgG Ab3 and more interestingly of IgE Ab3, i.e., the same isotype as Ab1. These IgE and IgG antibodies recognized predominantly the 26-kDa Ag and were cytotoxic for schistosomula in the presence of platelets for IgE Ab3 and eosinophils for IgG Ab3. Both IgE and IgG Ab3 conferred by passive transfer protective immunity to infected rats (up to 50%). Thus the immunization with an IgE mAb led in part to the production of Ab3 of the same isotype as Ab1. In conclusion, these results suggest that the isotype selection of the antibodies of the third generation (Ab3) might be influenced by the Ab1. The respective role of the idiotope and isotype of Ab1 in isotype regulation is discussed.