Tyoku Matuhasi

N-terminal amino acid sequence of a major allergen of Japanese cedar pollen (Cry j I)

A purified preparation of a major allergen of Japanese cedar pollen, sugi basic protein (SBP, Cry jI), was separated into 5 subtractions of 50‐45 kDa. All of the SBP subtractions were confirmed to be reactive to IgE antibodies from patients with Japanese cedar pollinosis, and also to mouse anti‐SBP monoclonal antibodies. The sequences of 20 N‐terminal amino acids of these 5 subtractions were found to be identical. Peptide mapping analyses of the SBP subtractions showed similar patterns, with some differences which might in part be due to the existence of an N‐linked carbohydrate chain. The N‐terminal amino acid sequence of SBP was identical to the reported sequence of an allergen of mountain cedar which vegetated in North America.

Antigenic Analyses of Sugi Basic Protein by Monoclonal Antibodies; I. Distribution and Characterization of B-Cell-Tropic Epitopes of Cry j I Molecules

Using 23 monoclonal antibodies raised against Sugi basic protein (SBP, major allergen of Japanese cedar pollen), composed of Cry j I and Cry j II, analyses of B-cell-tropic epitopes of Cry j I were performed. The following results were obtained. (1) As far as the mAbs were used, no major cross-reactive determinants were detected between Cry j I and Cry j II molecules. (2) 21 of the 23 mAbs were specific for Cry j I, and the anti-Cry j II mAbs were classified into four groups by their fine specificities, suggesting that Cry j I bears at least four antigenic determinant regions. (3) Cry j I molecules were found to take a monomeric form in solution and to display no repeating antigenic epitopes on their surfaces. (4) Some of the determinants seemed to be located in the interior of a Cry j I molecule, and when the Ag is coated on a plastic plate, the determinants become exposed on its surface. (5) Binding of human IgE antibodies to Cry j I and Cry j II was blocked by some of the obtained mAbs, suggesting that these epitopes recognized by the mAbs might have an important role in human allergic response against the cedar pollen.

Antigenic Analyses of Sugi Basic Protein by Monoclonal Antibodies: II. Detection of Immunoreactive Fragments in Enzyme-cleaved Cry j l

The 4 anti-Cry j I mAbs showing an epitope specificity different from each other, 046, 029, 026 and 027, were selected to analyze the structure of the antigenic determinant for each mAb on a Cry j I molecule. Immunoreactive fragments in enzyme-cleaved Cry j I were detected by means of the adsorption on the mAb column and of the binding to the mAbs on Elisa. The mAb 026 was found to be reactive to the fragments containing a Cry j I N-terminal region obtained by V8 protease or pepsin digestion, but not to those by lysylendopeptidase digestion. The mAb 027 was found to be capable of binding to the fragments containing a linear structure of Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser-Leu-Ser-Lys, which were generated by V8 protease, lysylendopeptidase or pepsin digestion. Furthermore, the synthetic peptide Asn-Ala-Gly-Val-Leu-Thr-Cys-Ser- Leu-Ser-Lys-Arg could bind to 027, but not to 026, and could inhibit the binding of 027 to Cry j I or to its immunoreactive fragments. No fragments capable of reacting to the mAbs 046 and 029 could be found in this study, suggesting that 046 and 029 recognize a conformationally constituted epitope of Cry j I molecule which is destroyed by enzymatic cleavage. The epitope recognized by the mAbs 027 or 026 was found to be located in conformationally hidden parts of the molecule which was exposed to react to the mAbs only after the physicochemical or enzymatic treatment.

Detection and Characterization of Polymers in Cephalothin by Passive Cutaneous Anaphylaxis in Mice

Immunization of BALB/c mice by repeated injections of cephalothin (CET) - Ascaris suum extract conjugate resulted in formation of IgE antibodies, which were able to sensitize syngeneic animals for passive cutaneous anaphylaxis (PCA). By means of high-pressure liquid chromatography, a fraction with extremely high PCA-eliciting activity, but without appreciable antimicrobial activity, was isolated from the CET preparation. Physicochemical analyses of the fraction identified the major component of polymer impurities as being a proteinaceous complex with a molecular weight of 6,580. Very little cross-reactivity of CET and potassium benzyl penicillin (PcG) was noted when these antibiotics were used as the challenge antigens for PCA induced by corresponding murine antisera. The results of the inhibition studies indicated, however, that at least two antigens were involved in the PCA induced by anti-CET antibodies, one strictly specific for CET and another shared by PcG. Evidence was also presented that the nucleus structure and acyl side-chain structure of CET play the major role in the PCA elicited by the challenge with CET and its polymer, respectively.

Growth of rat-mouse hybridoma cells in immunosuppressed hamsters: An easy and effective method to prepare monoclonal antibodies from heterohybridoma cell lines

Rat-mouse hybridoma cells producing anti-mouse IgE antibodies were intraperitoneally or subcutaneously inoculated into newborn or suckling hamsters receiving rabbit anti-hamster thymocyte globulin from the day of birth twice a week for at least 3 weeks. The hybridoma cells were found to grow in the abdominal cavity of the hamsters as ascites tumor or in subcutaneous tissue as solid tumor without loss of antibody-secreting activities. For the production of ascites, 2-week-old hamsters were preferable to newborn hamsters. In 3-week-old hamsters, the hybridoma cells could scarcely survive. The antibody titers of the ascites were determined to be 10(5)-10(6) in the ELISA and in the ability to neutralize the skin-sensitizing capacity of mouse IgE antibodies. The rat monoclonal antibodies were easily separated from ascites, serum or cell culture supernatant with affinity chromatography using Affigel protein A-Sepharose and anti-hamster IgG-Sepharose columns. The described method could be efficiently applicable for the proliferation of other hybridomas, such as human-human, human-mouse or hamster-mouse, etc.

Suppression of Murine IgE Responses with Ovalbumin-Pullulan Conjugates: Comparison of the Suppressive Effect of Different Conjugation Methods and Different Molecular Weights of Pullulan

Ovalbumin (OVA)-pullulan conjugates were made using four different conjugation methods and eight different molecular weights of pullulan ranging from 4,200 to 600,000. Pretreatment of mice by the administration of conjugates made by using cyanulic chloride or cyanogen bromide and pullulan of molecular weight 40,000 or more, anti-OVA IgE antibody response was suppressed completely and anti-OVA IgM and IgG antibody response was enhanced. In contrast, by the administration of conjugates made by using an oxidation or thiol activation method, only partial suppression of anti-OVA IgE antibody response was achieved and no enhancement of anti-OVA IgM and IgG antibody production was observed.

Fc receptors on IgM and IgG antibody-forming cells in an early immune response

Abstract Antibody-forming cells were studied to determine whether they had receptors for the Fc portion of IgG(FcR). Spleen cells from mice immunized with horse red blood cells(HRBC) were assayed for anti-HRBC plaque-forming cells (PFC) after they were depleted by Ficoll-Hypaque density sedimentation of cells forming rosettes with sheep red blood cells(SRBC) coated with IgG anti-SRBC. Direct PFC were shown to bear FcR but the receptor was not on indirect PFC. FcR on direct PFC were indicated as being occupied by antigen-antibody complexes at a certain stage of the immune response. Dose of antigen and immunization route were of crucial importance for the occupation of FcR on PFC.

p phenotype in two successive generations of a Japanese family.

Abstract. A remarkable Japanese family, in which the propositus, his mother, and his maternal aunt have the rare p phenotype, is reported. The anti‐P+P1 (anti‐Tja) antibodies were classified as IgM by the indirect antiglobulin technique. Red cells of the propositus were examined for the activity levels of several enzymes and glycolytic intermediates and were tested for glutathione stability, autohemolysis, osmotic fragility, and lactate production. All were within normal limits.

A new approach to make toxoid eliciting good IgM and/or IgG but little IgE antibody responses

We have found that the induction of IgE antibody response is suppressed selectively and antigen-specifically, whereas IgM and IgG antibody responses are enhanced in mice pretreated with an antigen conjugated with pullulan, a linear polymer of glucose [1]. Even when the conjugate is repeatedly injected into mice, the IgE antibody response is very poor but the IgM and IgG responses are well elicited as compared with those to the antigen immunized with alum adjuvant. These findings led us to the assumption that a toxin when conjugated with pullulan (or other polysaccharides) can be used as a toxoid; this is because the toxin cannot manifest its toxicity since the binding site of the toxin to a cell surface receptor is hindered stereochemically by the pullulan moiety of the conjugate [2]. The experiments revealing that toxins conjugated with pullulan are detoxicated and can suppress the induction of IgE antibody while enhancing the IgM and IgG antibody responses to the toxins will be briefly described here. Fifty mg of pullulan (345 000 daltons), dissolved in 24 ml of phosphate-buffered saline (PBS, pH 7.0), was cooled in an ice bath. Cyanuric chloride (20 mg) in 1 ml of cooled N,N-dimethylformamide was added to the solution. The mixture was stirred in the bath for 4h, keeping the pHat 7. Then, 12500 Lf(ca. 59.3 mg protein) of tetanus toxin in PBS were added to the mixture, which was stirred at 37 ~ for another 72 h. To the mixture was added 2 mg of glycine to stop the reaction of cyanuric chloride. It was then dialyzed against 0.05 M Tris-HC1 buffer, pH 7.5, overnight. The resulting mixture was chromatographed on DEAE sephacel equilibrated with the same buffer. The conjugate of toxin to pullulan was eluted gradually by 0.05M Tris-HC1 buffer containing NaC1 of increasing concentrations. The eluate was monitored by cellulose acetate membrane electrophoresis. The fractions by which the electrophoretic mobility was much smaller than that of toxin or of formalinized toxoid were pooled. The conjugation of toxin to pullulan was confirmed by simultaneous staining with nigrosin and with Schiffs reagent. The resulting preparation has practically no toxicity against mice. Two Lb per mouse of the preparation was injected subcutaneously to groups of 5 mice several times at an interval of 1-4 weeks. The sera taken at various intervals were titrated for the IgE antibody by passive cutaneous anaphylaxis (PCA) using Sprague-Dawley rats and for the IgM and/or IgG antibodies by passive hemagglutination (PHA). A representative data is illustrate in Fig. 1 a. The IgE antibody to tetanus toxin was well suppressed; but IgM and/or IgG antibodies responded fairly well compared with control groups which were injected in the same way with the traditional toxoid plus alum adjuvant or with plain toxoid. The habu (Trirnerusus flavoviridis) venom conjugated with pullulan by the same method revealed no toxicity in the rabbit skin hemarrhage test. The conjugate elicited the enhancement of IgM and/or IgG antibody responses with the suppression of IgE antibody response (Fig. 1 b). These results suggest that the conjugation IgE anti-tetanus toxin (PCA) 40 80 120 160 I I I I

Specificity of an Anti-Murine B Cell Serum

An antiserum (ABS) specific for murine B cells was prepared in rabbits by immunization with lymph node cells from nude mice as reported previously (1). To make the antiserum specific for antibody-forming cell precursors (AFCP) extensive absorption was required with mouse serum, erythrocytes and thymus cells. Even after ABS was no longer cytotoxic for thymus cells, further absorption with thymus cells was necessary to make it ineffective for plaque-forming cells (PFC). The correlation between cells bearing C3- or Fc-receptors and the cells bearing antigen(s) to which ABS reacts was studied. In the residual spleen cells treated with ABS and complement, there were some EAg and EAmCm rosette-forming cells. There were also some spleen cells reacting with ABS even after depletion of C3- and/or Fc-receptor-bearing cells. These results show that the antigen(s) to which ABS reacts could be a marker on B cells different from C3- or Fc-receptors. Spleen cells reacting with ABS were compared with cells bearing C3- or Fc-receptors various days after birth. The cells sensitive to ABS increased in spleens earlier after birth than those with C3- or Fc-receptors.