To determine the content of endogenous toxic substance Pinellia ternata lectin(PTL) protein in Pinelliae Rhizoma and the related processed products, this study prepared specific monoclonal antibodies against PTL by hybridoma cell technology, and established a quantitative double-antibody sandwich enzyme linked immunosorbent assay(ELISA) for PTL antigen. The detection conditions were 2.5 μg·mL~(-1) working concentration of the captured antibody and 1∶450 of the dilution multiple of detected antibody. The coating condition was staying overnight at 4 ℃. The blocking time and incubation times of antigen and detected antibody were all 90 minutes. The incubation time of horseradish peroxidase conjugated streptavidin-horseradish peroxidase(SA-HRP) was 15 minutes. The quantitative limit of the method for PTL antigen was 0.375 ng·mL~(-1). The linear range was 75.000-4 800.000 pg·mL~(-1), and R~2=0.997 1. The recovery rate was 90.0%-110.0%, and the variation coefficients of intra-test and inter-test precision were 2.0%-3.0% and 2.0%-8.5%.The content of PTL in three batches of Pinelliae Rhizoma and the related processed products was determined by the method, and the average content of PTL in Pinelliae Rhizoma was 35.42 mg·g~(-1). The average content of PTL in Pinelliae Rhizoma Praeparatum Cum Alumine, Pinelliae Rhizoma Praeparatum, and Pinelliae Rhizoma Praeparatum Cum Zingibere Et Alumine were 1.15 mg·g~(-1), 16.53 μg·g~(-1), and 122.63 ng·g~(-1), respectively, indicating that the content of PTL decreased significantly after processing. The quantitative double-antibody sandwich ELISA for PTL antigen established in this paper had good linearity, sensitive response, and high accuracy, which provided a simple and effective monitoring method for the detection of PTL content in the processing of Pinelliae Rhizoma.
Pinellia ; Drugs, Chinese Herbal ; Antibodies, Monoclonal ; Enzyme-Linked Immunosorbent Assay ; Horseradish Peroxidase

PURPOSE: Although the DTaP and Tdap vaccines used to prevent pertussis have been used for a long time, there is no standard method for measuring pertussis antigens. Therefore, this preliminary study was conducted to develop an enzyme-linked immunosorbent assay method using an animal model for measuring antibodies against pertussis toxin, the most important pertussis pathogenic antigen, in the sera of vaccinated mice. MATERIALS AND METHODS: Bordetella pertussis Tohama phase I was cultured for 24–30 hours, and then pertussis toxin was purified from the culture medium by chromatography. Purified pertussis toxin was diluted in phosphate-buffered saline-coating buffer, and 100 µL of diluted pertussis toxin was added to each well and reacted at room temperature for 4 hours. Positive serum was diluted to 1/1,250–1/80,000 and negative serum was diluted to 1/50 to determine the coating concentration with the optimal signal/noise ratio. Optimal test conditions were confirmed from the dilution factors of the secondary antibody and streptavidin horseradish peroxidase (SA-HRP). RESULTS: Optimal conditions were as follows: 4 µg/mL for coating antigen; 1/40,000 for secondary antibody; and 1/1,000 for the SA-HRP dilution factor. Comparison of the sera obtained from mice treated with a developing vaccine and commercial vaccine with National Institute for Biological Standard and Control standard serum under the established conditions showed the following results: 1,300.62, 534.94, and 34.85, respectively. CONCLUSION: The method developed in this study is suitable for measuring anti-pertussis toxin antibodies and may be applicable for clinical sample analysis or indirect diagnosis of pertussis.
Animals ; Antibodies ; Bordetella pertussis ; Chromatography ; Diagnosis ; Enzyme-Linked Immunosorbent Assay* ; Horseradish Peroxidase ; Methods ; Mice* ; Models, Animal ; Pertussis Toxin ; Streptavidin ; Vaccines ; Whooping Cough

Currently, compared to jaw-closing (JC) α-motoneurons, the information on the distribution and morphology of glutamatergic synapses on the jaw-closing (JC) γ-motoneurons, which may help elucidate the mechanism of isometric contraction of the JC muscle, is very limited. This study investigated the distribution and ultrastructural features of vesicular glutamate transporter 1 (VGLUT1)- and VGLUT2-immunopositive (+) axon terminals (boutons) on JC γ-motoneurons by retrograde tracing with horseradish peroxidase, electron microscopic immunocytochemistry, and quantitative analysis. About 35% of the boutons on identified JC γ-motoneurons were VGLUT+, and of those, 99% were VGLUT2+. The fraction of VGLUT1+ boutons of all boutons and the percentage of membrane of JC γ-motoneurons covered by these boutons were significantly lower than those for the JC α-motoneurons, revealed in our previous work. The bouton volume, mitochondrial volume, and active zone area of the VGLUT2+ boutons on the JC γ-motoneurons were uniformly small. These findings suggest that the JC γ-motoneurons, in contrast to the JC α-motoneurons, receive generally weak glutamatergic synaptic input almost exclusively from VGLUT2+ premotoneurons that form direct synapse with motoneurons.
Animals ; Horseradish Peroxidase ; Immunohistochemistry ; Isometric Contraction ; Membranes ; Microscopy, Electron ; Mitochondrial Size ; Motor Neurons ; Presynaptic Terminals ; Rats ; Synapses ; Vesicular Glutamate Transport Protein 1

Virus-like particles (VLPs) derived from human papillomavirus (HPV) L1 capsid proteins were used for HPV quadrivalent recombinant vaccine. The HPV quadrivalent vaccine is administrated in a 3-dose regimen of initial injection followed by subsequent doses at 2 and 6 months to prevent cervical cancer, vulvar, and vaginal cancers. The type 6, 11, 16, or 18 of HPV infection is associated with precancerous lesions and genital warts in adolescents and young women. The HPV vaccine is composed of viral L1 capsid proteins are produced in eukaryotic expression systems and purified in the form of VLPs. Four different the L1 protein of 3 different subtypes of HPV: HPV11, HPV16, and HPV18 were expressed in Escherichia coli divided into 2 fragments as N- and C-terminal of each protein in order to examine the efficacy of HPV vaccine. Vaccinated sera failed to recognize N-terminal L1 HPV type 16 and type 18 by western blot while they detected N-terminal L1 protein of HPV type 11. Moreover, the recombinant C-terminal L1 proteins of type 16 was non-specifically recognized by the secondary antibody conjugated with horseradish peroxidase. This expression and purification system may provide simple method to obtain robust recombinant L1 protein of HPV subtypes to improve biochemical analysis of antigens with immunized sera.
Adolescent ; Blotting, Western ; Capsid Proteins ; Condylomata Acuminata ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; Female ; Horseradish Peroxidase ; Humans ; Methods ; Papillomaviridae ; Recombinant Proteins* ; Uterine Cervical Neoplasms ; Vaginal Neoplasms

Several studies have reported a good correlation between levels of serum hepatitis B virus surface antigen (HBsAg) and covalently closed circular DNA (cccDNA) before and after antiviral therapy. As a result, the quantification of HBsAg levels has attracted much attention in recent years as an important approach to evaluate viral activity. In this study, mAbs against HBsAg were generated and 9 mAbs (H17, H30, H31, H67, H73, H97, H101, H118, and H128) were investigated for optimization of HBsAg quantitation ELISA. Determination of the best combinations of mAbs for sandwich ELISA identified H17 and H31 mAbs as the ideal capture and horseradish peroxidase (HRP) conjugate mAbs, respectively. A standard curve for the current assay system exhibited linearity up to 40 ng/ml of HBsAg while a detection limit of approximately 1 ng/ml of HBsAg was also estimated, which was comparable to that of the other commercial ELISA kits. The ELISA system established in this study is particularly differentiated from other commercial kits in using mAbs for both capture and HRP conjugate, which provides a solution to inconsistency of quality and ethical issues in polyclonal antibodies production using laboratory animals.
Animals, Laboratory ; Antibodies ; Antigens, Surface ; DNA, Circular ; Enzyme-Linked Immunosorbent Assay* ; Ethics ; Hepatitis B Surface Antigens ; Hepatitis B virus* ; Hepatitis B* ; Hepatitis* ; Horseradish Peroxidase ; Limit of Detection

BACKGROUND: Specific IgE antibodies against the low-molecular-weight carbohydrate antigen that does not bridge IgE molecules on mast cells are not associated with clinical symptoms. Cross reactivity can be determined in allergen-specific IgE detection assays when the carbohydrate structures between pollen allergens and plant derived food allergens are similar; in such cases, false positive results for grain or legume allergens can be reported for pollen allergic patients who are not sensitized to those allergens. This phenomenon arises owing to the presence of cross-reactive carbohydrate determinants (CCDs). OBJECTIVE: This study aimed to assess the impact of CCD interference on the results for pollen allergen-specific IgE antibodies in the general adult population and to perform CCD inhibition tests evaluating the involvement of CCD on samples positive to pollen allergens. METHODS: Serum samples from 322 subjects were tested for IgE antibodies to pollens and CCD. The research subjects were given questionnaires about pollen allergic symptoms to help assess the presence of allergies. Allergen IgE antibodies for Japanese cedar, Japanese cypress, orchard grass, ragweed, MUXF, bromelain, horseradish peroxidase (HRP), and ascorbate oxidase (ASOD) were analyzed. RESULTS: It was observed that among individuals who tested positive to any of the pollen allergens, the positive ratio of CCD-specific IgE antibody was the highest for HRP (13.5%–50.0%). The results from the inhibition tests revealed that CCD was marginally present. Although IgE antibodies for cedar pollen did not react with CCD, IgE antibodies for Japanese cypress, orchard grass, and ragweed might be detected by the presence of CCD. CONCLUSION: The results of the inhibition tests revealed the obvious presence of CCD suggesting its involvement. Considering these findings, careful evaluation of patient IgE results should be performed for Japanese cypress, orchard grass, and ragweed.
Adult ; Allergens ; Ambrosia ; Antibodies ; Ascorbate Oxidase ; Asian Continental Ancestry Group ; Bromelains ; Cryptomeria ; Cupressus ; Dactylis ; Fabaceae ; False Positive Reactions ; Horseradish Peroxidase ; Humans ; Hypersensitivity ; Immunoglobulin E ; Mast Cells ; Plants ; Pollen ; Research Subjects ; Rhinitis, Allergic ; Rhinitis, Allergic, Seasonal

The ultrastructural parameters related to synaptic release of endings which are presynaptic to tooth pulp afferent terminals (p-endings) were analyzed to understand the underlying mechanism for presynaptic modulation of tooth pulp afferents. Tooth pulp afferents were labelled by applying wheat-germ agglutinin conjugated horseradish peroxidase to the rat right lower incisor, whereafter electron microscopic morphometric analysis with serial section and reconstruction of p-endings in the trigeminal oral nucleus was performed. The results obtained from 15 p-endings presynaptic to 11 labeled tooth pulp afferent terminals were as follows. P-endings contained pleomorphic vesicles and made symmetrical synaptic contacts with labeled terminals. The p-endings showed small synaptic release-related ultrastructural parameters: volume, 0.82 ± 0.45 µm³ (mean ± SD); surface area, 4.50 ± 1.76 µm²; mitochondrial volume, 0.15 ± 0.07 µm³; total apposed surface area, 0.69 ± 0.24 µm²; active zone area, 0.10 ± 0.04 µm²; total vesicle number, 1045 ± 668.86; and vesicle density, 1677 ± 684/µm². The volume of the p-endings showed strong positive correlation with the following parameters: surface area (r=0.97, P<0.01), mitochondrial volume (r=0.56, P<0.05), and total vesicle number (r=0.73, P<0.05). However, the volume of p-endings did not positively correlate or was very weakly correlated with the apposed surface area (r=-0.12, P=0.675) and active zone area (r=0.46, P=0.084). These results show that some synaptic release-related ultrastructural parameters of p-endings on the tooth pulp afferent terminals follow the "size principle" of Pierce and Mendell (1993) in the trigeminal nucleus oralis, but other parameters do not. Our findings may demonstrate a characteristic feature of synaptic release associated with p-endings.
Animals ; Horseradish Peroxidase ; Incisor ; Mitochondrial Size ; Rats ; Tooth* ; Trigeminal Nuclei

PURPOSE: In the celery-mugwort-birch-spice syndrome, a significant proportion of IgE is directed against high molecular weight (HMW) glycoproteins, including the celery allergen Api g 5. BIP3, a monoclonal antibody originally raised against birch pollen, recognizes HMW allergens in birch and mugwort pollens, celery, and Apiaceae spices. Our aim was to generate mimotopes using BIP3 for immunization against the HMW allergens relevant in the celery-mugwort-birch-spice cross reactivity syndrome. METHODS: Mimotopes were selected from a random-peptide display library by BIP3 and applied in IgE inhibition assays. The 3 phage clones with the highest inhibitory capacity were chosen for immunization of BALB/c mice. Mouse immune sera were tested for IgG binding to blotted birch pollen extract and used for inhibiting patients' IgE binding. Furthermore, sera were tested for binding to Api g 5, to horseradish peroxidase (HRP) as a second glycoprotein, or to non-glycosylated control allergen Phl p 5 in ELISA, and the specific Api g 5-specific IgG titers were determined. RESULTS: Three rounds of biopanning resulted in phage clones exhibiting 7 different sequences including 1 dominant, 1-6-cyclo-CHKLRCDKAIA. Three phage clones had the capacity to inhibit human IgE binding and induced IgG to the HMW antigen when used for immunizing BALB/c mice. The induced BIP3-mimotope IgG reached titers of 1:500 specifically to Api g 5, but hardly reacted to glycoprotein HRP, revealing a minor role of carbohydrates in their epitope. CONCLUSIONS: The mimotopes characterized in this study mimic the epitope of BIP3 relevant for Api g 5, one of the cross-reactive HMW allergens relevant in the celery-mugwort-birch-spice syndrome. BIP3 mimotopes may be used in the future for hyposensitization in this clinical syndrome by virtue of good and specific immunogenicity.
Allergens ; Animals ; Apiaceae ; Apium graveolens ; Artemisia ; Bacteriophages ; Betula ; Carbohydrates ; Clone Cells ; Enzyme-Linked Immunosorbent Assay ; Food Hypersensitivity ; Glycoproteins ; Horseradish Peroxidase ; Humans ; Immune Sera ; Immunization ; Immunoglobulin E ; Immunoglobulin G ; Mice ; Molecular Weight ; Pollen ; Spices ; Vaccination ; Virtues

Previous studies suggested that myelinated axons innervating rat molar pulps undergo morphological changes in their peripheral course. However, little information is available on the morphological feature of the parent axons at the site of origin. We therefore investigated the size of the myelinated parent axons and their morphological features at the proximal sensory root of the trigeminal ganglion by horseradish peroxidase (HRP) injection into rat upper molar pulps and subsequent light and electron microscopy. A total of 248 HRP-labeled myelinated axons investigated were highly variable in the size. Fiber area, fiber diameter, axon area (axoplasm area), axon diameter (axoplasm diameter), and myelin thickness were 11.32 +/- 8.36 microm2 (0.80~53.17 microm2), 3.99 +/- 1.53 microm (1.08~9.26 microm), 8.70 +/- 6.30 microm2 (0.70~41.83 microm2), 3.13 +/- 1.13 microm (0.94~7.20 microm) and 0.43 +/- 0.23 microm (0.07~1.06 microm), respectively. The g-ratio (axon diameter / fiber diameter) of the labeled axons was 0.79 +/- 0.05 (0.61~0.91). Axon diameter was highly correlated with myelin thickness (correlation coefficients,r=0.83) but little correlated with g-ratio (r=-0.33) of individual myelinated parent axons. These results indicate that myelin thickness of the myelinated parent axons innervating rat molar pulps increase with increasing axon diameter, thus maintaining a constant g-ratio.
Animals ; Axons* ; Dental Pulp ; Horseradish Peroxidase ; Humans ; Microscopy, Electron ; Molar* ; Myelin Sheath* ; Parents* ; Rats* ; Trigeminal Ganglion*

BACKGROUND/AIMS: Although mucosal mast cell tryptase is known to significantly increase intestinal permeability, the relationship between mucosal mast cells and intestinal permeability remains unclear. The objective of this study was to evaluate the correlation among intestinal permeability, tryptase activity and mucosal mast cell count. METHODS: Rectal biopsies from 16 patients with diarrhea-predominant irritable bowel syndrome (IBS-D) and 7 normal subjects were assessed for tryptase activity and macromolecular permeability using horseradish peroxidase in Ussing chambers. In addition, mucosal mast cell levels were immunohistochemically quantified via image analysis. RESULTS: Rectal biopsy of tissues from IBS-D patients showed significantly increased permeability compared with those from normal controls (0.644 +/- 0.08 and 0.06 +/- 0.00 ng/2 hr/mm2, P < 0.01). Tryptase activity was also substantially higher in rectal biopsy samples from IBS-D patients than those from normal controls (0.86 +/- 0.18 and 0.28 +/- 0.04 mU/mg protein, P < 0.05). Mucosal mast cell counts were not significantly different between the 2 groups (P > 0.05). However, correlation analysis revealed that only mucosal mast cell count was significantly correlated with intestinal permeability in IBS-D patients (r = 0.558, P < 0.05). CONCLUSIONS: This study demonstrated a positive correlation between the number of mucosal mast cells and intestinal permeability, suggesting that mucosal mast cells play an important role for increased intestinal permeability in patients with IBS-D.
Biopsy ; Diarrhea ; Horseradish Peroxidase ; Humans ; Irritable Bowel Syndrome ; Mast Cells ; Permeability ; Tryptases