No abstract available.
B-Lymphocytes* ; Lymph Nodes* ; Pertussis Toxin* ; Whooping Cough*

OBJECTIVETo develop a time-resolved fluoroimmunoassay (TRFIA) for detection of pertussis toxin (PT) S1 subunit for quality control of human PT vaccine.

METHODSA double antibody sandwich one-step method was used to establish the TRFIA for detecting PT S1 subunit in the vaccine.

RESULTSThe sensitivity of c peptide analysis reached 2.5 ng/ml without cross-reactions with other antigens. This assay could be used in detecting S1 subunit in the vaccine.

CONCLUSIONThe TRFIA for detecting PT S1 subunit is simple, sensitive and rapid for quality control of the PT vaccine.

Cross Reactions ; Fluoroimmunoassay ; methods ; Pertussis Toxin ; analysis ; Pertussis Vaccine ; chemistry ; standards ; Quality Control ; Sensitivity and Specificity

PURPOSE: We conducted the study to evaluate the immunogenicity and safety of three component DTaP vaccine (Infanrix(R)) in a group of Korean healthy infants on a three-dose primary vaccination. And we compared the immunogenicity of this DTaP vaccine with two component DTaP vaccine which has been widely used in Korea. METHODS: We enrolled one hundred fifty one healthy infants aged 8-9 weeks. These infants were vaccinated at age 2, 4 and 6 months of age with three component DTaP vaccine. Solicited adverse events were actively monitored for 72 hours following each vaccination, and all adverse events after each vaccination were observed for three weeks. Anti-diphtheria toxoid Ab., anti-tetanus toxoid Ab., anti-pertussis toxin Ab., anti-filamentous hemagglutinin Ab., and anti-pertactin Ab. were measured using ELISA for assessing immunogenicity of study vaccine in 60 infants. Immunogenicity analysis of two component DTaP vaccine was performed with same methods in 14 infants as control. RESULTS: The seroconversion rates of anti-diphtheria toxoid Ab, anti-tetanus toxoid Ab. anti- filamentous hemagglutinin Ab. were 100% in both group. Seroconversion rate of anti-pertactin Ab in study group was 100%, but the rate in control group was 50%. However, geometric mean concentration of anti-pertussis toxin Ab. was higher in control group. Mild local and systemic reactions were observed within three days after vaccination, and no serious adverse events related study vaccine were happened during study period. CONCLUSIONS: Our study results suggest that three component DTaP vaccine (Infanrix(R)) is a well- tolerable and high immunogenic vaccine, especially anti-Pertactin Ab. of the study vaccine is very immunogenic. It can be available as routine DTaP vaccination in our infants.
Diphtheria-Tetanus-acellular Pertussis Vaccines* ; Enzyme-Linked Immunosorbent Assay ; Hemagglutinins ; Humans ; Infant* ; Korea ; Pertussis Toxin ; Vaccination

PURPOSE: Active reduced dose tetanus-diphtheria-acellular pertussis (Tdap) vaccination for adolescents and adults is necessary because waning immunity after primary diphtheria-tetanus-pertussis vaccination is related to the recent emergence of pertussis. This study was conducted to compare the immunogenicity and protection efficacy against Bordetella pertussis between a new GCC Tdap vaccine and a commercially available Tdap vaccine in a murine model. MATERIALS AND METHODS: BALB/c mice were immunized with two doses of diphtheria-tetanus-acellular pertussis (DTaP) vaccine for priming and a subsequent Tdap booster vaccination. According to the type of booster vaccine, mice were divided into four groups: commercially available Tdap vaccine in group 1 and GCC Tdap vaccines of different combinations of pertussis antigens in groups 2 to 4. Humoral and cell-mediated immune responses and protection efficacy using a murine intranasal challenge model after booster vaccination were compared among the four groups. RESULTS: Every group showed significant increases in antibody titers against pertussis antigens such as pertussis toxin, filamentous hemagglutinin, and pertactin after booster vaccination. Spleen cells showed both Th1 and Th2 cell-mediated immune responses stimulated by pertussis antigens in all groups without any significant difference. In the intranasal B. pertussis infection model, bacteria were eradicated in all groups five days after challenge infection. CONCLUSION: This preliminary study did not show significantly different immunogenicity or protection efficacy of the new GCC Tdap vaccines compared to the commercially available Tdap vaccine, although a more extensive study is necessary to assess the differing efficacies of the new GCC Tdap vaccines.
Adolescent ; Adult ; Animals ; Bacteria ; Bordetella pertussis* ; Hemagglutinins ; Humans ; Mice ; Pertussis Toxin ; Republic of Korea ; Spleen ; Vaccination ; Vaccines ; Whooping Cough

BACKGROUND: Thyroid goiters are very common, however, the mechanism of development is not fully understood. A TSH receptor has been known to activate two different signaling pathways the cAMP/protein kinase A(PKA) and phospholipase C(PLC)/protein kinase C(PKC) systems. However, both systems are limited in the degree to which they explain the discrepancy between a goiter and TSH receptor activation. It has recently been reported that the expression of p66 Shc was increased by TSH stimulation in thyrocytes, suggesting that the p66 Shc molecule may play a critical role in the transition of the TSH-induced growth signals. METHODS AND RESULTS: In this study, we examined the expression of p66 Shc by stimulation of TSH, and the regulatory mechanisms of the TSH-induced expression of the p66 Shc in FRTL-5 cells. In FRTL-5 cells, TSH could increase the expression of the p66 Shc, and the this expression was decreased to basal levels after the removal of TSH. The TSH-induced p66 Shc expression was competitively inhibited by TSH receptor blocking antibodies. The increments of the expression of the p66 Shc protein caused by TSH were both time and concentration dependent, and it was same in the mRNA levels. Cholera toxin increased the expression of the p66 Shc, while pertussis toxin did not. The activators of the cAMP/PKA pathway (8-bromo-cAMP and forskolin) also stimulated the expression of p66 Shc, and the PKA inhibitor H89 decreased the expression, while the inhibition of the PKC pathway by GF109203X, or PMA, affected the expression of p66 Shc very little. CONCLUSION: Our data suggests that p66 Shc may play an important role in regulating the growth of thyrocytes. The TSH receptor - Gs protein - adenylate cyclase - cAMP - PKA pathway mainly mediates the TSH effects on the expression of p66 Shc molecules.
Adenylyl Cyclases ; Antibodies, Blocking ; Cholera Toxin ; Goiter ; Pertussis Toxin ; Phospholipases ; Phosphotransferases ; Receptors, Thyrotropin ; RNA, Messenger ; Thyroid Gland

Acellular pertussis vaccine has been used widely in Korea since 1984. However, because many of the former generations were not inoculated with pertussis vaccine, they may infect infants with pertussis. With this background, we investigated the prevalence of pertussis antibodies in all age groups. Enzyme-linked immunosorbent assay (ELISA) to assess IgG antibodies to pertussis toxin (PT) and filamentous hemagglutinin (FHA) and bacterial agglutination (BA) to assess antibodies to agglutinogen were compared on 842 serum samples which were donated from 11 hospitals in Seoul area. In comparison with age groups under 20 years, antibodies of adults against PT and FHA were maintained. But antibodies against agglutinogen showed no pattem in all age groups. Antibodies to PT were correlated with antibodies to FHA. There was no significant difference in antibody levels between male and female (p<0.05).
Adult ; Agglutination ; Antibodies ; Bordetella pertussis ; Enzyme-Linked Immunosorbent Assay ; Family Characteristics ; Female ; Hemagglutinins ; Humans ; Immunoglobulin G ; Infant ; Korea* ; Male ; Pertussis Toxin ; Pertussis Vaccine ; Prevalence ; Seoul ; Whooping Cough*

OBJECTIVE: This study is to explore the effects on specific bindings between [ 3H]RX821002, alpha-2 adrenergic receptor antagonist and alpha-2 adrenergic receptor in rat brain by G-protein modulation. METHODS: The radioligand binding receptor study was conducted with [ 3H]RX821002, a new alpha-2 adrenergic receptor antagonist, in the presence or absence of Gpp(NH)p and pertussis toxin. RESULTS: The alpha-2 adrenergic receptors were saturated with [ 3H]RX821002 in the fashion of the single binding site. The dissociation constant (Kd) was 0.70+/-0.30 nM, and maximum binding (Bmax) was 599.9+/-283.4 fmol/mg protein. The saturation study showed that the maximum binding (B max ; 668.0+/-50.1 fmol/mg protein) was increased and the dissociation constant (Kd ; 0.61+/-0.14 nM) was decreased significantly in the presence of Gpp (NH)p compared to those (B max ; 559.8+/-81.9 fmol/mg protein, Kd ; 0.87+/-0.14 nM) in the absence of Gpp (NH)p (by paired t-test ; B max, p=0.023, Kd, p=0.005). In the presence of pertussis toxin, the maximum binding (B max ; 617.0+/-58.5 fmol/mg protein) was increased significantly (by paired t-test ; B max, p=0.001) but the issociation constant (Kd ; 0.92+/-0.24 nM) was not decreased compared to those (B max ; 554.1+/-66.1 fmol/mg protein, Kd ; 0.89+/-0.24 nM) in the absence of pertussis toxin. CONCLUSION: These results confirm that the binding profiles between [ 3H]RX821002 and alpha-2 adrenergic receptors be modified by G-protein modulation. This suggests that the drug effects on receptors be influenced by various conditions such as G-protein modulation.
Animals ; Binding Sites ; Brain* ; GTP-Binding Proteins* ; Guanylyl Imidodiphosphate ; Pertussis Toxin ; Rats* ; Receptors, Adrenergic, alpha-2

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

PURPOSE: There is no standard method for confirming the immunogenicity of acellular pertussis vaccines. We tried to develop a local standard method for evaluating the immunogenicity of the three-component of acellular pertussis vaccines which was developed by a Korean local company. MATERIALS AND METHODS: The developed pertussis antigens (pertussis toxin, filamentous hemagglutinin, pertactin) were evaluated by in-house enzyme-linked immunosorbent assay (ELISA) using 189 negative sera, 25 positive sera, and 73 paired sera (pre- and post-Tdap [tetanus, diphtheria, and acellular pertussis] vaccinated sera). ELISA units were calculated by the reference line method, compared with World Health Organization reference sera, and the cut-off value was calculated using negative sera. RESULTS: When compared to National Institute for Biological Standards and Control control antigen (NIBSC) control antigens, the developed pertussis toxin (PT) and filamentous haemagglutinin (FHA) antigens were 203.48 and 61.60 IU/µg, respectively. Each in-house ELISA was established by validating the coefficients of variation % (PT, 11.53%; FHA, 8.60%; pertactin [PRN], 9.86%) obtained from the results of inter- and intra-assay variation. Also, the cut-off values of PT, FHA, and PRN were 11.65, 38.95, and 5.66 EU/mL, respectively. The distributions of antibody levels in paired showed that 93.15% (68/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were higher than a 100% increase after vaccination. Additionally, the values of 89.04% (65/73) in anti-PT IgG, 97.26% (72/73) in anti-FHA IgG, and 100% in anti-PRN IgG were below each cut-off point. CONCLUSION: We established an in-house ELISA method using self-developed antigens, and these immunoassays have provided a way to standardize measuring the immunogenicity of newly developed vaccines, through single- and dual-serology.
Diphtheria ; Enzyme-Linked Immunosorbent Assay ; Hemagglutinins ; Immunoassay ; Immunoglobulin G ; Korea* ; Methods* ; Pertussis Toxin ; Pertussis Vaccine ; Vaccination ; Vaccines* ; Whooping Cough* ; World Health Organization

BACKGROUND: Extracellular nucleotides act as agonists to regulate a broad range of physiological processes by interacting with P2 receptors in various tissues including the kidney tubules. This study was undertaken to evaluate the effect of P2 receptor activation on PTH-dependent regulation of phosphate transport in the renal proximal tubular cells. METHODS: Proximal tubular cells were isolated from the rabbit kidney and grown as monolayers on 24 well culture plates. Phosphate uptake was determined by measuring the uptake of radiolabeled phosphate into cell monolayers. Cyclic AMP content was determined by radioimmunoassay using [3H]cAMP assay kit. RESULTS: Activation of P2 receptors with ATP exerted differential effects on phosphate uptake and cAMP generation. In the absence of PTH, it inhibited phosphate uptake and stimulated cAMP generation. In contrast, in the presence of PTH, it attenuated PTH-induced stimulation of cAMP generation and inhibition of phosphate uptake. The profile of the effects of different P2 agonists suggested that P2Y1- and P2Y2-like receptors are involved in the effects of ATP. The effect of ATP to interfere with the PTH-induced regulation was significantly blocked by calphostin C, pertussis toxin or PKC-depletion, whereas, the effects of ATP in the absence of PTH were abolished by indomethacin. CONCLUSION: Our results suggest that PKC-dependent modification of Gi proteins and, subsequently, reduced responsiveness of adenylate cyclases is responsible for the attenuating effect of ATP on the PTH-dependent regulation of phosphate transport in rabbit proximal tubule cells.
Adenosine Triphosphate ; Adenylyl Cyclases ; Cyclic AMP ; Indomethacin ; Kidney ; Kidney Tubules ; Nucleotides ; Parathyroid Hormone ; Pertussis Toxin ; Physiological Processes ; Radioimmunoassay