Micro cell culture method for determination of diphtheria toxin and antitoxin titres using VERO cells

Miyamura, Kikuko; Nishio, Shigeko; Ito, Akiharu; Murata, Ryosuke; Kono, Reisaku

doi:10.1016/0092-1157(74)90015-8

Cited by 210 publications

(57 citation statements)

References 4 publications

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“…More recently, an in vitro neutralization assay that uses Vero cells has shown promise as an alternative to these in vivo assays (23,26). In the present study, we utilized both in vitro and in vivo approaches to assign the potency of 315C4.…”

Section: Fig 3 315c4 Inhibits Diphtheria Toxin Cytotoxicity On Vero Cmentioning

confidence: 99%

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

et al. 2013

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Diphtheria antitoxin (DAT) has been the cornerstone of the treatment of Corynebacterium diphtheriae infection for more than 100 years. Although the global incidence of diphtheria has declined steadily over the last quarter of the 20th century, the disease remains endemic in many parts of the world, and significant outbreaks still occur. DAT is an equine polyclonal antibody that is not commercially available in the United States and is in short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we obtained human monoclonal antibodies (hMAbs) directly from antibody-secreting cells in the circulation of immunized human volunteers. We isolated a panel of diverse hMAbs that recognized diphtheria toxoid, as well as a variety of recombinant protein fragments of diphtheria toxin. Forty-five unique hMAbs were tested for neutralization of diphtheria toxin in in vitro cytotoxicity assays with a 50% effective concentration of 0.65 ng/ml for the lead candidate hMAb, 315C4. In addition, 25 g of 315C4 completely protected guinea pigs from intoxication in an in vivo lethality model, yielding an estimated relative potency of 64 IU/mg. In comparison, 1.6 IU of DAT was necessary for full protection from morbidity and mortality in this model. We further established that our lead candidate hMAb binds to the receptor-binding domain of diphtheria toxin and physically blocks the toxin from binding to the putative receptor, heparin-binding epidermal growth factor-like growth factor. The discovery of a specific and potent human neutralizing antibody against diphtheria toxin holds promise as a potential therapeutic.

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Section: Fig 3 315c4 Inhibits Diphtheria Toxin Cytotoxicity On Vero Cmentioning

confidence: 99%

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

et al. 2013

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“…Antibodies to PT and FHA were measured using standard enzyme-linked immunosorbent assays (ELISA), and pertussis antibody titers were expressed as ELISA units (EU)/ml (15). Diphtheria antitoxin titers were examined using the micro cell culture method with Vero cells and expressed as international units (IU)/ml (16). Tetanus antitoxin titers were determined using a KPA kit (Kaketsuken, Japan) and expressed as international units (IU)/ml (17).…”

Section: Methodsmentioning

confidence: 99%

Immunogenicity and Safety after Booster Vaccination of Diphtheria, Tetanus, and Acellular Pertussis in Young Adults: an Open Randomized Controlled Trial in Japan

Hara

Okada

Yamaguchi

et al. 2013

Clin Vaccine Immunol

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The recent increase of pertussis in young adults in Japan is hypothesized to be due in part to waning protection from the acellular pertussis vaccine. While a booster immunization may prevent an epidemic of pertussis among these young adults, little is known about the safety and immunogenicity of such a booster with the diphtheria, tetanus, and acellular pertussis vaccine (DTaP), which is currently available in Japan. One hundred and eleven medical students with a mean age of 19.4 years were randomly divided into 2 groups of 55 and 56 subjects and received, respectively, 0.2 or 0.5 ml of DTaP. Immunogenicity was assessed by performing the immunoassay using serum, and the geometric mean concentration (GMC), GMC ratio (GMCR), seropositive rate, and booster response rate were calculated. Adverse reactions and adverse events were monitored for 7 days after vaccination. After booster vaccination in the two groups, significant increases were found in the antibodies against pertussis toxin, filamentous hemagglutinin, diphtheria toxoid, and tetanus toxoid, and the booster response rates for all subjects reached 100%. The GMCs and GMCRs against all antigens were significantly higher in the 0.5-ml group than in the 0.2-ml group. No serious adverse events were observed. Frequencies of local reactions were similar in the 2 groups, although the frequency of severe local swelling was significantly higher in the 0.5-ml group. These data support the acceptability of booster immunization using both 0.2 and 0.5 ml of DTaP for young adults for controlling pertussis. (This study was registered at UMIN-CTR under registration number UMIN000010672.)

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“…Neutralizing antibody assay. Serum samples were tested for specific antidiphtheria toxin-neutralizing antibodies using a method similar to Miyamura et al (26) as described previously (24). Briefly, serial twofold or threefold dilutions of serum or standard antitoxin for diphtheria (equine antisera from CBER, FDA, Bethesda, Md.…”

Section: Methodsmentioning

confidence: 99%

Induction of Protective Serum Meningococcal Bactericidal and Diphtheria-Neutralizing Antibodies and Mucosal Immunoglobulin A in Volunteers by Nasal Insufflations of theNeisseria meningitidisSerogroup C Polysaccharide-CRM197 Conjugate Vaccine Mixed with Chitosan

et al. 2005

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Thirty-six healthy volunteers received either a single intramuscular injection of Neisseria meningitidis serogroup C polysaccharide (MCP)-CRM197 conjugate vaccine in alum or two nasal insufflations 28 days apart of the same vaccine powder, without alum, mixed with chitosan. Nasal immunization was well tolerated, with fewer symptoms reported than after intramuscular injection. The geometric mean concentrations of MCPspecific immunoglobulin G (IgG) after one nasal immunization were 3.25 g/ml in naïve subjects and 14.4 g/ml in subjects previously immunized parenterally, compared with 4.30 g/ml in naïve subjects immunized intramuscularly. The geometric mean titer of serum bactericidal antibody (SBA) rose 24-fold after two nasal immunizations in naïve subjects and was comparable to parenteral immunization (1,080 versus 1,625). All subjects achieved SBA titers associated with protection after two nasal immunizations: even those with titers of <8 at entry. A single nasal immunization boosted the SBA titer to >128 in 96% of previously immunized subjects, and two immunizations achieved this level in 92% of naive subjects. MCP-specific IgG levels were ϳ70% IgG2 and ϳ20% IgG1 after nasal or intramuscular immunization. Increases in CRM197-specific IgG and diphtheria toxin-neutralizing activity were observed after nasal or intramuscular immunization, with balanced IgG1/IgG2 and higher IgG4. Significant MCP-specific secretory IgA was detected in nasal wash only after nasal immunization and predominantly on the immunized side. Simple nasal insufflation of existing MCP-CRM197 conjugate vaccines in chitosan offers an inexpensive but effective needle-free prime and boost against serogroup C N. meningitidis and diphtheria.

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Micro cell culture method for determination of diphtheria toxin and antitoxin titres using VERO cells

Cited by 210 publications

References 4 publications

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

Identification of a Human Monoclonal Antibody To Replace Equine Diphtheria Antitoxin for Treatment of Diphtheria Intoxication

Immunogenicity and Safety after Booster Vaccination of Diphtheria, Tetanus, and Acellular Pertussis in Young Adults: an Open Randomized Controlled Trial in Japan

Induction of Protective Serum Meningococcal Bactericidal and Diphtheria-Neutralizing Antibodies and Mucosal Immunoglobulin A in Volunteers by Nasal Insufflations of theNeisseria meningitidisSerogroup C Polysaccharide-CRM197 Conjugate Vaccine Mixed with Chitosan

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