The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells

Gillenius, Pekka; Jäätmaa, Eva; Askelöf, Per; Granström, Marta; Tiru, Mandayam

doi:10.1016/s0092-1157(85)80034-2

Cited by 170 publications

(74 citation statements)

References 4 publications

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“…Neutralizing antibodies to PTx were measured as the dilution of serum that inhibited complete PTx-induced clustering of Chinese hamster ovary (CHO) cells (12,18). Test sera were diluted directly in wells of sterile 96-well tissue culture plates in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) using 1:10 dilutions.…”

Section: Measurement Of Neutralizing Antibodies By Cho Cell Assaymentioning

confidence: 99%

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Sutherland

Chang

Yoder

et al. 2011

Clin. Vaccine Immunol.

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Despite more than 50 years of vaccination, disease caused by the bacterium Bordetella pertussis persists, with rates increasing in industrialized countries over the past decade. This rise may be attributed to several factors, including increased surveillance, emergence of vaccine escape variants, waning immunity in adults, and the introduction of acellular subunit vaccines, which include chemically detoxified pertussis toxin (PTd). Two potently protective epitopes on pertussis toxin (PTx) are recognized by the monoclonal antibodies 1B7 and 11E6, which inhibit catalytic and cell-binding activities, respectively. In order to determine whether the PTx exposure route affects antibody responses to these epitopes, we analyzed sera from 30 adults with confirmed pertussis exposure and from 30 recently vaccinated adults for specific anti-PTx antibody responses and in vitro CHO cell neutralization titers. While overall titers against PTx and the genetically detoxified variant, PTg, containing the R9K and E129G substitutions, were similar in the two groups, titers against specific epitopes depended on the exposure route. Natural infection resulted in significantly higher titers of anti-PTx-subunit 1, 1B7-like, and 11E6-like antibodies, while acellular vaccination resulted in significantly higher titers of antibodies recognizing PTd. We also observed a correlation between in vitro protection and the presence of 1B7-like and 11E6-like antibodies. Notably, chemical detoxification, as opposed to genetic inactivation, alters the PTx tertiary and quaternary structure, thereby affecting conformational epitopes and recognition of PTx by 1B7 and 11E6. The lower levels of serum antibodies recognizing clinically relevant epitopes after vaccination with PTd support inclusion of PTg in future vaccines.Pertussis vaccines, widely introduced as an inactivated whole-cell vaccine in 1950, have been responsible for a dramatic decline in pertussis-related morbidity and mortality but have been unable to eradicate disease despite 95% coverage in many areas. Disturbingly, rates of confirmed pertussis cases in industrialized countries have increased steadily

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Section: Measurement Of Neutralizing Antibodies By Cho Cell Assaymentioning

confidence: 99%

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Sutherland

Chang

Yoder

et al. 2011

Clin. Vaccine Immunol.

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“…The clustering effect induced by pertussis toxin (PT) can be used when samples are analyzed in clinical trials of vaccines containing pertussis toxoid, since neutralizing antibodies against pertussis toxoid have been observed to inhibit this effect (1).…”

mentioning

confidence: 99%

“…This method works well with a few samples, but with multiple samples to be analyzed, like those in clinical trials, it is convenient to titrate serum samples with pertussis toxin in the titer wells and subsequently add cells to the mixture in the wells (1). This sequence of additions to titer wells is the same as that used in the methods to determine protection against diphtheria toxin and poliovirus, where serum samples are titrated with diphtheria toxin or poliovirus in titer wells before the addition of Vero cells (5,6).…”

mentioning

confidence: 99%

Elimination of Interfering Activity in Serum Samples in the Chinese Hamster Ovary Pertussis Serology Assay

Østergaard

Sørensen

Nielsen

et al. 2008

Clin Vaccine Immunol

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An interfering substance in various frozen serum samples was observed to inhibit the adhesion of Chinese hamster ovary (CHO) cells to microplate surfaces during a CHO pertussis neutralization test, resulting in wells that lacked cells or wells with dead cells after 2 days of incubation. The interfering activity in the serum could be eliminated by (i) transferring cells to other wells after their initial incubation, (ii) adding fetal calf serum (FCS) to the sample dilution buffer, (iii) precoating microplates with FCS, or (iv) preincubating the samples at 4°C for 5 days. Preincubating the samples at 4°C for 5 days reduced the interfering activity in only some of the samples. Adding serum to the sample dilution buffer or precoating the microplates with serum did not influence the antibody titers in the serum samples. The method described may be used for routine applications.Toxin from Bordetella pertussis has been observed to induce clustering of Chinese hamster ovary (CHO) cells (4). The clustering effect induced by pertussis toxin (PT) can be used when samples are analyzed in clinical trials of vaccines containing pertussis toxoid, since neutralizing antibodies against pertussis toxoid have been observed to inhibit this effect (1).Cells are initially seeded into wells of titer plates, and subsequently, various concentrations of serum samples that have been preincubated with pertussis toxin are added to the adherent cells in the wells, according to the method described by Hewlett et al. (4). This method works well with a few samples, but with multiple samples to be analyzed, like those in clinical trials, it is convenient to titrate serum samples with pertussis toxin in the titer wells and subsequently add cells to the mixture in the wells (1). This sequence of additions to titer wells is the same as that used in the methods to determine protection against diphtheria toxin and poliovirus, where serum samples are titrated with diphtheria toxin or poliovirus in titer wells before the addition of Vero cells (5, 6).When serum samples are analyzed for neutralizing antibodies to pertussis toxin by the method where cells are added to preincubated mixtures of serum samples and pertussis toxin, we have observed that interfering activity in quantities ranging from a few percent to 50% of the samples interferes with reading of the cells. This is in agreement with observations by others who have observed toxic activity in serum samples (1-3, 7, 8). The interfering activity could be abolished by using the original method described by Hewlett, but since this method is too laborious to use in clinical trials, we have, instead, improved the method where cells are added to the wells subsequent to the serum samples and the pertussis toxin. MATERIALS AND METHODSCells and media. CHO cells purchased from ATCC (CHO-K1, ATCC CCL-61) were cultured in a culture medium consisting of Ham's F12K (Gibco) supplemented with 8% heat-inactivated fetal calf serum (FCS; Gibco), 0.001% gentamicin, and 0.15% sodium bicarbonate in 175-cm 2 flasks at...

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“…Neutralization of pertussis toxin toxicity to Chinese hamster ovary (CHO) cells was determined as previously described (7,10). An enzyme-linked immunosorbent assay (ELISA) was used to measure immunoglobulin G (IgG) antibodies to pertussis toxin.…”

mentioning

confidence: 99%

“…The CHO cell cytotoxicity assay was the first in vitro assay for assessing neutralizing antibodies to pertussis toxin (7,10). However, neutralization of pertussis toxin with the CHO cell assay is a poor predictor of protection from disease.…”

mentioning

confidence: 99%

Antibody-Mediated Neutralization of Pertussis Toxin-Induced Mitogenicity of Human Peripheral Blood Mononuclear Cells

et al. 2004

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Antibody-mediated neutralization of pertussis toxin-induced proliferation of human peripheral blood mononuclear cells (PBMC) was assessed using alamarBlue and compared with results from the Chinese hamster ovary (CHO) cell assay using sera from vaccinated adults and convalescent children. Neutralization values for the CHO assay were similar for vaccinated and convalescent subjects; however. the convalescent group had higher titers in the PBMC assay. Results for pertussis toxin neutralization with the CHO assay appear to be distinct from those with the PBMC assay.

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The standardization of an assay for pertussis toxin and antitoxin in microplate culture of Chinese hamster ovary cells

Cited by 170 publications

References 4 publications

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Antibodies Recognizing Protective Pertussis Toxin Epitopes Are Preferentially Elicited by Natural Infection versus Acellular Immunization

Elimination of Interfering Activity in Serum Samples in the Chinese Hamster Ovary Pertussis Serology Assay

Antibody-Mediated Neutralization of Pertussis Toxin-Induced Mitogenicity of Human Peripheral Blood Mononuclear Cells

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