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

Abstract

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...

Liposome and QS-21 Combined Adjuvant Induces theHumoral and Cellular Responses of Acellular Pertussis Vaccine in a Mice Model

“…The PT-functional IgG-titers were measured on day 24 after immunization. The PT neutralization assay was carried out in line with principles described previously with slight modification [ 23 ]. Briefly, two-fold serially diluted sera (CD1) were mixed with 4 CTU 100 (the minimal dose of active PT needed to cause 100% cell clustering in 2 h) of JNIH-5 (2.5 ng/mL) (NIBSC) and incubated for 2 h at 37 °C.…”

“…The PT-functional IgG-titers were measured on day 24 after immunization. The PT neutralization assay was carried out in line with principles described previously with slight modification [23]. Briefly, two-fold serially diluted sera (CD1) were mixed with 4 CTU 100 (the minimal dose of active PT needed to cause 100% cell clustering in 2 h) of JNIH-5 (2.5 ng/mL) (NIBSC) and incubated for 2 h at 37 • C. A total of 50 µL of serum/PT mixtures was added to CHO-K1 cells (3 × 10 4 cells/well) (National Institutes for Food and Drug Control, China) incubated for 48 h at 37 • C. Cells were stained with crystal violet and evaluated for morphological alterations (clustered phenotype) by light microscopy.…”

Pertussis serology: assessment of IgG anti‐PT ELISA for replacement of the CHO cell assay*

“…The CHO cell assay was performed as described by Østergård et al ( 5 ). In brief, 50 μL of a dilution series of serum (1:2 to 1:4096) was mixed with 0.25 ng of PT (Statens Serum Institut, Copenhagen, Denmark) in a microplate and subsequently, 100 μL of CHO cells in culture medium (1 × 10 5 cells/mL) was added.…”

“…Thus, in the IgG anti-PT ELISA, the PT is immobilized on a plastic surface and the amount of human IgG antibodies binding to the toxin is measured colorimetrically ( 4 ). In the CHO cell assay, neutralization of the biological activity of the PT by human antibodies is evaluated by the inhibition of toxin-mediated clustering of CHO cells ( 5 ). In the IgG anti-PT ELISA, the anti-PT antibodies are quantified by automated reading of the amount of produced colour, whereas in the CHO cell assay, the neutralizing anti-PT antibodies are quantified by the use of a dilution series and evaluation of the clustering of the CHO cells by microscopy.…”

“…However, both the IgG anti-PT ELISA and the CHO cell assay have been altered recently. The CHO cell assay was improved by precoating of microplates with foetal calf serum to avoid problems occurring from sera with interfering activity ( 5 ). The IgG anti-PT ELISA was improved by addition of a blocking step including powdered skimmed milk to avoid problems occurring when heat-treated sera were tested ( 4 ).…”