High-throughput detection and multiplex identification of cell contaminations

Schmitt, Markus; Pawlita, Michael

doi:10.1093/nar/gkp581

Cited by 125 publications

(127 citation statements)

References 18 publications

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“…Cells were kept under standard conditions in a humidified incubator at 37 °C and 5% CO 2 . They were authenticated28 and regularly checked for contaminations by multiplex PCR29 by the DKFZ Genomics and Proteomics core facility.…”

Section: Methodsmentioning

confidence: 99%

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

et al. 2018

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For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS3‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS3 detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E711–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.

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Section: Methodsmentioning

confidence: 99%

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

et al. 2018

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“…18 A cross-neutralizing monoclonal anti-L2 antibody, K4L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] , highly potent at neutralizing HPV 16 and other high-risk HPV pseudovirions (PSV), fails to neutralize HPV 31. Vice versa, an anti-HPV 31 L2 neutralizing antibody (K3L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] ) is not able to neutralize HPV 16 PSV. Also, anti-HPV 16 L2 polyclonal sera neutralize HPV 31 pseudovirions with a low efficiency (Seitz et al, unpublished data).…”

Section: Resultsmentioning

confidence: 99%

“…Absence of cell-culture contaminations was confirmed by the Multiplex cell Contamination Test. 23 For pseudovirion extraction, 293TT cells were harvested, and the cell pellet was resuspended in 150 ml of lysis buffercontaining 140 ml DBPS, 9 ml 10% Brij58 (w/v; Sigma) and 1 ml RNAse A/T (Fermentas). Cells were rotated overnight at 37 C to induce pseudovirion maturation.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

“…Two monoclonal antibodies, K18L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] and K3L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] , as well as five polyclonal sera were tested on the HPV 31 reference and variant pseudovirions in a neutralization assay. Although K18L2 [20][21][22][23][24][25][26][27][28][29][30][31][32]…”

Section: Neutralization Of Hpv 31 D31n and P34t L2 Variantsmentioning

confidence: 99%

“…Neutralization of HPV 18 P33H, P33N and P29S 1 P33N L2 variants HPV 18 L2 reference, P33H, P33N and P29S þ P33N pseudovirions were tested in an in vitro neutralization assay with cross-neutralizing antibodies K4L2 [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38] and K18L2 20-38 and 10 polyclonal sera.…”

Section: Neutralization Of Hpv 31 D31n and P34t L2 Variantsmentioning

confidence: 99%

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Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2

Seitz

Schmitt

Böhmer³

et al. 2012

Intl Journal of Cancer

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The amino terminus of the human papillomavirus minor capsid protein L2 contains a major cross-neutralizing epitope that provides the basis for the development of a broadly protective HPV vaccine. This attainable broad protection would eliminate one of the major drawbacks of the commercial L1-based prophylactic vaccines. In this study, we asked whether there are natural variants of the L2 cross-neutralizing epitope and if these variants provide means for immune escape from vaccineinduced anti-L2 antibodies. For this, we isolated in silico and in vitro, a total of 477 L2 sequences of HPV types 16, 18, 31, 45, 51, 52 and 58. We identified natural L2 epitope variants for HPV 18, 31, 45 and 51. To determine whether these variants escape L2-directed neutralization, we generated pseudovirions encompassing the natural variants and tested these in an in vitro neutralization assay using monoclonal and polyclonal antibodies. Our results indicate that natural variants of the L2 major neutralizing epitope are frequent among two different study populations from Germany and Mongolia and in the GenBank database. Of two identified HPV 31 L2 single amino acid variants, one could be neutralized well, while the other variant was neutralized very poorly. We also observed that single amino acid variants of HPV 18 and 45 are neutralized well while a HPV 18 double variant was neutralized at significantly lower rates, indicating that L2 variants have to be accounted for when developing HPV L2-based prophylactic vaccines.Human papillomaviruses (HPVs) are the causative agents for the development of cervical cancer. Close to 100% of tumors from the uterine cervix harbor sequences of HPVs; at least 15 different types are linked to cancer, and these are termed as ''oncogenic HPV types.'' 1 Worldwide, the 10 most frequently isolated HPV types in cervical cancer are HPV 16,18,33, 45,31, 58, 52,35, 59 and 56. 2 In general, however, infection with genital HPV is common in adults, and most infections are cleared by the immune system. The most frequent HPV types isolated in women with normal cytology are, in order of prevalence, HPV 16, 42, 58,31,18, 56, 81,35,33, 45 and 52. 3 Although other factors contribute to the malignant transformation process, it is generally accepted that HPV infection is an absolute requirement. 4,5 Built on the recognition of HPV infection in the etiology of cervical cancer, two commercial vaccines, Gardasil V R and Cervarix V R , have been licensed in 2006 and 2007, respectively. 6,7 Both vaccines are based on the L1 major capsid protein and are highly effective in preventing HPV infection and HPV-induced cervical lesions. 8,9 It is expected that both vaccines will reduce the cervical cancer rate in vaccinated women by 70-80%.Both L1-based vaccines have some limitations including costly production, invasive administration and limited protection against nonvaccine HPV types. The latter is due to the fact that neutralizing anti-L1 binds to surface loops on the viral capsids, which are highly heterogeneous among the dif...

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Cell Culture Contamination

Gollin

Lewis

2017

Encyclopedia of Life Sciences

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Cell cultures are valuable models of health and disease, especially in translational research. Cell culture is important for genome editing, stem cell technologies, cancer research, investigating the basic mechanisms involved in development and differentiation, elucidating the role of genetic alterations in normal and abnormal cellular behaviour and developing and testing therapeutics. The clinically important field of classical cytogenetics requires cell culture to obtain metaphase chromosomes from dividing cells at the mitotic stage of the cell cycle when the chromosomes are most compact and visible as distinct entities. Further, culture in vitro facilitates analysis of cells in a controlled environment; minimises morbidity and mortality of human, animal or plant subjects and enables replicate studies over long periods of time. Cell lines are essential models for testing therapeutic efficacy, including drug screening and sensitivity studies. The main difficulty with cell cultures in the clinical or research laboratory is their propensity to contamination, whether by microbes or stray cells from other cultures. It is imperative to verify the authenticity of (authenticate) cell lines and rule out microbial contamination of cell cultures before initiating a series of experiments, at regular intervals during each experimental study and at the end of the study to assure the quality and validity of experimental results. Key Concepts Cell cultures are valuable models in biomedical research. Cell cultures have a propensity to become contaminated by microbes. Intra‐ or interspecies cross‐contamination of cell lines by mislabelling or stray cells from other cell lines results in misidentified cell cultures. High‐quality research results rely on (1) cell line authentication to rule out cross‐contamination and (2) testing cell cultures for and avoiding microbial contamination, at the beginning, at regular intervals during and at the end of a research study. Contaminated or misidentified cell cultures waste both time and money and produce invalid research results.

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High-throughput detection and multiplex identification of cell contaminations

Cited by 125 publications

References 18 publications

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

A Targeted LC‐MS Strategy for Low‐Abundant HLA Class‐I‐Presented Peptide Detection Identifies Novel Human Papillomavirus T‐Cell Epitopes

Natural variants in the major neutralizing epitope of human papillomavirus minor capsid protein L2

Cell Culture Contamination

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