Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

Kuhne, Maren; Dippong, Martin; Flemig, Sabine; Hoffmann, Katrin; Petsch, Kristin; Schenk, Jörg A.; Kunte, Hans-Jörg; Schneider, R.

doi:10.1016/j.jim.2014.07.004

Cited by 14 publications

(11 citation statements)

References 46 publications

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“…To study the surface expression of TREM2, HeLa cells expressed with TREM2 constructs were washed with cold PBS and were stained with goat anti‐TREM2 and secondary antibody coupled with Alexa Fluor ® 488. Cells were fixed for 30 min with 2% buffered formalin and washed with PBS . Expression of TREM2 on the cell membrane was measured by flow cytometry using a BD FACSCanto™ II Flow Cytometry System.…”

Section: Methodsmentioning

confidence: 99%

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Disease‐Associated Mutations of TREM2 Alter the Processing of N‐Linked Oligosaccharides in the Golgi Apparatus

Park

et al. 2015

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The triggering receptor expressed on myeloid cells 2 (TREM2) is an immune-modulatory receptor involved in phagocytosis and inflammation. Mutations of Q33X, Y38C and T66M cause Nasu-Hakola disease (NHD) which is characterized by early onset of dementia and bone cysts. A recent, genome-wide association study also revealed that single nucleotide polymorphism of TREM2, such as R47H, increased the risk of Alzheimer's disease (AD) similar to ApoE4. However, how these mutations affect the trafficking of TREM2, which may affect the normal functions of TREM2, was not known. In this study, we show that TREM2 with NHD mutations are impaired in the glycosylation with complex oligosaccharides in the Golgi apparatus, in the trafficking to plasma membrane and further processing by γ-secretase. Although R47H mutation in AD affected the glycosylation and normal trafficking of TREM2 less, the detailed pattern of glycosylated TREM2 differs from that of the wild type, thus suggesting that precise regulation of TREM2 glycosylation is impaired when arginine at 47 is mutated to histidine. Our results suggest that the impaired glycosylation and trafficking of TREM2 from endoplasmic reticulum/Golgi to plasma membrane by mutations may inhibit its normal functions in the plasma membrane, which may contribute to the disease.

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Section: Methodsmentioning

confidence: 99%

“…Cells were fixed for 30 min with 2% buffered formalin and washed with PBS (35). Expression of TREM2 on the cell membrane was measured by flow cytometry using a BD FACSCanto ™ II Flow Cytometry System.…”

Section: Flow Cytometrymentioning

confidence: 99%

Disease‐Associated Mutations of TREM2 Alter the Processing of N‐Linked Oligosaccharides in the Golgi Apparatus

Park

et al. 2015

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“…Identification of antibodies with the desired specificity is frequently accomplished by immunological techniques such as the enzyme-linked immunosorbent assay (ELISA). Alternative protocols that optimize the screening for antibody-producing cells using flow cytometry are also available [ 14 , 15 ]. These techniques do not, however, provide complete information regarding the heterogeneity of the hybridoma clones.…”

Section: Introductionmentioning

confidence: 99%

Production of highly and broad-range specific monoclonal antibodies against hemagglutinin of H5-subtype avian influenza viruses and their differentiation by mass spectrometry

Sa̧czyńska

Bierczyńska-Krzysik

Cecuda-Adamczewska

et al. 2018

Virol J

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BackgroundThe highly pathogenic avian influenza viruses of the H5 subtype, such as the H5N1 viral strains or the novel H5N8 and H5N2 reassortants, are of both veterinary and public health concern worldwide. To combat these viruses, monoclonal antibodies (mAbs) against H5 hemagglutinin (HA) play a significant role. These mAbs are effective diagnostic and therapeutic agents and powerful tools in vaccine development and basic scientific research. The aim of this study was to obtain diagnostically valuable mAbs with broad strain specificity against H5-subtype AIVs.ResultsWe applied the hybridoma method to produce anti-HA mAbs. The cloning and screening procedures resulted in the selection of 7 mouse hybridoma cell lines and their respective antibody clones. Preliminary immunoreactivity studies showed that these newly established mAbs, all of the IgG1 isotype, had high specificity and broad-range activities against the H5 HAs. However, these studies did not allow for a clear distinction among the selected antibodies and mAb-secreting hybridoma clones. To differentiate the analyzed mAbs and determine the exact number of hybridoma clones, peptide mapping of the Fc and Fab fragments was performed using a Matrix-Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF/TOF) mass spectrometer. Detailed analyses of the acquired MS and MS/MS spectra confirmed that the Fc fragments constituted highly conserved species- and isotype-immunoglobulin components, whereas the Fab fragments exhibited considerable variation in the sequences that determine antibody specificity. This approach enabled unambiguous characterization of the selected mAbs according to their peptide composition. As a result, 6 different clones were distinguished.ConclusionsOur work provided a unique panel of anti-H5 HA mAbs, which meets the demand for novel, high-specificity analytical tools for use in serologic surveillance. Applications of these mAbs in areas other than diagnostics are also possible. Moreover, we demonstrated for the first time that peptide mapping of antibody fragments with mass spectrometry is an efficient method for the differentiation of antibody clones and relevant antibody-producing cell lines. The method may be successfully used to characterize mAbs at the protein level.Electronic supplementary materialThe online version of this article (10.1186/s12985-017-0886-2) contains supplementary material, which is available to authorized users.

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“…However, in the present study, no hybridoma secreting anti-BSA mAb was screened out using the SAL-BSA conjugate for antibody screening, even though the FBS content in the supernatant was reduced from 10% to 1.0%. Typically, the mAb content in the supernatant of hybridoma is less than 10 μg/mL, which is equivalent to 70 nmol mAb per liter (Kuhne et al 2014; Tomita et al 2011). Meanwhile, the average content of albumin in FBS is about 23g/L, which is equivalent to 350 μmol BSA per liter of the hybridoma culture supernatant (10% FBS) (Yang and Xiong 2012).…”

Section: Discussionmentioning

confidence: 99%

Bovine serum albumin as an immunogenic carrier facilitating the development of hapten-specific monoclonal antibodies

Zhao

Jiang²

2020

Preprint

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There is a common misconception that the generation of hapten-specific monoclonal antibodies (mAbs) requires the use of a heterologous conjugate to ensure carrier-specific antibodies not being detected. In this study, salbutamol (SAL) was used as a model hapten to exhibit the benefits of bovine serum albumin (BSA) as a carrier for developing hapten-specific mAbs. SAL-BSA conjugate would serve as both an immune antigen and a screening antigen during the preparation of SAL-specific mAbs. Six hybridomas were identified to secret mAbs specific for free SAL with minor or negligible cross-reactivity with other β-agonists. Meanwhile, none of hybrodomas secreting anti-BSA antibodies were screened out even though the fetal bovine serum (FBS) added to the medium decreased from 10% to 1% (v/v). Based on one of the six mAbs, 3F12, a direct competitive enzyme-linked immunosorbent assay (dcELISA) was developed for meausring SAL. Under the optimized assay, the quantitative working range was from 312.5 to 20,000 pg/mL (R2 = 0.9959), with a limit of detection (LOD) of 142.9 pg/mL. The results showed that BSA is an efficient and suitable protein carrier for facilitating the development of hapten-specific mAbs.

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Comparative characterization of mAb producing hapten-specific hybridoma cells by flow cytometric analysis and ELISA

Cited by 14 publications

References 46 publications

Disease‐Associated Mutations of TREM2 Alter the Processing of N‐Linked Oligosaccharides in the Golgi Apparatus

Disease‐Associated Mutations of TREM2 Alter the Processing of N‐Linked Oligosaccharides in the Golgi Apparatus

Production of highly and broad-range specific monoclonal antibodies against hemagglutinin of H5-subtype avian influenza viruses and their differentiation by mass spectrometry

Bovine serum albumin as an immunogenic carrier facilitating the development of hapten-specific monoclonal antibodies

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