Hybridoma Technology for the Generation of Monoclonal Antibodies

Zhang, Chonghui

doi:10.1007/978-1-61779-931-0_7

Cited by 96 publications

(62 citation statements)

References 40 publications

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“…Hybridomas were derived by conventional procedure (66). Briefly, myeloma partner cells (P3X63Ag8.653, ATCC) were maintained in RPMI 1640 (Corning) supplemented with 10% FBS.…”

Section: Cellsmentioning

confidence: 99%

“…Selected hybridoma clones were grown in RPMI 1640 supplemented with 15% FBS and 10% HCS. Each clone underwent two rounds of single cell subcloning to ensure the clonality of the antibody (66). Collected hybridoma supernatant was added with 20 mM sodium phosphate buffer (pH 7.0) and NAb purified using a HiTrap Protein G HP 5-ml column (GE Healthcare) according to the manufacturer's instructions.…”

Section: Cellsmentioning

confidence: 99%

See 1 more Smart Citation

Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

Chiuppesi

Wussow

Johnson

et al. 2015

J Virol

127

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Human cytomegalovirus (HCMV) elicits neutralizing antibodies (NAb) of various potencies and cell type specificities to prevent HCMV entry into fibroblasts (FB) and epithelial/endothelial cells (EpC/EnC). NAb targeting the major essential envelope glycoprotein complexes gB and gH/gL inhibit both FB and EpC/EnC entry. In contrast to FB infection, HCMV entry into EpC/EnC is additionally blocked by extremely potent NAb to conformational epitopes of the gH/gL/UL128/130/131A pentamer complex (PC). We recently developed a vaccine concept based on coexpression of all five PC subunits by a single modified vaccinia virus Ankara (MVA) vector, termed MVA-PC. Vaccination of mice and rhesus macaques with MVA-PC resulted in a high titer and sustained NAb that blocked EpC/EnC infection and lower-titer NAb that inhibited FB entry. However, antibody function responsible for the neutralizing activity induced by the MVA-PC vaccine is uncharacterized. Here, we demonstrate that MVA-PC elicits NAb with cell type-specific neutralization potency and antigen recognition pattern similar to human NAb targeting conformational and linear epitopes of the UL128/130/ 131A subunits or gH. In addition, we show that the vaccine-derived PC-specific NAb are significantly more potent than the anti-gH NAb to prevent HCMV spread in EpC and infection of human placental cytotrophoblasts, cell types thought to be of critical importance for HCMV transmission to the fetus. These findings further validate MVA-PC as a clinical vaccine candidate to elicit NAb that resembles those induced during HCMV infection and provide valuable insights into the potency of PC-specific NAb to interfere with HCMV cell-associated spread and infection of key placental cells. IMPORTANCEAs a consequence of the leading role of human cytomegalovirus (HCMV) in causing permanent birth defects, developing a vaccine against HCMV has been assigned a major public health priority. We have recently introduced a vaccine strategy based on a widely used, safe, and well-characterized poxvirus vector platform to elicit potent and durable neutralizing antibody (NAb) responses targeting the HCMV envelope pentamer complex (PC), which has been suggested as a critical component for a vaccine to prevent congenital HCMV infection. With this work, we confirm that the NAb elicited by the vaccine vector have properties that are similar to those of human NAb isolated from individuals chronically infected with HCMV. In addition, we show that PCspecific NAb have potent ability to prevent infection of key placental cells that HCMV utilizes to cross the fetal-maternal interface, suggesting that NAb targeting the PC may be essential to prevent HCMV vertical transmission. Human cytomegalovirus (HCMV) is the most common infectious cause of permanent births defects worldwide, often resulting in auditory and cognitive abnormalities and in rare cases even in multiorgan failure and death (1-4). Congenital HCMV infection occurs in 0.05 to 1% of all pregnancies, and 10 to 25% of congenitally infected newborns d...

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“…Hybridomas were derived by conventional procedure (66). Briefly, myeloma partner cells (P3X63Ag8.653, ATCC) were maintained in RPMI 1640 (Corning) supplemented with 10% FBS.…”

Section: Cellsmentioning

confidence: 99%

Section: Cellsmentioning

confidence: 99%

Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

Chiuppesi

Wussow

Johnson

et al. 2015

J Virol

127

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show abstract

“…Monoclonal antibodies (mAbs) were prepared with standard Hybridoma technology (Zhang, 2012). Balb/c mice were immunized with three injections (0, 2, 4 weeks) of simian rotavirus SA11.…”

Section: Monoclonal Antibodies and Seramentioning

confidence: 99%

Development of an enzyme-linked immunospot assay for determination of rotavirus infectivity

Lin

et al. 2014

Journal of Virological Methods

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“…For mammalian cell applications, immortalized rat, mouse, hamster and human hybrids have been developed and utilized since the 1960s (1)(2)(3)(4). These hybrid eukaryotic cell lines have been useful scientific tools for chromosomal and genomic mapping of many genes including tumor suppressor genes and also important for monoclonal antibody production (5,6). In some of these hybrids, the presence of a selection marker (i.e., antibiotic resistance genes) on a specific human chromosome allowed for preferential loss of a majority of the other human chromosomes in the hybrid and the retention of marked human chromosome.…”

Section: Introductionmentioning

confidence: 99%

The CGL1 (HeLa × Normal Skin Fibroblast) Human Hybrid Cell Line: A History of Ionizing Radiation Induced Effects on Neoplastic Transformation and Novel Future Directions in SNOLAB

2017

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Hybridoma Technology for the Generation of Monoclonal Antibodies

Cited by 96 publications

References 40 publications

Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

Vaccine-Derived Neutralizing Antibodies to the Human Cytomegalovirus gH/gL Pentamer Potently Block Primary Cytotrophoblast Infection

Development of an enzyme-linked immunospot assay for determination of rotavirus infectivity

The CGL1 (HeLa × Normal Skin Fibroblast) Human Hybrid Cell Line: A History of Ionizing Radiation Induced Effects on Neoplastic Transformation and Novel Future Directions in SNOLAB

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