An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity

Rath, Satyajit; Stanley, Carolynne; Steward, M. W.

doi:10.1016/0022-1759(88)90204-9

Cited by 128 publications

(66 citation statements)

References 8 publications

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“…The affinity of the scFvs for E7 protein was determined by a competitive ELISA. 20 The affinity of scFv43 was, with a close approximation, 10 6 M 21 whereas that of scFvs 32 and 51 was estimated to be lower. The low affinity could be one of the reasons why scFvs 32 and 51 failed to recognize E7 in Western blotting on cellular lysates from HPV16-positive cells.…”

Section: Characterization and Affinity Determination Of The Anti-hpv1mentioning

confidence: 94%

“…20 The different antibodies at concentration of 0.7 mg=ml, were incubated in solution with His-E7 (competitor) at different concentrations, ranging from 10 210 -10 26 M, for 2 hr. The presence of unbound antibodies was investigated by incubating the samples in microtiter wells coated with 300 ng=well of His-E7, overnight at 4°C.…”

Section: Scfv Affinity Determinationmentioning

confidence: 99%

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Intracellular anti‐E7 human antibodies in single‐chain format inhibit proliferation of HPV16‐positive cervical carcinoma cells

Accardi

Donà

Bonito

et al. 2005

Intl Journal of Cancer

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The E7 tumor antigen of human papillomavirus type 16 (HPV16) is a validated target for immunodiagnosis and immunotherapy of HPV-associated cervical cancer. Anti-HPV16 E7 antibodies in scFv format were isolated from a human antibody phage display library and characterized. With the aim of interfering with the oncogenic activity of E7 protein, the most reactive of the selected antibody fragments was expressed by eukaryotic vectors in different compartments of the HPV16-positive cervical carcinoma SiHa cell line. The intracellular antibodies (intrabodies) were tested for their ability of inhibiting cell proliferation. A significant inhibition was obtained targeting the intrabodies to the nuclear and secretory compartments whereas no significant effect was observed in case of cytoplasmic localization. Inhibition was highly specific as no antiproliferative effect was obtained either with the E7-specific intrabodies in HPV-negative cells nor with irrelevant intrabodies in SiHa cells. ' 2005 Wiley-Liss, Inc.Key words: human papillomavirus; E7 protein; intrabodies; singlechain variable fragment; antiproliferative effect; cervical cancer The HPV16 is involved in the aetiology of cervical cancer, the second main cause of cancer mortality in women worldwide.1,2 A real need exists to counter HPV infections either by prevention or by therapy. Many prophylactic and therapeutic vaccine strategies are currently under development or in clinical trial. 3,4The HPV16 oncoproteins E6 and E7, expressed in cervical carcinoma, are crucial for the development and the maintenance of the malignant phenotype.1 These antigens are valuable targets for immunotherapy of tumors because they are able to elicit CTLmediated tumor rejection.5 The HPV16 E7 has been shown recently to elicit antitumoral immunity in mice even when expressed in Nicotiana benthamiana plants. 6 This protein binds several tumour suppressors and promotes keratinocyte transformation by favoring DNA synthesis or inhibiting cellular differentiation.7-9 Several approaches have been explored to interfere with the HPV-induced abnormal proliferation by blocking the E7 activity at either gene or protein level, e.g., by phosphorothioate oligonucleotides, 10 short interfering RNA 11 and aptamers. 12 Intracellular immunization has provided new chances for interfering with the function of predetermined intracellular targets by directing antibodies in scFv format to specific compartments of mammalian cells. 13 The intracellular antibodies (intrabodies) can specifically recognize the target antigen in the intracellular environment and counteract its function with a not yet clarified mechanism of action, spanning from forming antigen-antibody complexes to diverting the target-antigen to unusual compartments.14 This approach has been employed already to achieve antineoplastic effects using intrabodies targeting oncogenic proteins.15 Evidence of antineoplastic effects was obtained in HPV16-positive human carcinoma cell lines by a hybridoma-derived recombinant scFv specific for the HPV16 E7...

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Section: Characterization and Affinity Determination Of The Anti-hpv1mentioning

confidence: 94%

Section: Scfv Affinity Determinationmentioning

confidence: 99%

Intracellular anti‐E7 human antibodies in single‐chain format inhibit proliferation of HPV16‐positive cervical carcinoma cells

Accardi

Donà

Bonito

et al. 2005

Intl Journal of Cancer

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“…The relative avidities of anti-PspA 3-286 monoclonal and polyclonal Abs were determined by competition ELISA using protocols described and validated by Rath et al (29) and Devey et al (30), respectively. This assay has been shown to rank a panel of mAbs of different affinities to the hapten dinitrophenyl in the same order as equilibrium dialysis, and its use and limitations have previously been discussed in detail (31,32).…”

Section: Determination Of Relative Avidity Of Anti-pspa 3-286 Monoclomentioning

confidence: 99%

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Rohatgi

Dutta²,

Tahir³

et al. 2009

The Journal of Immunology

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Immunization of human volunteers with a single dose of pneumococcal surface protein A (PspA) stimulates broad cross-reactive Abs to heterologous PspA molecules that, when transferred, protect mice from fatal infection with Streptococcus pneumoniae. In this study, we report the molecular characterization of 36 mouse mAbs generated against the extracellular domain of PspA (PspA 3-286 ) from strain R36A. Abs to PspA 3-286 were encoded by diverse V H and V families/genes. The H chain CDR3 and L chain CDR3 lengths were 3-13 (7.8 ؎ 0.5) and 8 -9 (8.7 ؎ 0.2) codons, respectively. Unexpectedly, seven hybridomas expressed H chains that lack D H gene-derived amino acids. Nontemplate-encoded addition(s) were observed in the H chain expressed in six of these seven hybridomas; Palindromic addition(s) were absent. Absence of D H gene-derived amino acids did not prevent antiPspA 3-286 mAbs from attaining average relative avidity. Avidity maturation occurred during primary IgG anti-PspA 3-286 polyclonal Ab response in PspA 3-286 -and R36A-immunized mice. Compared with PspA 3-286 -immunized mice, the relative avidity of the primary polyclonal IgG Abs was higher in R36A immunized mice on days 72, 86, and 100. Two pairs of clonally related hybridomas were observed. D H genes expressed in the majority (75.9%) of the hybridomas used reading frame 3. Analysis of replacement/silent mutation ratio in the CDR and framework regions provided evidence for Ag-driven selection in 11 mAbs. Based on epitope localization experiments, the mAbs were classified into 12 independent groups. ELISA additivity assay indicated that members within a group recognized topographically related epitopes. This study provides molecular insights into the biology of D H -less Abs.

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“…The relative affinities of each Ab were compared using an inhibition ELISA, as described elsewhere (19).…”

Section: Ab Charge and Affinitymentioning

confidence: 99%

Glomerular Deposition of Immune Complexes Made with IgG2a Monoclonal Antibodies

Gonzalez

Waxman

2000

The Journal of Immunology

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The factors that determine whether immune complexes (IC) are cleared safely from the circulation or are deposited in vulnerable tissues such as glomeruli are not well defined. To better understand how IC are handled, the present study examined the fate in vivo of three model IC preparations with different immunochemical characteristics. Radiolabeled IC were constructed with murine IgG1, IgG2a, or IgG3 anti-DNP mAbs bound to DNP-BSA, designated IgG1 IC, IgG2a IC, and IgG3 IC, respectively. The IC were infused i.v. into BALB/c mice, and clearance and tissue localization of the three IC probes were compared. The results indicate that the major portion of each IC preparation was cleared from the circulation by the liver. However, compared with the other two probes, IgG2a IC were preferentially deposited in the kidney. Histologic examination revealed the presence of IgG2a IC in glomeruli. The enhanced renal uptake of IgG2a IC could not be attributed solely to such characteristics as IC size, Ag/Ab ratio, Ab charge, or affinity. However, the preferential renal deposition of IgG2a IC was abrogated by complement depletion. Thus, enhanced renal uptake in normal mice was complement dependent. These data suggest that interactions between IC and the complement system can influence the propensity of IC to deposit in tissues susceptible to IC-mediated injury.

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An inhibition enzyme immunoassay for estimating relative antibody affinity and affinity heterogeneity

Cited by 128 publications

References 8 publications

Intracellular anti‐E7 human antibodies in single‐chain format inhibit proliferation of HPV16‐positive cervical carcinoma cells

Intracellular anti‐E7 human antibodies in single‐chain format inhibit proliferation of HPV16‐positive cervical carcinoma cells

Molecular Dissection of Antibody Responses against Pneumococcal Surface Protein A: Evidence for Diverse DH-Less Heavy Chain Gene Usage and Avidity Maturation

Glomerular Deposition of Immune Complexes Made with IgG2a Monoclonal Antibodies

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