Molecular engineering: applications to the clinical laboratory

1993 ◽  
Vol 39 (9) ◽  
pp. 1988-1997
Author(s):  
R G Hamilton
Keyword(s):  
Clinical Laboratory ◽  
Genetically Engineered ◽  
Molecular Engineering ◽  
Total Serum ◽  
Chimeric Antibody ◽  
Human Igg ◽  
Chimeric Antibodies ◽  
Human Igg1 ◽  
V Region ◽  
Volume Estimates

Abstract Advances in cellular and molecular biology methods have led to the molecular engineering of novel human biomolecules, some of which have been successfully applied to the documentation of clinical laboratory assays. Here I describe the use of engineered chimeric antibodies in the clinical immunology laboratory in three principal applications: (a) as reference proteins to document the specificity of clinical assay reagents, be used as reagent-grade purified antigens, and facilitate the epitope mapping of antibody reagents; (b) as calibration proteins to assign mass/volume estimates to proposed antibody standards; and (c) as interference proteins to study the effects of naturally occurring autoantibodies on the accuracy and sensitivity of current clinical assays. The model recombinant proteins used for these illustrations are chimeric antibodies with a defined V-region specificity for one of two haptens (nitrophenyl or dansyl) and C-region domains covering a spectrum of human isotypes. I also describe a panel of mutant human IgG1-4 anti-dansyl chimeric antibodies that have been genetically engineered with swapped, deleted, or point-mutated wild-type C-region exons and used as specialized reagents for mapping the epitopes to which clinically used human IgG-specific monoclonal antibodies bind. Finally, the use of a recombinant human IgG1 anti-human IgE Fc chimeric antibody to simulate human IgG anti-IgE autoantibody interference in assays of total serum IgE is investigated.

scholarly journals The immunogenicity of chimeric antibodies.

1989 ◽  
Vol 170 (6) ◽  
pp. 2153-2157 ◽  
Author(s):  
M Brüggemann ◽  
G Winter ◽  
H Waldmann ◽  
M S Neuberger
Keyword(s):  
The Self ◽  
Chimeric Antibody ◽  
Human Origin ◽  
Strong Response ◽  
Chimeric Antibodies ◽  
V Region

Mice were immunized with model xenogeneic (both the VH frameworks and the CH domains of human origin), chimeric (just VH frameworks human), or self antibodies, and the antiantibody responses were dissected. Only the self antibody did not elicit a response. A strong response was elicited by the most xenogeneic antibody with approximately 90% against the C and approximately 10% against the V. The anti-V response was not attenuated in the chimeric antibody, demonstrating that foreign VH frameworks can be sufficient to lead to a strong antiantibody response. The magnitude of this xenogeneic anti-VH response was similar to that of the allotypic response elicited by immunizing mice of the Igha allotype with an Ighb antibody. Thus, although chimerization can diminish antiantibody responses, attention should be paid both to V region immunogenicity and to polymorphism.

Genetically Engineered (Chimeric) Antibodies

1989 ◽  
Vol 24 (10) ◽  
pp. 65-80 ◽  
Author(s):  
Sherie L. Morrison
Keyword(s):  
Genetically Engineered ◽  
Chimeric Antibodies

Implications for the monitoring of patients with multiple myeloma undergoing treatment with the anti-CD38 monoclonal daratumumab

2019 ◽  
Vol 57 (2) ◽  
pp. 178-181 ◽  
Author(s):  
Sally Thirkettle ◽  
Joanne Russell ◽  
Sarah Wilson ◽  
Tasneem Ganijee ◽  
Samar Kulkarni ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Healthcare Workers ◽  
Plasma Cells ◽  
Clinical Laboratory ◽  
Response To Treatment ◽  
Serological Response ◽  
The Novel ◽  
Remission Rates ◽  
Human Igg1 ◽  
Single Laboratory

Background The novel daratumumab immunotherapy is a human IgG1 kappa antibody targeted against CD38, which is almost universally expressed on myeloma plasma cells. Daratumumab has efficacy in clinical trials for the treatment of multiple myeloma; however, it complicates laboratory monitoring of the serological response to treatment, as it is detected by serum electrophoresis and/or immunofixation. Methods Laboratory reports of electrophoresis patterns serially performed in a single laboratory of six patients with relapsed multiple myeloma receiving daratumumab therapy as part a clinical trial were reviewed retrospectively. Results Post administration of daratumumab therapy, an additional band was visible by serum electrophoresis, migrating to the mid-gamma region, which was confirmed as IgG kappa by immunofixation. In five out of the six patients, this band was quantified at <2.0 g/L. For one patient, this band co-migrated with the patient’s disease paraprotein band, so both bands were quantified together. The appearance of an apparent second paraprotein band while receiving treatment for multiple myeloma can cause anxiety for patients, confusion for healthcare workers and may also underestimate complete remission rates. Conclusions The clinical laboratory must be aware of the interference of daratumumab in serum electrophoresis. Effective communication between clinicians and the laboratory is essential for the production of clinically valuable, non-misleading reports for these patients.

IgG heavy-chain subclass typing of myeloma paraproteins by isoelectric focusing immunoblot analysis.

1989 ◽  
Vol 35 (3) ◽  
pp. 364-368 ◽  
Author(s):  
F J Fasullo ◽  
H A Fritsche ◽  
F J Liu ◽  
R G Hamilton
Keyword(s):  
Monoclonal Antibodies ◽  
Bovine Serum Albumin ◽  
Serum Proteins ◽  
Clinical Laboratory ◽  
Immunoblot Analysis ◽  
Laboratory Method ◽  
Therapy Program ◽  
Myeloma Proteins ◽  
Chain Composition ◽  
Human Igg1

Abstract The objective of this study was to develop a clinical laboratory method for subclass typing of human immunoglobulin G (IgG) paraproteins. Serum proteins were isoelectrically focused (IEF) in a mini-gel and passively blotted by capillary diffusion onto untreated nitrocellulose. Unreacted sites on the nitrocellulose were blocked with bovine serum albumin and the bound IgG was detected with peroxidase-conjugated anti-human IgG1-4 monoclonal antibodies from WHO/IUIS clones. The IEF immunoblot specificity was demonstrated by analysis of documented IgG, IgA, and IgM myeloma proteins of known subclass and light-chain composition. IEF immunoblots of sera from 18 myeloma patients who had an above-normal total IgG concentration produced IEF immunoblot patterns composed of five to 10 discrete bands (pI range 6.0 to 8.4). In contrast, no detectable IgG bands were observed with sera containing IgA and IgM paraproteins. The observed subclass frequencies of IgG paraproteins were 56% IgG1 (10/18), 28% IgG2 (5/18), 11% IgG3 (2/18), and 5% IgG4 (1/18). IEF immunoblot analysis permits the monitoring of changes in the pI and subclass of an IgG paraprotein over the course of a myeloma patient's therapy program.

Shared V-Region Antigens and Cross-Reacting Specificities of Human IgG Anti-F(ab′)2and Anti-DNA Antibodies

1996 ◽  
Vol 80 (2) ◽  
pp. 194-203 ◽  
Author(s):  
Ralph C. Williams, Jr. ◽  
Christine C. Malone ◽  
Francesco Silvestris
Keyword(s):  
Human Igg ◽  
Dna Antibodies ◽  
V Region

scholarly journals Aglycosylation of human IgG1 and IgG3 monoclonal antibodies can eliminate recognition by human cells expressing FcγRI and/or FcγRII receptors

1989 ◽  
Vol 259 (2) ◽  
pp. 347-353 ◽  
Author(s):  
M R Walker ◽  
J Lund ◽  
K M Thompson ◽  
R Jefferis
Keyword(s):  
Monoclonal Antibodies ◽  
K562 Cells ◽  
Red Cells ◽  
Rosette Formation ◽  
Human Monoclonal Antibodies ◽  
Human Igg ◽  
Igg Binding ◽  
Human Igg1 ◽  
Murine Monoclonal Antibodies ◽  
Human Red Cells

Aglycosylated human IgG1 and IgG3 monoclonal anti-D (Rh) and human IgG1 and IgG3 chimaeric anti-5-iodo-4-hydroxy-3-nitrophenacetyl (anti-NIP) monoclonal antibodies produced in the presence of tunicamycin have been compared with the native glycosylated proteins with respect to recognition by human Fc gamma RI and/or Fc gamma RII receptors on U937, Daudi or K562 cells. Human red cells sensitized with glycosylated IgG3 form rosettes via Fc gamma RI with 60% of U937 cells. Inhibition of rosette formation required greater than 35-fold concentrated more aglycosylated than glycosylated human monoclonal anti-D (Rh) antibody. Unlabelled polyclonal human IgG and glycosylated monoclonal IgG1 and anti-D (Rh) antibody inhibited the binding of 125I-labelled monomeric human IgG binding by U937 Fc gamma RI at concentrations greater than 50-fold lower than the aglycosylated monoclonal IgG1 anti-D (Rh) (K50 approximately 3 x 10(-9) M and approximately 6 x 10(-7) M respectively). Similar results were obtained using glycosylated and aglycosylated monoclonal human IgG1 or IgG3 chimaeric anti-NIP antibody-sensitized red cells rosetting with Fc gamma RI-/Fc gamma RII+ Daudi and K562 cells. Rosette formation could be inhibited by the glycosylated form (at greater than 10(-6) M) but not by the aglycosylated form. Haemagglutination analysis using a panel of murine monoclonal antibodies specific for epitopes located on C gamma 2, C gamma 3 or C gamma 2/C gamma 3 interface regions did not demonstrate differences in Fc conformation between the glycosylated or aglycosylated human monoclonal antibodies. These data suggest that the Fc gamma RI and Fc gamma RII sites on human IgG are highly conformation-dependent and that the carbohydrate moiety serves to stabilize the Fc structure rather than interacting directly with Fc receptors.

scholarly journals Recombinant Mouse-Human Chimeric Antibodies as Calibrators in Immunoassays That Measure Antibodies toToxoplasma gondii

1998 ◽  
Vol 36 (5) ◽  
pp. 1277-1284 ◽  
Author(s):  
John Hackett ◽  
Jane Hoff-Velk ◽  
Alan Golden ◽  
Jeff Brashear ◽  
John Robinson ◽  
...  
Keyword(s):  
Human Plasma ◽  
High Titer ◽  
Variable Region ◽  
Immunoglobulin M ◽  
Expression Vectors ◽  
Chimeric Antibody ◽  
Chimeric Mouse ◽  
Variable Regions ◽  
Chimeric Antibodies ◽  
Igg1 Antibody

In the present study, we examined the feasibility of using recombinant antibodies containing murine variable regions and human constant regions as calibrators or controls in immunoassays. As a model system, we chose the Abbott IMx Toxo immunoglobulin M (IgM) and Toxo IgG assays designed to detect antibodies to Toxoplasma gondii. Two mouse monoclonal antibodies were selected based on their reactivity to the T. gondii antigens P30 and P66. Heavy- and light-chain variable-region genes were cloned from both hybridomas and transferred into immunoglobulin expression vectors containing human kappa and IgG1 or IgM constant regions. The constructs were stably transfected into Sp2/0-Ag14 cells. In the IMx Toxo IgG assay, immunoreactivity of the anti-P30 chimeric IgG1 antibody paralleled that of the positive human plasma-derived assay calibrators. Signal generated with the anti-P66 chimeric IgG1 antibody was observed to plateau below the maximal reactivity observed for the assay calibrator. Examination of the IgM chimeric antibodies in the IMx Toxo IgM assay revealed that both the anti-P30 and anti-P66 antibodies matched the assay index calibrator manufactured with human Toxo IgM-positive plasma. When evaluated with patient samples, the correlation between results obtained with the chimeric antibody calibrators and the positive human plasma calibrators was ≥0.985. These data demonstrate that chimeric mouse-human antibodies are a viable alternative to high-titer positive human plasma for the manufacture of calibrators and controls for diagnostic assays.

scholarly journals Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: an Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

2021 ◽  
Author(s):  
Fatemeh Khademi ◽  
Pantea Mohammadi ◽  
Ali Mostafaei
Keyword(s):  
Polymerase Chain Reaction ◽  
Cell Viability ◽  
B Cell Lymphoma ◽  
Chimeric Antibody ◽  
Chimeric Mouse ◽  
Chain Reaction ◽  
Chimeric Antibodies ◽  
Overlap Extension ◽  
Polymerase Chain ◽  
Anti Cd20

Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized. We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1 mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies. The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab. The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

Drug Dependent Immune Destruction of Human Platelets in the NOD/SCID Mouse.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3419-3419
Author(s):  
Daniel W. Bougie ◽  
Brian Boylan ◽  
Dhirendra Nayak ◽  
Peter J. Newman ◽  
Richard H. Aster
Keyword(s):  
Human Blood ◽  
Blood Cells ◽  
Scid Mouse ◽  
Human Platelets ◽  
Human Igg ◽  
Human Blood Cells ◽  
Rapid Clearance ◽  
Human Igg1 ◽  
Immune Destruction ◽  
Drug Dependent

Abstract Animal models have been of limited value for studying survival and immune destruction of human blood cells because of xenoantibodies that mediate rapid elimination of the transfused cells. The NOD/SCID mouse is a genetic variant that lacks immunoglobulins, including xenoantibodies, and may therefore be useful for studying survival and immune destruction of human blood cells (Newman, PJ et al. J Thromb Haemost.2007; 5 Suppl 1:305–9). Human platelets, infused into the retroorbital plexus of NOD/SCID mice survive about three days, nearly as long as mouse platelets themselves. Injection of IgG from a patient with quinine-induced immune thrombocytopenia (TP) and a newly developed quinine-dependent murine monoclonal antibody (mAb) were without effect on platelet survival. However, both antibodies caused rapid platelet clearance after IP injection of quinine. 500 μg, but not 50 μg, of IgG from serum containing an anti-HPA-1a (PlA1) antibody also caused rapid clearance of HPA-1a positive platelets. These findings indicate that the NOD/SCID mouse can be valuable for the study of immune clearance of human platelets and perhaps other unresolved problems in transfusion medicine. However, it is not known whether mice recognize cells opsonized with human IgG or mouse IgG equally well. To examine this, we compared the survival of human platelets sensitized with 1) mAb 7E3 (IgG1) specific for an epitope in the beta A domain of GPIIIa or 2) c7E3 (from Centocor Inc), which recognizes the same epitope but possesses a human IgG1 Fc. In titration studies, we found that about four times as much c7E3 as 7E3 is needed to produce equivalent clearance of human platelets, indicating that mouse IgG is a more efficient opsonin than human IgG. However, this difference should not limit use of the model for studies of antibody-mediated blood cell destruction, since mg quantities of IgG can easily be isolated from small amounts (0.1– 0.2 ml) of human serum.

Quantitative analysis of multiple V-region interactions among normal human IgG

1996 ◽  
Vol 26 (3) ◽  
pp. 710-716 ◽  
Author(s):  
Ahidjo Ayouba ◽  
Gabriel Peltre ◽  
Antonio Coutinho
Keyword(s):  
Quantitative Analysis ◽  
Human Igg ◽  
Normal Human ◽  
V Region
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