Novel murine mAbs define specific and cross-reactive epitopes on the latex profilin panallergen Hev b 8

Abstract Approximately 30% of patients with severe hemophilia A will develop inhibitory antibodies to factor VIII (fVIII inhibitors). The immune response to fVIII currently is the most significant complication in the management of patients with hemophilia A. In addition, autoimmune antibodies to fVIII can develop in non-hemophiliacs, producing acquired hemophilia A, which frequently produces life- or limb-threatening bleeding. These inhibitors primarily are directed against the A2 or C2 domains of fVIII. The human response to the C2 domain of fVIII classically has been thought to inhibit fVIII activity by blocking its binding to phospholipid. We recently characterized the antibody response to the C2 domain of human fVIII in a murine hemophilia model and described 5 structural groups of antibodies. Groups A, AB, and B are classical anti-C2 antibodies. Groups BC and C consist of non-classical anti-C2 antibodies that inhibit the proteolytic activation of fVIII but do not block the binding of fVIII to phospholipid. Most non-classical antibodies have inhibitor titers greater than 10,000 Bethesda units/mg IgG. To determine if non-classical antibodies are present in fVIII inhibitor patients, patient plasmas were tested in an ELISA for their ability to block the binding of representative antibodies from the different anti-human fVIII C2 antibody groups. Classical and non-classical monoclonal antibodies (MAbs) were biotinylated and serially diluted into either fVIII deficient plasma or patient inhibitor plasma and then added to microtiter wells coated with fVIII. The ability of patient plasma to block the binding of the murine MAbs to fVIII was determined. A total of 16 patient plasmas were assessed: 4 from patients with a C2 predominant response, 2 with a non-C2 predominant response, and 10 with unknown specificities. Three of the 4 patients with C2 predominant responses had non-classical anti-C2 antibodies, while the 2 with non-C2 predominant responses did not. In the unknown plasmas, 6 of 10 had evidence of non-classical antibodies. Figure 1 shows representative results of the effect of 3 patient plasmas on the binding of a biotinylated non-classical MAb to fVIII. Patient plasmas 1 and 2 blocked MAb binding while patient plasma 3 did not. This study indicates that the majority of patients with fVIII inhibitors have non-classical anti-C2 antibodies in their response to fVIII. Figure Figure

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Epitope mapping of functional domains of human factor Va with human and murine monoclonal antibodies. Evidence for the interaction of heavy chain with factor Xa and calcium

Blood ◽

10.1182/blood.v70.1.139.139 ◽

1987 ◽

Vol 70 (1) ◽

pp. 139-146 ◽

Cited By ~ 34

Author(s):

AE Annamalai ◽

AK Rao ◽

HC Chiu ◽

D Wang ◽

AK Dutta-Roy ◽

...

Keyword(s):

Heavy Chain ◽

Factor Xa ◽

Direct Interaction ◽

Enzyme Linked Immunosorbent Assay ◽

Factor V ◽

Factor Va ◽

Direct Binding ◽

First Time ◽

Murine Monoclonal Antibodies ◽

Murine Mabs

Abstract We have purified a unique neutralizing IgG1, kappa monoclonal antibody (MAb) against factor V (F-V) from a patient's plasma. This MAb (H2) demonstrated specificity for human F-V heavy chain (D), mol wt 105,000. Using an enzyme-linked immunosorbent assay (ELISA) we assessed the competitive binding to F-Va of H2, H1 (human MAb directed to light chain, F1F2), and two murine MAbs, B38 (to F1F2) and B10 (to activation peptide C1). All four antibodies are of high affinity with KD varying from 0.17 to 1.17 X 10(-10) mol/L. They recognized distinct epitopes in F-V. F-Xa competed in a concentration-dependent fashion for binding of H1, H2, and B38 but not B10 to F-V/Va in the absence of phospholipids or platelets. Thus both F1F2 and D polypeptides of F-Va but not C1 interacted with F-Xa. All MAbs bound to F-V/Va in the absence of Ca++. However, free Ca++ (0.1 to 4.0 mmol/L) increased the amount of H1 and H2 bound to factor V/Va, 1.65-fold and 3.65-fold, respectively but had little effect on the binding of either murine MAbs. Prothrombin (20 micrograms/mL to 400 micrograms/mL) in the absence of phospholipid did not inhibit the binding of MAbs. These studies provide evidence for the first time for a direct interaction between human F-Va heavy chain and F-Xa and Ca++ and for the direct binding of F-Xa to F-Va in the absence of phospholipids or platelets and enhance our understanding of functional F-V domains.

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Specificity and Diversity of Antibodies to Mycobacterium tuberculosis Arabinomannan

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.10.1.88-94.2003 ◽

2003 ◽

Vol 10 (1) ◽

pp. 88-94 ◽

Cited By ~ 14

Author(s):

Josephine Anne D. Navoa ◽

Suman Laal ◽

Liise-Anne Pirofski ◽

Gary R. McLean ◽

Zhongdong Dai ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Human Serum ◽

Cross Reactivity ◽

Human Infection ◽

Serum Samples ◽

Purified Protein Derivative ◽

Human Antibodies ◽

Human Serum Samples ◽

Negative Controls ◽

Murine Mabs

ABSTRACT Arabinomannan (AM) is a polysaccharide antigen of the mycobacterial capsule. However, it is uncertain whether AM constitutes an immunologically distinct fraction of Mycobacterium tuberculosis. In this study, we analyzed the repertoire and specificity of antibodies to AM by using AM-binding murine monoclonal antibodies (MAbs) and human serum samples. Murine MAbs were found to be diverse in their specificity to AM and cross-reactivity with other arabinose-containing mycobacterial polysaccharides, with MAb 9d8 binding exclusively to AM. Human antibodies to AM were detected in serum samples from patients with pulmonary tuberculosis (TB), as well as in those from healthy, purified protein derivative-negative controls, with significantly higher titers among patients. The binding of human antibodies to AM was inhibited by MAb 9d8 in three patients with TB but not in controls. MAb 5c11, which recognizes other mycobacterial arabinose-containing carbohydrates in addition to AM, inhibited the binding of serum samples from 75% of patients and 76% of controls. Analysis of human antibodies with murine MAbs to human VH determinants demonstrated diversity among antibodies to AM with qualitative and quantitative differences compared with antibodies to lipoarabinomannan. In summary, our study suggests that antibodies to AM are diverse and heterogeneous with respect to antigen recognition and VH determinant expression, with human serum samples containing different subsets of antibodies to AM with the specificities of AM-binding murine MAbs. One MAb and a subset of human antibodies bind AM specifically, suggesting that this polysaccharide is antigenically distinct and is expressed in human infection.

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Introduction on Monoclonal Antibodies

10.5772/intechopen.98378 ◽

2021 ◽

Author(s):

Mona Sadeghalvad ◽

Nima Rezaei

Keyword(s):

Monoclonal Antibodies ◽

Protein Sequence ◽

First Generation ◽

Polyclonal Antibodies ◽

High Specificity ◽

Repeated Administration ◽

Antigen Binding Site ◽

Hybridoma Technology ◽

Mouse Antibody ◽

Murine Mabs

Monoclonal antibodies (mAbs) are a group of antibodies produced by identical clones of B lymphocytes against a particular antigen. mAbs are identical in several properties such as protein sequence, antigen-binding site region, binding affinity for their targets, and identical downstream functional effects. These characteristics of mAbs highlight their differences with the polyclonal antibodies which have heterogenous activities and recognize different epitopes on an antigen. Murine mAbs was the first generation of mAbs developed by hybridoma technology however, because of their murine origin, they can trigger the anti-mouse antibody response in the host which could accelerate mAb clearance and undesirable allergic reactions upon repeated administration. This issue was resolved by developing engineering methods toward producing less immunologic chimeric or humanized antibodies. mAbs applications have become a novel way of targeting antigens in a wide variety of diseases such as autoimmunity, malignancies, and asthma. In addition, high specificity and high affinity binding properties of mAbs make them effective biological reagents in immunodiagnostic assays. They can be used in diagnosis of infectious diseases and detection of certain antigens or in serological assessments for detection of antibodies against a certain antigen. This chapter summarizes the general properties of mAbs, their production processes, and their important diagnostic and therapeutic applications.

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Antibodies to synthetic peptides based on band 3 motifs react specifically with Plasmodium falciparum (humanmalaria)-infected erythrocytes and block cytoadherence

Parasitology ◽

10.1017/s0031182000075934 ◽

1994 ◽

Vol 108 (4) ◽

pp. 389-396 ◽

Cited By ~ 20

Author(s):

I. Crandall ◽

I. W. Sherman

Keyword(s):

Amino Acids ◽

Plasmodium Falciparum ◽

Synthetic Peptides ◽

Protein Band ◽

Significant Degree ◽

Band 3 ◽

Anion Transport ◽

Amelanotic Melanoma ◽

Murine Mabs ◽

Antibodies To Synthetic Peptides

SUMMARYRabbit polyclonal and mouse monoclonal antibodies (Mabs) prepared against synthetic peptides patterned on exofacialloops 3 (amino acids 546–555) and 7 (821–834) of the human anion transport protein band 3 inhibited the cytoadherence of Plasmodium falciparum-infected erythrocytes to C32 amelanotic melanoma cells. Mabs directed against exofacial loop4 (amino acids 628–642) did not inhibit adherence to a significant degree. The murine Mabs recognized only P. falciparum- infected erythrocytes suggesting that the epitopes of loops 3, 4 and 7 are normally cryptic in uninfected erythrocytes.

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Functional Activities and Immunoglobulin Variable Regions of Human and Murine Monoclonal Antibodies Specific for the P1.7 PorA Protein Loop of Neisseria meningitidis

Infection and Immunity ◽

10.1128/iai.68.4.1871-1878.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 1871-1878 ◽

Cited By ~ 9

Author(s):

Jianfu Wang ◽

Gary A. Jarvis ◽

Mark Achtman ◽

Einar Rosenqvist ◽

Terje E. Michaelsen ◽

...

Keyword(s):

Human Monoclonal Antibody ◽

Variable Region ◽

Effector Function ◽

Enzyme Linked Immunosorbent Assay ◽

Molecular Characteristics ◽

Fab Fragment ◽

Region Gene ◽

Variable Regions ◽

Group B ◽

Murine Mabs

ABSTRACT The meningococcal PorA protein is considered a promising vaccine candidate. Although much is understood regarding the structure of PorA proteins, little is known about the structure-function relationships of PorA antibodies. The aim of this study was to compare the functional and molecular characteristics of a human monoclonal antibody (MAb) and three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs 207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated neither effector function. Epitope mapping with synthetic peptides revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ, which is the same specificity motif that a previous study had established for SS269 and MN14C11.6. Nucleotide and amino acid sequence analyses of the variable regions of the four MAbs showed that the SS269 VH region belonged to the VH3 family and was approximately 70% homologous to those of the murine MAbs which were all from the 7183 family, whereas the SS269 VL region belonged to the Vλ1-b family and was less than 40% homologous to those of the murine MAbs which were all members of the Vκ1 family. The Fab fragment of SS269 was cloned and expressed in Escherichia coli and was shown by enzyme-linked immunosorbent assay analyses to bind as well as intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We conclude that distinct differences exist in the effector function activities and variable region gene sequences of human and murine P1.7-specific MAbs despite their recognition of similar epitopes.

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Identification of a novel nuclear domain.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.785 ◽

1991 ◽

Vol 112 (5) ◽

pp. 785-795 ◽

Cited By ~ 176

Author(s):

C A Ascoli ◽

G G Maul

Keyword(s):

Mrna Processing ◽

Nuclear Bodies ◽

Liver Parenchymal ◽

Quiescent Cells ◽

Nuclear Domain ◽

Interphase Cells ◽

Nuclease Digestion ◽

Nuclear Domains ◽

Parenchymal Cells ◽

Murine Mabs

For most known nuclear domains (ND), specific functions have been identified. In this report we used murine mAbs and human autoantibodies to investigate precisely circumscribed structures 0.2-0.3 micron in diameter which appear as "nuclear dots" distributed throughout the nucleoplasm. Nuclear dots are metabolically stable and resistant to nuclease digestion and salt extraction. The localization of nuclear dots is separate from kinetochores, centromeres, sites of mRNA processing and tRNA synthesis, nuclear bodies, and chromosomes. The nuclear dots, therefore, represent a novel ND. Nuclear dots break down as cells enter metaphase and reassemble at telophase. In interphase cells, nuclear dots are frequently "paired," and some are visible as "doublets" when stained with one particular antiserum. The number of dot doublets increased when quiescent cells were stimulated with serum although the total number of dots did not change substantially. One of the antigens was identified as a protein with a molecular mass of approximately 55 kD showing three charge isomers in the pI range of 7.4 to 7.7. Autoantibodies affinity purified from this nuclear dot protein (NDP-55) show nuclear dots exclusively. Nuclear dot-negative rat liver parenchymal cells became positive after chemical hepatectomy, suggesting involvement of the NDP-55 in the proliferative state of cells.

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Human anti-murine antibody interference in measurement of carcinoembryonic antigen assessed with a double-antibody enzyme immunoassay

Clinical Chemistry ◽

10.1093/clinchem/37.1.51 ◽

1991 ◽

Vol 37 (1) ◽

pp. 51-57 ◽

Cited By ~ 18

Author(s):

Theresa Price ◽

Barbara G Beatty ◽

J David Beatty ◽

Alan J McNally

Keyword(s):

Monoclonal Antibody ◽

Plasma Concentration ◽

Carcinoembryonic Antigen ◽

Enzyme Immunoassay ◽

Surgical Resection ◽

Clinical Evidence ◽

Murine Mabs ◽

Murine Antibody

Abstract Fifty-eight plasma specimens from 30 patients who had undergone presurgical radioimmunoscintigraphy with 111In-labeled anti-carcinoembryonic antigen (CEA) murine monoclonal antibody (Mab) and who had no clinical evidence of disease after surgical resection showed increased concentrations of CEA (greater than or equal to 5 micrograms/L) in plasma when studied with the previously available commercial CEA enzyme immunoassay (EIA) from Roche. The possible role of anti-murine antibody (HAMA) interference was addressed by adding mouse IgG (mIgG) to the plasma (2 g/L) before assay. Fifteen specimens (26%) showed no change in CEA (reflecting a true increase as shown by the original results), 22 (38%) showed a decrease in CEA of greater than 15% but remained positive (reflecting an artefactual increase), and 21 (36%) became CEA-negative (less than or equal to 5 micrograms/L; reflecting a false increase). Subsequently, we assayed the same samples with a modified version of this CEA EIA kit and 47 specimens remained CEA positive (greater than 5 micrograms/L): 25 (53%) were truly increased, 12 (26%) remained artefactually increased, and 10 (21%) continued to show a false increase. The degree of interference in the original EIA kit correlated with the plasma concentration of HAMA (P less than 0.005). All artefactually and falsely increased CEA values observed in both kits were corrected by addition of polyclonal mIgG or of a mixture of IgG1, IgG2a, and IgG2b Mabs before assay. This correction is important in the follow-up of patients who receive murine Mabs for treatment or diagnosis.

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Production and Characterization of Murine mAbs to the Extracellular Domain of Human Neu Oncogene Product GP185HER2

Hybridoma ◽

10.1089/hyb.1992.11.519 ◽

1992 ◽

Vol 11 (4) ◽

pp. 519-527 ◽

Cited By ~ 29

Author(s):

GIOVANNA DIGIESI ◽

PATRIZIO GIACOMINI ◽

ROCCO FRAIOLI ◽

MASSIMO MARIANI ◽

MARIA RITA NICOTRA ◽

...

Keyword(s):

Extracellular Domain ◽

Neu Oncogene ◽

Murine Mabs

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Human autoantibody-reactive epitopes of SS-B/La are highly conserved in comparison with epitopes recognized by murine monoclonal antibodies.

Journal of Experimental Medicine ◽

10.1084/jem.166.6.1627 ◽

1987 ◽

Vol 166 (6) ◽

pp. 1627-1640 ◽

Cited By ~ 67

Author(s):

E K Chan ◽

E M Tan

Keyword(s):

Nuclear Protein ◽

Rna Binding ◽

Mammalian Species ◽

Rna Polymerase Iii ◽

Target Antigen ◽

Cellular Functions ◽

Calf Thymus ◽

Catalytic Sites ◽

Murine Monoclonal Antibodies ◽

Murine Mabs

SS-B/La, an ubiquitous nuclear protein of 46-48 kD, is a target antigen of autoantibodies in SLE and Sjogren's syndrome and is involved in the maturation of RNA polymerase III transcripts such as 5S RNA and tRNAs. We have previously shown (14, 15) that SS-B consists of two protease-resistant domains of 23 and 28 kD, with the latter containing the RNA binding site. The epitopes of SS-B/La reactive with human autoantibodies are conserved among several mammalian species examined. BALB/c mice immunized with affinity-purified calf thymus SS-B produce IgG anti-SS-B/La antibodies, which reacted with bovine, human, and rabbit SS-B but not with mouse SS-B/La. The spleen of a mouse with the highest antibody titer was selected for fusion with P3 myeloma. Five IgG1k mAbs (A1-5) were selected by ELISA and immunoblotting. All except A3 reacted with the 28-kD domain. A1, A2, and A3 were capable of immuno-precipitating the 48-kD SS-B protein and its associated RNAs. A1, A2, and A3 also gave fine nuclear speckled staining on human, monkey, bovine, and rabbit cells that was similar in appearance to that with human autoantibodies, but in contrast to staining with human autoantibodies, they did not stain cells from rat, mouse, or rat kangaroo. It appears that human autoantibodies target highly conserved epitopes that can be distinguished from epitopes recognized by immunization-induced murine mAbs. Taken together with other data, it appears that human autoantibodies may be recognizing epitopes that are active or catalytic sites of molecules subserving important cellular functions.

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