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Introduction: Globally, breast carcinoma is the most prevalent and lethal form of cancer in women. Breast cancer is no longer considered a single disease but a complex heterogeneous disease with multiple genetic and epigenetic alterations. The prognosis and management of the disease depend on histological stage, type, grade, tumour size, lymph node status, and the status of hormonal receptors like ER, PR, and Her2/neu. Recently, more attention has been given to the molecular classification of breast cancer. Aim: To analyse and compare the clinicopathological characteristics of invasive breast cancer in the four breast carcinoma subtypes defined by the immunohistochemical expression of Estrogen Receptor (ER), Progesterone Receptor (PR), and Human Epidermal Growth Factor Receptor 2 (Her2/neu). Materials and Methods: The cross-sectional study was conducted on 64 primary invasive breast carcinoma cases diagnosed on mastectomy specimens between February 1, 2018, and July 30, 2022, in the Department of Pathology at the histopathology section of Silchar Medical College and Hospital, Silchar, Assam, India. Age and tumour characteristics (morphology, grade, stage, and size) and nodal disease status were included in the data for analysis. Immunohistochemical markers were analysed on the sections of these diagnosed cases. IBM Statistical Packages for Social Sciences (SPSS) software was used for data analysis. Qualitative data was presented as frequency and percentage, while quantitative data was presented as mean±{Standard Deviation (SD)}. The Chi-square test was used to determine the statistical significance of hormonal receptors with the various clinicopathological features. A p-value of

Myoglobin (Mb) is the main constituent of vertebrate skeletal muscle and myocardium and plays an essential role in oxygen binding, storage, transport, and earliest disease diagnosis. This study focuses on preparing the novel recombinant rabbit anti-Mb monoclonal antibody and applying it to a diagnosis of Mb deposition in rhabdomyolysis-associated acute kidney injury (RM-AKI). The full-length coding sequence of rat Mb was cloned and expressed, and the high-quality and titer rabbit anti-Mb polyclonal antibodies were produced by the immunogen His-Mb fusion protein. A new hybridoma cell was obtained by hybridoma screening technology. With the help of DNA sequencing and a molecular clonal, anti-Mb monoclonal antibody heavy and light chains expression plasmid was constructed. Finally, the recombinant rabbit anti-Mb monoclonal antibody with extraordinarily high affinity (KD = 1.21 pM) was obtained. Meanwhile, it had broad species reactivity (mouse, rat, human, and horse) and good tissue specificity (skeletal muscle and myocardium). It also had a very good performance in western blotting, immunohistochemistry, and immunofluorescence assay to detect the Mb level in the kidney, myocardium, and skeletal muscle of RM-AKI. This study will be significantly helpful for Mb-associated disease diagnosis, and pathogenesis exploration, and further may act as a neutralizing antibody for disease treatment. [ABSTRACT FROM AUTHOR]

A dual monoclonal antibody sandwich enzyme-linked immunosorbent assay (mAb sandwich ELISA) has been developed using rabbit monoclonal antibodies generated by Ecobody technology, which includes the isolation of single B cells binding to a specific antigen, amplification of the heavy and light chains of these immunoglobulins, and expression of the fragment of antigen binding (Fab) by cell-free protein synthesis (CFPS). A rabbit was immunized with swine influenza virus (SIV) vaccine, from which single B cells binding to the antigen were isolated. Then, immunoglobulin mRNA was amplified from single cells by reverse transcription-polymerase chain reaction, followed by the attachment of a T7 promoter, appropriate tags, and a T7 terminator for the expression of the Fab portion by CFPS. By taking advantage of two different peptide tags fused to the same Fab, optimal combinations for coating Fab on assay plates and detecting Fab, both synthesized by CFPS, were investigated for mAb sandwich ELISA. Pairs of Fab detected 0.5 ng SIV in the assay. In summary, this result showed the applicability of Ecobody technology for a variety of immunodetection kits for high throughput analyses. [ABSTRACT FROM AUTHOR]

To date, SARS-CoV-2 pandemic has caused more than 188 million infections and 4.06 million deaths worldwide. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein has been regarded as an important target for vaccine and therapeutics development because it plays a key role in binding the human cell receptor ACE2 that is required for viral entry. However, it is not easy to detect RBD in Western blot using polyclonal antibody, suggesting that RBD may form a complicated conformation under native condition and bear rare linear epitope. So far, no linear epitope on RBD is reported. Thus, a monoclonal antibody (mAb) that recognizes linear epitope on RBD will become valuable. In the present study, an RBD-specific rabbit antibody named 9E1 was isolated from peripheral blood mononuclear cells (PBMC) of immunized rabbit by RBD-specific single B cell sorting and mapped to a highly conserved linear epitope within twelve amino acids 480CNGVEGFNCYFP491 on RBD. 9E1 works well in Western blot on S protein and immunohistochemistry on the SARS-CoV-2 infected tissue sections. The results demonstrated that 9E1 can be used as a useful tool for pathological and functional studies of SARS-CoV-2.

Rhamnogalacturonan II (RG-II) is a region of pectin macromolecules that is present in plant primary cell walls. RG-II can be solubilized from cell walls as a borate-RG-II complex (B-RG-II), where two RG-II fragments are cross-linked via a borate diester linkage. Here, a rabbit monoclonal antibody against B-RG-II was prepared, which recognized both B-RG-II and RG-II monomers without borate ester-crosslinking. A pectic fragment with unknown structure was also recognized by the antibody, but neither homogalacturonan nor rhamnogalacturonan I was recognized. Immunoelectron microscopic analyses of Arabidopsis root tip cells were performed using this antibody. The signal was detected in developing cell plates and cell walls, which were denser in longitudinal walls than in transverse walls. These results coincide with our previous results obtained in suspension cultured tobacco cells, confirming that RG-II is present in cell plates at an early stage of their assembly. Abbreviations: B: boron; B-RG-II: borate-RG-II complex; ELISA: enzyme-linked immunosorbent assay; IgG: immunoglobulin G; mBSA: methylated bovine serum albumin; PGA: polygalacturonic acid; PLL: poly-L-lysine; RG-I: rhamnogalacturonan I; RG-II: rhamnogalacturonan II [ABSTRACT FROM AUTHOR]

Abstract Rabbit monoclonal antibodies (mAbs) have many advantages over mouse antibodies in biological research and diagnostics applications because they exhibit high affinity and specificity. However, the methods of recombinant rabbit mAb production have not been optimized to the same extent as techniques used to produce mouse and human mAbs. In this study, we sought to optimize the production of a recombinant rabbit mAb against human plexin domain containing protein 2 (PLXDC2), a known cell surface antigen, by culturing HEK293-6E cells transfected with antibody-encoding genes at two different temperatures and by purifying the end-product by three different chromatography methods. The quality and function of purified antibody preparations were checked by electrophoresis and western blot analysis. The secreted rabbit mAb produced by a combination of culturing at 32 °C, purification by ammonium sulfate fractionation, and diethylaminoethyl resin (DEAE) ion exchange chromatography was of high quality. In contrast, the antibody produced by the cells grown at 37 °C for 6 days after transfection and purified by Protein A/G affinity method was low quality. Hypothermic conditions during production reduced protein heterogeneity probably by favorably affecting the levels of glycosylation and aggregation. In particular, according to western blotting data, CIMmultus DEAE chromatography that utilizes monolithic columns not only excluded inferior charge variants resulting from nonspecific reactions but also yielded rabbit mAb that was of better quality than commercially available rabbit polyclonal antibodies. The combination of techniques suggested by us may be a general approach to enhance product quality of rabbit mAbs produced by transient expression systems. Highlights • Optimized method of rabbit monoclonal antibodies by HEK293-6E cells is proposed. • HEK293-6E cells grown hypothermically at 32 °C produced best quality antibodies. • CIMmultus DEAE chromatography was the optimal method of antibody purification. [ABSTRACT FROM AUTHOR]

The rabbit monoclonal antibodies (mAbs) have advantages in pharmaceuticals and diagnostics with high affinity and specificity. During the past decade, many techniques have been developed for isolating rabbit mAbs, including single B cell antibody technologies. This review describes the basic characterization of rabbit antibody repertoire and summarizes methods of hybridoma technologies, phage display platform, and single B cell antibody technologies. With advances in antibody function and repertoire analysis, rabbit mAbs will be widely used in therapeutic applications in the coming years.

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.

Aberrant ETV1 overexpression arising from gene rearrangements or mutations occur frequently in prostate cancer, round cell sarcomas, gastrointestinal stromal tumors, gliomas, and other malignancies. The absence of specific monoclonal antibodies (mAb) has limited its detection and our understanding of its oncogenic function. An ETV1 specific rabbit mAb (29E4) was raised using an immunogenic peptide. Key residues essential for its binding were probed by ELISA and its binding kinetics were measured by surface plasmon resonance imaging (SPRi). Its selective binding to ETV1 was assessed by immunoblots and immunofluorescence assays (IFA), and by both single and double-immuno-histochemistry (IHC) assays on prostate cancer tissue specimens. Immunoblot results showed that the mAb is highly specific and lacked cross-reactivity with other ETS factors. A minimal epitope with two phenylalanine residues at its core was found to be required for effective mAb binding. SPRi measurements revealed an equilibrium dissociation constant in the picomolar range, confirming its high affinity. ETV1 (+) tumors were detected in prostate cancer tissue microarray cases evaluated. IHC staining of whole-mounted sections revealed glands with a mosaic staining pattern of cells that are partly ETV1 (+) and interspersed with ETV1 (−) cells. Duplex IHC, using ETV1 and ERG mAbs, detected collision tumors containing glands with distinct ETV1 (+) and ERG (+) cells. The selective detection of ETV1 by the 29E4 mAb in immunoblots, IFA, and IHC assays using human prostate tissue specimens reveals a potential utility for the diagnosis, the prognosis of prostate adenocarcinoma and other cancers, and the stratification of patients for treatment by ETV1 inhibitors. • Aberrant ETV1 expression occur in many cancers due to gene mutations or fusions. • Lack of ETV1 specific antibodies prevents understanding of its oncogenic function. • Characterization of a rabbit ETV1 mAb we developed highlights its specificity. • IHC detection of ETV1 in prostate cancer tissues showed a unique staining pattern. • Results support a potential use of the mAb for cancer diagnosis and prognosis. [ABSTRACT FROM AUTHOR]

Rabbit monoclonal antibodies (mAbs) have advantages over mouse antibodies in diagnostics and biotechnological applications owing to higher affinity and specificity. We developed a platform to generate rabbit mAbs by a novel monoclonal antibody generation method named “Single-Cell Reverse Transcription-PCR linked in vitro-Expression (SICREX)” system. In this method, we use single-cell based RT-PCR followed by sequential PCR steps of mAb genes and subsequent cell-free protein synthesis (CFPS) by using linear DNA fragments of mAbs. This platform enables the rapid generation and evaluation of mAbs derived from antigen-specific single B cells in the peripheral blood of immunized animals without mammalian cell cultivation. In this study, the antigen used was a food-borne gram-positive pathogen, Listeria monocytogenes , that is known to cause serious infection. Three active mAbs in CFPS were obtained by constructing the single chain of variable fragment (scFv) form. These scFvs were produced in the cytoplasm of E. coli Shuffle T7 Express strain as an active form and used for further investigation. [ABSTRACT FROM AUTHOR]

The hepatocyte growth factor and its receptor c-Met are correlated with castration-resistance in prostate cancer. Although HGF has been considered as an attractive target for therapeutic antibodies, the lack of cross-reactivity of monoclonal antibodies with human/mouse HGFs is a major obstacle in preclinical developments. We generated a panel of anti-HGF RabMAbs either blocking HGF/c-Met interaction or inhibiting c-Met phosphorylation. We selected one RabMAb with mouse cross-reactivity and demonstrated that it blocked HGF-stimulated downstream activation in PC-3 and DU145 cells. Anti-HGF RabMAb inhibited not only the growth of PC-3 cells but also HGF-dependent proliferation in HUVECs. We further demonstrated the efficacy and potency of the anti-HGF RabMAb in tumor xenograft mice models. Through these in vitro and in vivo experiments, we explored a novel therapeutic antibody for advanced prostate cancer. [ABSTRACT FROM AUTHOR]

Highly sensitive and accurate screening of ractopamine (RAC) residue in animal urine is greatly needed to ensure food security. The detection performance of immunoassay for RAC was always seriously harmed by the antibody inactivation derived from urea. Here, we first discovered one rabbit monoclonal antibody (RmAb) to RAC with a high affinity of 0.007 ng mL−1 and a surprising urea tolerance of 3 M urea, which is beneficial for developing robustly developed immunoassay in urine without sample pretreatment. The limits of detection of developed indirect competitive enzyme-linked immunosorbent assay based on RmAb1 for RAC were 0.0042–0.014 μg L−1 with the coefficient of variation below 11.7% in swine, sheep, and cow urine, significantly improved 10–100-fold in sensitivity. Moreover, the urea-tolerant mechanism of RmAb1 showed that more non-polar amino acids, more hydrogen bond donors on the surface, and preponderant Pi interaction of antibody-RAC all contributed to the stability of the RmAb1 in a high concentration of urea. [Display omitted] • An extremely urea-tolerance and high-affinity RAC-specific RmAb1 was successfully produced. • The sensitivity of icELISA based on RmAb1 significantly improved 10–100-fold. • Urine samples in the icELISA based on RmAb1 did not need pretreatment. • The MD analysis guided a better understanding of the urea-tolerant mechanism of RmAb1. [ABSTRACT FROM AUTHOR]

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from non-murine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A sensitive competitive indirect enzyme-linked immunoassay (ciELISA) based on a rabbit monoclonal antibody (RabMAb) against chloramphenicol (CAP) has been developed and validated in this study. After optimisation of several key physicochemical factors, such as Tween-20 percentage, pH value and ionic strength, the immunoassay showed excellent performance within the linear range of 0.18–6.37 ng mL−1, with the 50% inhibition concentration (IC50) of 1.06 ng mL−1and the limit of detection (LOD) of 0.1 ng mL−1. In addition, the cross-reactivities of RabMAb towards chloramphenicol succinate, florfenicol and thiamphenicol were 2.09, 12.45 and 18.10%, respectively. Finally, the developed method was applied in spiked swine urine, milk and honey samples, with recoveries ranging from 71.03 to 109.62%. The result demonstrated that the developed immunoassay is a valuable method for screening and quantitation of CAP residues in real samples. [ABSTRACT FROM AUTHOR]

Highlights: [•] Our study suggests that RabMAbs are highly relevant for therapeutic applications. [•] We generated a panel of 30 neutralization anti-KDR rabbit monoclonal antibodies. [•] More than half of these anti-VEGFR2 RabMAbs was cross-reactive with mouse VEGFR2. [•] We humanized one RabMAb with cross-reactivity by Mutational Lineage Guided method. [•] Humanized RabMAb inhibit tumor growth and angiogenesis in xenograft mice. [ABSTRACT FROM AUTHOR]

Background Monoclonal antibodies generally obtained through the classic mouse hybridoma system were requisite for the establishment of various immunoassays. In this study, a new rabbit monoclonal antibody ( RabMAb) against sulfonamides ( SAs) was first produced via hybridoma technique in rabbit. The related enzyme-linked immunosorbent assay ( ELISA) was then developed and applied to real sample analysis. Results A sensitive competitive indirect ELISA method based on a novel RabMAb for rapid detection of sulfonamides was first established. The obtained half-maximum inhibition concentration ( IC50) values for four SAs were all below 10 ng mL−1, with 0.68 ng mL−1 sulfathiazole ( STZ), 1.11 ng mL−1 sulfadiazine ( SD), 1.15 ng mL−1 sulfapyridine ( SP) and 5.27 ng mL−1 sulfamethoxazole ( SMX). Desirable recoveries when detecting different spiked swine urine and milk samples were achieved ranging from 92.6% to 104.3% and from 61.1% to 81.6%, respectively. Conclusion The proposed immunoassay with the newly developed RabMAb is capable of detection of four SAs ( STZ, SD, SP and SMX) with proven satisfactory performance and is applicable for routine large-scale analysis in practical uses.© 2012 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Based on the available rabbit monoclonal antibody (RabMAb), a rapid and sensitive lateral flow immunoassay (LFA) platform has been developed for quantitative detection of four sulfonamide residues(SRs) of sulfadiazine (SD), sulfathiazole (STZ), sulfapyridine (SP), and sulfamethoxazole (SMX).Within the designed LFA competitive format assay, which was based on antigen-antibody properties, the hapten conjugate N1-[4-(carboxymethyl)-2-thiazolyl] sulfanilamide linked to protein ovalbumin (TS-OVA) and goat anti-rabbit antibody were sprayed as capture and control reagents, respectively, and then the antibody was conjugated to colloidal gold particles as the detection reagent. With quantitative assessment aided by a colorimetric strip reader, the sensitivities of the established LFA method for SD, STZ, SP, and SMX were 0.91 ng mL−1, 0.10ng mL−1,0.12ng mL−1, and 2.13ng mL−1, and the half-maximum inhibition concentrations (IC50) were 5.19 ng mL−1, 1.25 ng mL−1, 0.66 ng mL−1, and 24.14 ng mL−1, respectively. The recoveries at three spiked levels (5, 20, 50 ng mL−1for SD, STZ, and SP; 20, 50, 100 ng mL−1for SMX) were in the range of 78.02–135.10% and 76.40–137.16% for milk and swine urine, respectively. More importantly, the detection performance of the established platform was consistent with that of in-parallel LC-MS/MS analysis. In conclusion, the proposed LFA platform has showed the potential for fast, sensitive and relatively accurate quantification of four sulfonamide residues in practical uses. [ABSTRACT FROM PUBLISHER]

Immunohistochemical detection of cluster of differentiation 3 (CD3) expression is important for the diagnosis of peripheral T-cell and NK-cell lymphomas. We compared CD3 staining intensity and percentage of CD3-positive cells in 28 T-cell lymphomas was compared using mouse anti-human CD3 antibody (clone F7·2·38) and rabbit anti-human CD3 antibody (clone 2GV6). Analysis was performed on tissue microarray sections containing 12 peripheral T-cell lymphomas unspecified, 4 angioimmunoblastic T-cell lymphomas, 6 adult T-cell leukemia/lymphomas, and 6 anaplastic large T-cell lymphomas. Compared to mouse antibody, CD3 staining intensity and the fraction of CD3-positive cells increased significantly with rabbit antibody in the vast majority of the T-cell lymphomas analyzed. The mean staining intensity of positive cells (weak: +1; moderate: +2; strong: +3) of all 28 lymphomas rose from 2·1 with mouse antibody to 2·5 with rabbit antibody. Notably, the mean fraction of strongly positive cells (+3) was 17·7% with mouse antibody and increased considerably to 49·1% with rabbit antibody. In addition, there was an overall 8% increase in CD3-positive cells if rabbit antibody was used. The results show that detection of CD3 expression in T-cell lymphomas with rabbit antibody represents an improved standard by increasing sensitivity and staining intensity. [ABSTRACT FROM AUTHOR]

Agostinelli C, Paterson J C, Gupta R, Righi S, Sandri F, Piccaluga P P, Bacci F, Sabattini E, Pileri S A & Marafioti T (2012) Histopathology 61, 33-46 Detection of LIM domain only 2 (LMO2) in normal human tissues and haematopoietic and non-haematopoietic tumours using a newly developed rabbit monoclonal antibody Aims: We describe a new rabbit monoclonal antibody, raised against a fixation-resistant epitope of the transcription regulator LIM domain only 2 (LMO2). Methods and results: Lymphoma cell lines and a large series of normal and neoplastic samples were investigated by Western blot and immunohistochemistry. The antibody detected nuclear positivity for the protein, with the exception of a proportion of classical Hodgkin lymphomas (HLs), peripheral T cell lymphomas (PTCLs) and solid tumours that showed granular cytoplasmic staining. In normal lympho-haematopoietic tissues, LMO2 was expressed at different intensities by CD34+ blasts, haematopoietic precursors, germinal centre (GC), mantle and splenic marginal zone B cells. While reactive with only scattered elements in the thymus and nine of 237 PTCLs, the antibody stained 31 of 39 T-acute lymphoblastic lymphoma/leukaemias (T-ALLs) and the T-ALL-derived human leukaemic cell line, CCRF-CEM. LMO2 was found in 88% of B-acute lymphoblastic lymphoma/leukaemias (B-ALLs), 5% chronic lymphocytic leukaemias (CLLs) and 14%, 57% and 41% of mantle, follicular and Burkitt lymphomas, respectively. In the setting of diffuse large B cell lymphomas (DLBCLs), LMO2-positivity was related strongly to a GC phenotype. LMO2 was found in 83% primary mediastinal large B cell lymphomas (PMBLs) and 100% nodular lymphocyte predominant Hodgkin lymphomas (NLPHLs), whereas only 10% of classical HLs were stained. Acute and chronic myeloid leukaemias were usually positive. Conclusions: The new anti-LMO2 antibody can be applied confidently to routine sections, contributing to the differential diagnosis of several lymphoma subtypes, subtyping of DLBCLs and potential development of innovative therapies. [ABSTRACT FROM AUTHOR]

Abstract: Embryonic stem (ES) cells are pluripotent stem cells derived from the inner cell mass of the blastocyst. ES cell surface molecules are important for the identification, labeling, sorting, quality control and functional studies of ES cells. Currently, knowledge of ES surface molecules is limited. To identify new surface molecules, we generated a panel of rabbit monoclonal antibodies (rMabs) against mouse ES (mES) cells. We identified three monoclonal antibodies that interact with molecules on the mES cell surface and found that the expression of their respective antigens decreased upon mES cell differentiation. The antigen of the rMab ZjuESrMab29 was identified as granulocyte macrophage colony-stimulating factor receptor α (GM-CSFR α) by liquid chromatography–tandem mass spectrometry (LC–MS/MS). This study demonstrated that rabbit monoclonal antibody production via whole-cell immunization could be a practical method for the discovery of stem cell surface antigens. [Copyright &y& Elsevier]

To find a better condition for KIT immunostaining, five antibodies against KIT were compared, including a widely used polyclonal antibody (pAb) A4502, three mouse-derived mAb (MMA; T595, 1DC3, K45), and a newly developed rabbit-derived mAb (RabMA; Y145). Twenty-three gastrointestinal stromal tumors (GIST) were stained, including five KIT-weak or -negative GIST with PDGFRA gene mutations from a previous report, six Ewing/malignant primitive peripheral neuroectodermal tumor, six malignant melanomas, two neuroblastomas, six seminomas, seven thymic carcinoma and seven small cell carcinomas of the lung as KIT-expressing tumors, and four leiomyomas, six leiomyosarcomas, five gastric schwannomas, four solitary fibrous tumors, one inflammatory fibroid polyp and six desmoid tumors as KIT-non-expressing tumors. The positive rates of RabMA Y145 in KIT-expressing tumors were almost equal to pAb A4502, whereas those of three MMA had lower rates. MMA T595 was positive for mast cells, but negative for interstitial cells of Cajal and some GIST. None of the KIT-non-expressing tumors was positive for Y145, whereas some leiomyosarcomas and desmoid tumors were positive for A4502. At present, pAb A4502 or RabMA Y145 seems to be suitable for KIT immunostaining in formalin-fixed paraffin-embedded tumor specimens, especially in the differential diagnosis of GIST from other mesenchymal tumors. [ABSTRACT FROM AUTHOR]

Abstract: Rabbit antibodies may have favorable properties compared to mouse antibodies, including high affinities and better antigen recognition. We used a biochemical and reverse immunologic approach to generate and characterize rabbit anti-phospho-keratin and anti-keratin monoclonal antibodies (MAb). Human keratins 8 and 18 (K8/K18) were used as immunogens after isolation from cells pretreated with okadaic acid or pervanadate to promote Ser/Thr or Tyr hyperphosphorylation, respectively. Selected rabbit MAb were tested by immunofluorescence staining, immunoprecipitation, and 2-dimensional gels. Keratin phospho and non-phospho-mutants were used for detailed characterization of two unique antibodies. One antibody recognizes a K8 G61-containing epitope, an important epitope given that K8 G61C is a frequent mutation in human liver diseases. This antibody binds K8 that is not phosphorylated on S73, but its binding is ablated by G61 but not S73 mutation. The second antibody is bispecific in that it simultaneously recognizes two epitopes: one phospho (K8 pS431) conformation-independent and one non-phospho conformation-dependent, with both epitopes residing in the K8 tail domain. Therefore, a reverse immunologic and biochemical approach is a viable tool for generating versatile rabbit MAb for a variety of cell biologic applications including the potential identification of physiologic phosphorylation sites. [Copyright &y& Elsevier]

To date, SARS-CoV-2 pandemic has caused more than 188 million infections and 4.06 million deaths worldwide. The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein has been regarded as an important target for vaccine and therapeutics development because it plays a key role in binding the human cell receptor ACE2 that is required for viral entry. However, it is not easy to detect RBD in Western blot using polyclonal antibody, suggesting that RBD may form a complicated conformation under native condition and bear rare linear epitope. So far, no linear epitope on RBD is reported. Thus, a monoclonal antibody (mAb) that recognizes linear epitope on RBD will become valuable. In the present study, an RBD-specific rabbit antibody named 9E1 was isolated from peripheral blood mononuclear cells (PBMC) of immunized rabbit by RBD-specific single B cell sorting and mapped to a highly conserved linear epitope within twelve amino acids 480CNGVEGFNCYFP491 on RBD. 9E1 works well in Western blot on S protein and immunohistochemistry on the SARS-CoV-2 infected tissue sections. The results demonstrated that 9E1 can be used as a useful tool for pathological and functional studies of SARS-CoV-2. [ABSTRACT FROM AUTHOR]

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease. [ABSTRACT FROM AUTHOR]

Single domain antibodies have certain advantages including their small size, high stability and excellent tissue penetration, making them attractive drug candidates. Rabbit antibodies can recognize diverse epitopes, including those that are poorly immunogenic in mice and humans. In the present study, we established a method to isolate rabbit VH single domain antibodies for potential cancer therapy. We immunized rabbits with recombinant human B7-H3 (CD276) protein, made a phage-displayed rabbit VH single domain library with a diversity of 7 × 10 9 , and isolated two binders (A1 and B1; also called RFA1 and RFB1) from phage panning. Both rabbit VH single domains exhibited antigen-dependent binding to B7-H3-positive tumor cell lines but not B7-H3 knockout tumor cell lines. Our study shows that protein immunization followed by phage display screening can be used to isolate rabbit single domain antibodies. The two single domain antibodies reported here may have potential applications in cancer immunotherapy., (© The Author(s) 2020. Published by Oxford University Press on behalf of Antibody Therapeutics.)

Rabbits have distinct advantages over mice as a source of target-specific antibodies. They produce higher affinity antibodies than mice, and may elicit strong immune response against antigens or epitopes that are poorly immunogenic or tolerated in mice. However, a great majority of currently available monoclonal antibodies are of murine origin because of the wider availability of murine fusion partner cell lines and well-established tools and protocols for fusion and cloning of mouse hybridoma. Phage-display selection of antibody libraries is an alternative method to hybridoma technology for the generation of target-specific monoclonal antibodies. High-affinity monoclonal antibodies from nonmurine species can readily be obtained by constructing immune antibody libraries from B cells of the immunized animal and screening the library by phage display. In this article, we describe the construction of a rabbit immune Fab library for the facile isolation of rabbit monoclonal antibodies. After immunization, B-cell cDNA is obtained from the spleen of the animal, from which antibody variable domain repertoires are amplified and assembled into a Fab repertoire by PCR. The Fab genes are then cloned into a phagemid vector and transformed to E. coli, from which a phage-displayed immune Fab library is rescued. Such a library can be biopanned against the immunization antigen for rapid identification of high-affinity, target-specific rabbit monoclonal antibodies.

Rabbit monoclonal antibodies (RabMAbs) can recognize diverse epitopes, including those poorly immunogenic in mice and humans. However, there have been only a few reports on RabMAb humanization, an important antibody engineering step usually done before clinical applications are investigated. To pursue a general method for humanization of RabMAbs, we analyzed the complex structures of 5 RabMAbs with their antigens currently available in the Protein Data Bank, and identified antigen-contacting residues on the rabbit Fv within the 6 Angstrom distance to its antigen. We also analyzed the supporting residues for antigen-contacting residues on the same heavy or light chain. We identified "HV4" and "LV4" in rabbit Fvs, non-complementarity-determining region (CDR) loops that are structurally close to the antigen and located in framework 3 of the heavy chain and light chain, respectively. Based on our structural and sequence analysis, we designed a humanization strategy by grafting the combined Kabat/IMGT/Paratome CDRs, which cover most antigen-contacting residues, into a human germline framework sequence. Using this strategy, we humanized 4 RabMAbs that recognize poorly immunogenic epitopes in the cancer target mesothelin. Three of the 4 humanized rabbit Fvs have similar or improved functional binding affinity for mesothelin-expressing cells. Interestingly, 4 immunotoxins composed of the humanized scFvs fused to a clinically used fragment of Pseudomonas exotoxin (PE38) showed stronger cytotoxicity against tumor cells than the immunotoxins derived from their original rabbit scFvs. Our data suggest that grafting the combined Kabat/IMGT/Paratome CDRs to a stable human germline framework can be a general approach to humanize RabMAbs.

HER2/neu is an efficient target for cancer therapy. However, reports about its overexpression rate in colorectal carcinomas showed wide variability. This study aims to investigate HER2/neu expression in colorectal carcinomas using these two rabbit monoclonal HER2/neu antibodies, and to clarify the relationship between protein overexpression and gene amplification of HER2/neu and their clinicopathologic importance. Tissue microarray was performed from sections of 106 cases colorectal carcinomas. Their clinical data, including gender, age, stage, recurrence, lymph node metastasis, and follow-ups were collected. Immunohistochemistry for rabbit monoclonal antibody SP3 and 4B5 were performed, Fluorescent in situ hybridization was applied to detect the amplification of HER2/neu gene. The HER2/neu overexpression of (2+ and 3+) in our results were seen in 7.5% (8/106) for 4B5 and 3.8% (4/106) for SP3 respectively, the HER2/neu amplification was in 2.8% (3/106). All cases of overexpression for SP3 were included by those for 4B5. Both antibodies stained 3 cases of HER2/neu 3+, and FISH confirmed HER2/neu amplification did occurred in these cases. In our study, 4B5 was more sensitive to detect HER2/neu of colorectal carcinoma than SP3. 2.8% patients with colorectal patients might benefit from anti-HER2/neu therapy.

By targeting antigens specifically, monoclonal antibodies represent a new class of therapeutic agents for the clinical management of various diseases including cancers. Monoclonal antibody technology has been greatly developed by reducing murine content in antibodies to minimize side effects in clinical applications. However, several intrinsic disadvantages of antibodies with murine origin limit the clinical efficacy of monoclonal antibodies based targeted therapy. The development of rabbit monoclonal antibody technology provides an alternative source of monoclonal antibodies with higher specificity and less cost for the development of routine targeted therapy against cancers.