Hybridoma Cell Lines Research Articles

Identification of new antigenic epitopes of porcine reproductive and respiratory syndrome virus nsp12 protein using monoclonal antibodies

Porcine reproductive and respiratory syndrome virus (PRRSV), a member of the arteritis virus family, significantly impacts the swine industry due to its high infectiousness. nsp12, the nonstructural protein encoded by PRRSV, is a membrane-associated protein, with limited knowledge about its antigenic properties and functions. In this study, we used the expressed and purified nsp12 protein as antigen to immunize mice, and successfully screened five positive hybridoma cell lines that stably secrete anti-nsp12 monoclonal antibodies using immunological assays such as indirect ELISA and IFA. The antigenic epitopes recognized by the five monoclonal antibodies were identified using the fusion expression of peptides derived from the overlapping truncators of the nsp12 gene. The results showed that monoclonal antibodies 3G11 and 9C2 recognized the antigenic epitope 93TWGFESDTAY102, 2E3 recognized 115DYNDAFRARQ124, and 10G6 and 2A4 recognized 142PGPVIEPTL150. Furthermore, the three newly identified antigenic epitopes were all immunodominant and located on the surface of nsp12 protein. Notably, the antigenic epitopes 93–102 aa are all highly conserved across PRRSV-2 strains, making them suitable targets for PRRSV-2 detection. In conclusion, our findings advance the understanding of the antigenic properties of the PRRSV nsp12 protein and facilitate the development of assays for PRRSV detection.

Establishment of a Human Hybridoma Cell Line That Produces IgG Autoantibody Involved in the Pathogenesis of IgA Nephropathy

Establishment of a Human Hybridoma Cell Line That Produces IgG Autoantibody Involved in the Pathogenesis of IgA Nephropathy

A Lincomycin-Specific Antibody Was Developed Using Hapten Prediction, and an Immunoassay Was Established to Detect Lincomycin in Pork and Milk.

Prolonged consumption of animal-derived foods containing high levels of lincomycin (LIN) residues can adversely impact human health. Therefore, it is essential to develop specific antibodies and immunoassay methods for LIN. This study utilized computational chemistry to predict the efficacy of LIN haptens prior to chemical synthesis, with subsequent confirmation obtained through an immunization experiment. A hybridoma cell line named LIN/1B11 was established, which is specific to LIN. The optimized indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) method exhibited high specificity for detecting LIN residues, with an IC50 value of 0.57 ± 0.03 µg/kg. The method effectively detected LIN residues in pork and milk samples, achieving a limit of detection (LOD) ranging from 0.81 to 1.20 µg/kg and a limit of quantification (LOQ) ranging from 2.09 to 2.29 µg/kg, with recovery rates between 81.9% and 108.8%. This study offers a valuable tool for identifying LIN residues in animal-derived food products. Furthermore, the efficient hapten prediction method presented herein improves antibody preparation efficiency and provides a simple method for researchers in screening haptens.

Precise location of three novel linear epitopes using the generated monoclonal antibodies against the Knob domain of FAdV-4 surface structural protein, fiber1.

Fowl adenovirus serotype 4 (FAdV-4) is the main pathogen of hepatitis-hydropericardium syndrome (HHS), which brings huge economic losses to the poultry industry worldwide. Fiber-1 protein plays an important role in viral infection and pathogenesis by binding directly to cellular receptors of FAdV-4. In particular, the knob domain of fiber-1 protein has been reported to induce the production of neutralizing antibodies and arouse protection against the lethal challenge of chickens with FAdV-4. The fiber-1 knob (F1K) protein was expressed in a prokaryotic expression system and purified using Ni-NTA affinity chromatography. Monoclonal antibodies (mAbs) against FAdV-4 were generated by immunizing BALB/c mice with the purified F1K protein and screened using a series of immunoassays. Potential B cell epitopes on the knob domain of fiber-1 protein were mapped using enzyme-linked immunosorbent assay (ELISA) and dot-blot. Precious location and crucial amino acids of the identified epitopes were determined using peptide array scanning, truncations and alanine-scanning mutagenesis. The epitopes were analyzed and visualized on the knob trimer of FAdV-4 fiber-1 protein using the PyMOL software. Water-soluble recombinant fiber-1 knob (F1K) protein was obtained with the assistance of chaperone. Four monoclonal antibodies (5C10, 6F8, 8D8, and 8E8) against FAdV-4 were generated and characterized using indirect ELISA, Western blot, dot-blot, and immunological fluorescence assay (IFA). The mAbs were demonstrated to be from different hybridoma cell lines based on the sequences of the variable regions. Meanwhile, three distinct novel linear B-cell epitopes (319SDVGYLGLPPH329, 328PHTRDNWYV336, and 407VTTGPIPFSYQ417) on the knob domain of fiber-1 protein were identified and the key amino acid residues in the epitopes were determined. Structural analysis showed that the two adjacent epitopes 319SDVGYLGLPPH329 and 328PHTRDNWYV336 were exposed on the surface of the fiber-1 knob trimer, whereas the epitope 407VTTGPIPFSYQ417 was located inside of the spatial structure. This was the first identification of B-cell epitopes on the knob domain of fiber-1 protein and these findings provided a sound basis for the development of subunit vaccines, therapeutics, and diagnostic methods to control FAdV infections.

Expression of goat poxvirus P32 protein and monoclonal antibody preparation.

P32 protein serves as a crucial structural component of Goat pox virus (GTPV), which causes a highly virulent infectious disease in sheep and goats. Despite the fact that P32 has been widely expressed in the previous studies, it is difficult to obtain recombinant P32 efficiently. This study aimed to achieve soluble expression of P32 recombinant protein and to develop its specific monoclonal antibody. The gene fragment of P32Δ (GP32Δ) was synthesized by optimizing the coding sequence of amino acids 1-246 of the known goatpox P32 protein. Subsequently, GP32Δ was cloned into a prokaryotic expression vector for expression and purification, resulting in the successful production of soluble recombinant protein rP32Δ. Utilizing rP32Δ, an indirect ELISA method was established by immunizing 6-week-old BALB/c mice with inactivated GTPV as the antigen. Through hybridoma technology, three monoclonal antibody hybridoma cell lines secreting anti-goat pox virus rP32Δ were screened, designated as 2F3, 3E8, and 4H5, respectively. These monoclonal antibodies, classified as IgG1, IgG2a, and IgG2b, respectively, with κappa light chains, were characterized following ascites preparation and purification. Indirect ELISA results demonstrated that the ELISA potency of the three monoclonal antibodies exceeded 1:12800. Furthermore, Western blot analysis revealed specific reactivity of both 3E8 and 4H5 with rP32Δ, while immunofluorescence assays confirmed 3E8's ability to specifically recognize GTPV in cells. The preceding findings demonstrate the successful acquisition of the soluble expressed recombinant P32 protein and its specific monoclonal antibody 3E8 in this study, thereby laying a foundational material basis for the establishment of a GTPV detection method.

Preparation and Antigenic Site Identification of Monoclonal Antibodies against PB1 Protein of H9N2 Subtype AIV.

Recently, low pathogenic avian influenza virus (LPAIV), including H9N2 subtype, has been common clinical epidemic strains, and is widely distributed globally. The PB1 protein is a key component of the viral RNA polymerase complex (vRNP), and is vital to viral transcription and translation. In this study, to investigate the antigenic determinants in the PB1 protein, the truncated PB1 sequence (1bp-735bp) from H9N2 subtype AIV was amplified with PCR, and expressed in plasmid pET-28a (+). After purification, the recombinant PB1 protein was used to immunize BALB/c mice. Following immunization, hybridoma cells producing PB1-specific monoclonal antibodies were generated through the fusion of splenic lymphocytes with SP2/0 cells. Then, four stable hybridoma cell lines (5F12, 5B3, 2H9, and 3E6) were screened using indirect ELISA and Western blotting. Furthermore, two antigenic sites, 67NPIDGPLPED76 and 97ESHPGIFENS106, were identified through the construction of truncated overlapping fragments of the PB1 protein. These sites were conserved among 28 AIV strains, and were located on the PB1 protein surface. The findings offer a theoretical reference for the development and improvement of H9N2 vaccines and offer biological materials for virus detection during AIV infection mechanisms.

A simple procedure for preparing biotinylated immunoglobulin M from hybridoma culture medium

We previously reported a chromatography system for purifying immunoglobulin M (IgM) using N,N,N′,N′-ethylenediaminetetrakis(methylenephosphonic acid)-modified zirconia particles that selectively absorb immunoglobulins. Here, we report a simple procedure for preparing biotinylated IgM from hybridoma culture medium using this zirconia-based chromatography system. The culture medium of an IgM-producing hybridoma cell line was used as the starting sample solution, and the IgM in the medium was concentrated and partially purified by zirconia chromatography. Next, 9-(biotinamido)-4,7-dioxanonanoic acid N-succinimidyl ester was added to react with the proteins in the sample. Subsequently, only the biotinylated IgM was isolated by Capto Core 400 polishing column chromatography. The entire process was easy to perform, could be completed within 2 h, and provided highly pure biotin-labeled IgM. This procedure is expected to be applicable to the labeling of IgM with various compounds and drugs.

Monoclonal Antibodies for Rift Valley Fever Virus Nucleocapsid: Application in IgG/IgM ELISA for Sero-Diagnosis.

Rift Valley fever virus (RVFV) belonging to the Phenuiviridae family is responsible for a zoonotic disease called Rift Valley fever (RVF). Currently, RVFV has spread from Africa to Asia, and due to its ability to cause high mortality rates, it has significantly impacted human health and economic development in many societies. Highly specific and sensitive systems for sero-diagnosis of RVFV infection are needed for clinical use. BALB/c mice were immunized with recombinant RVFV nucleocapsid (rRVFV-N) protein and the spleen cells fused with SP2/0 myeloma cells to create hybridoma cell lines. The secreted monoclonal antibodies (MAbs) were purified and characterized. Enzyme-linked immunosorbent assay (ELISA) systems for the detection of IgG and IgM using the new MAbs were established and evaluated. Serum samples from 96 volunteers and 93 patients of suspected RVF from Kenya were tested compared with the ELISA systems based on inactivated viruses and the rabbit polyclonal antibody. Three monoclonal antibodies against rRVFV-N protein were established. The performance of the MAb-based sandwich IgG ELISA and the IgM capture ELISA perfectly matched the ELISA systems using the inactivated virus or the polyclonal antibody. Recombinant RVFV-N protein-specific MAbs were developed and they offer useful tools for RVFV studies. The MAb-based ELISA systems for detecting IgG and IgM offer safe and useful options for diagnosing RVFV infections in humans.

Prokaryotic expression of N protein of akabane disease virus and preparation of monoclonal antibody

In order to generate monoclonal antibodies against the akabane virus (AKAV) N protein, this study employed a prokaryotic expression system to express the AKAV N protein. Following purification, BALB/c mice were immunized, and their splenocytes were fused with mouse myeloma cells (SP2/0) to produce hybridoma cells. The indirect ELISA method was used to screen for positive hybridoma cells. Two specific hybridoma cell lines targeting AKAV N protein, designated as 2C9 and 5E9, were isolated after three rounds of subcloning. Further characterization was conducted through ELISA, Western blotting, and indirect immunofluorescence assay (IFA). The results confirmed that the monoclonal antibodies specifically target AKAV N protein, exhibiting strong reactivity in IFA. Subtype analysis identified the heavy chain of the 2C9 mAb's as IgG2b and its light chain as κ-type; the 5E9 mAb's heavy chain was determined to be IgG1, with a κ-type light chain. Their ELISA titers reached 1:4 096 000. This study successfully developed two monoclonal antibodies targeting AKAV N protein, which lays a crucial foundation for advancing diagnostic methods for akabane disease prevention and control, as well as for studying the function of the AKAV N protein.

Construction of CD138-targeted chimeric antigen receptor- modified T cells and their effect in multiple myeloma therapy

Objective: To construct a novel chimeric antigen receptor T (CAR-T) cell targeting CD138 and to investigate its cytotoxicity against myeloma cells. Methods: The hybridoma strain that can stably secrete the CD138 monoclonal antibody (mAb) was prepared and obtained through monoclonal antibody screening technology. The hybridoma strain cells were intraperitoneally injected into mice to produce ascites containing monoclonal antibodies, which were then collected and purified to obtain pure CD138 mAb. Further examinations were performed to assess the biological characteristics of CD138 mAb. The variable region sequence of this antibody was amplified through reverse transcription polymerase chain reaction and was used as the antigen recognition domain of CD138 CAR, which was subsequently expressed on the surface of T cells by lentiviral infection. Flow cytometry was employed to assess the phenotype of CD138 CAR-T cells. In vitro cytotoxicity and degranulation assays were performed to evaluate their antitumor effects. Results: ① We successfully prepared anti-human CD138 antibody hybridoma cell lines and screened a hybridoma cell strain, 5G2, which could persistently and stably secrete the anti-CD138 antibody. ② The purified CD138 (5G2) mAb can especially recognize CD138(+) cells with a binding affinity constant (K(D)) of 6.011×10(-9) mol/L and showed no significant binding activity with CD138(-) cells. ③The variable region sequence of the CD138 (5G2) antibody was obtained using molecular cloning technology, and CD138 (5G2) CAR was successfully constructed and expressed on T cells through lentivirus infection and, concurrently, demonstrated effective binding to recombinant human CD138 protein.④ The proliferation of T cells transduced with the CD138 (5G2) CAR was highly efficient. The phenotype analysis revealed that CD138 (5G2) CAR-T cells exhibited a greater tendency to differentiate into central memory T cells and memory stem T cells, with a reduced proportion of terminally differentiated effector memory subsets. ⑤CD138 (5G2) CAR-T cells demonstrated specific cytotoxicity against CD138(+) myeloma cell line H929, whereas CD138(-) cell line K562 remained unaffected. The percentage of residual H929 cells was (12.92±8.02) % after co-culturing with CD138 (5G2) CAR-T cells, while (54.25±15.79) % was left in the Vector-T group (E∶T=1∶2; P<0.001). ⑥Results of degranulation assays demonstrated a significant activation of CD138 (5G2) CAR-T cells after co-culture with the H929 cell line, whereas no significant activation was observed in Vector-T cells [ (25.78±3.35) % vs (6.13±1.30) %, P<0.001]. ⑦After co-culturing with CD138(+) cells, CD138 (5G2) CAR-T cells exhibited a significant increase in cytokine secretion compared to the Vector-T group [interleukin-2: (1 697.52±599.05) pg/ml vs (5.07±1.17) pg/ml, P<0.001; interferon-γ: (3 312.20±486.38) pg/ml vs (9.28±1.46) pg/ml, P<0.001; and tumor necrosis factor-α: (1 837.43±640.49) pg/ml vs (8.75±1.65) pg/ml, P<0.001]. However, no significant difference was observed in cytokine secretion levels between the two groups after co-culturing with CD138(-) cells. Conclusion: This study successfully prepared a novel monoclonal antibody against CD138, and CAR-T cells constructed with the antigen recognition domain derived from this 5G2 mAb demonstrated effective antitumor activity against myeloma cells. This can be used as a new option for the detection of the CD138 antigen and proposes a novel strategy for multiple myeloma immunotherapy.

Identification of B-cell epitopes located on the surface in the PB2 protein of the H9N2 subtype avian influenza virus

ABSTRACT Avian influenza (AI), caused by H9N2 subtype avian influenza virus (AIV), poses a serious threat to poultry farming and public health due to its transmissibility and pathogenicity. The PB2 protein is a major component of the viral RNA polymerase complex. It is of great importance to identify the antigenic determinants of the PB2 protein to explore the function of the PB2 protein. In this study, the PB2 sequence of H9N2 subtype AIV, from 1090 to 1689 bp, was cloned and expressed. The recombinant PB2 protein with cutting gel was used to immunize BALB/c mice. After cell fusion, the hybridoma cell lines secreting monoclonal antibodies (mAbs) targeting the PB2 protein were screened by indirect ELISA and western blotting, and the antigenic epitopes of mAbs were identified by constructing truncated overlapping fragments in the PB2 protein of H9N2 subtype AIV. The results showed that three hybridoma cell lines (4B7, 4D10, and 5H1) that stably secreted mAbs specific to the PB2 protein were screened; the heavy chain of 4B7 was IgG2α, those of 4D10 and 5H1 were IgG1, and all three mAbs had kappa light chain. Also, the minimum B-cell epitope recognized was 475LRGVRVSK482 and 528TITYSSPMMW537. Homology analysis showed that these two epitopes were conserved among the different subtypes of AIV strains and located on the surface of the PB2 protein. The above findings provide an experimental foundation for further investigation of the function of the PB2 protein and developing monoclonal antibody-based diagnostic kits.

Diversity and affinity from plasma vs. B cells for monoclonal antibody development

Abstract Diagnostic assay development requires a diverse panel of high affinity antibodies. Historically, our lab has used splenic CD19+ B-cells to generate mouse hybridoma cell lines. However, literature suggests that, as a group, terminally differentiated, CD138+ plasma cells secrete the highest affinity antibodies, making these cells an attractive target for hybridoma development. To compare the use of these two lymphocyte populations for generating mouse hybridomas, three fusion experiments were completed whereby cells were separately isolated and fused. Hybridomas were plated in semi-solid media, expanded, and selected based on size and antibody secretion. An average of 246 (+ 73) plasma cell hybridoma colonies were available for selection, per 1x106 specific lymphocytes used for fusion, compared to 19 (+ 13) B cell hybridoma colonies. Cells from selected colonies were transferred to 96-well plates, grown for 7 days and the antibodies screened for antigen binding by enzyme immunoassay. Across the three fusion experiments, 40 (+19)% of the wells with growth from the plasma cell hybridomas were antigen reactive, vs. 6 (+ 0.5)% from the B cell hybridomas. Our data suggests a higher fusion efficiency from the plasma cell population, which allows for a greater sampling of the mouse immune repertoire, leading to identification of more antigen specific antibodies with a wider range of affinities and greater sequence diversity.

THE RESTORATION OF HYBRIDOMA CELLS LINES AFTER UNSUITABLE STORAGE

The ongoing war in Ukraine has disrupted the infrastructure of cryopreservation, compromising the viability of hybridoma cell lines that are critical to therapeutic antibody production. This study focuses on hybridoma cells that produce antibodies targeting protein C. They were damaged by wrong preservation conditions: in a freezer at -80 °C. The aim of the study was to identify optimal culture conditions for restoring the functionality and antibody production capacity of these compromised hybridoma cells. Materials and methods. The cryopreserved hybridoma clone, producing the IV-6A antibody, was used. After thawing, the cells were cultured in RPMI medium supplemented with 10% of three different commercially available Fetal Bovine Serum (FBS) variants: F7524 and F2442 (Sigma, USA) and One Shot format A5256701 (Gibco, USA). Cell vitality was monitored through daily observations with a light microscope. Quality assessment of antibody production was conducted through ELISA analysis. The binding capacity of the purified antibody was compared against a reference. Results. Hybridoma cells cultured in RPMI medium with F7524 or F2442 FBS exhibited a significant decrease in viability by day 4. Conversely, cells cultured with A5256701 FBS displayed high viability and an exponential growth pattern. ELISA confirmed that these cells retained the ability to produce the monoclonal antibody IV-6A. However, the purified antibody from the restored cell line showed a slightly less intensive binding capacity compared to the reference antibody. Conclusions. Despite the lasting improper storage conditions, the hybridoma cells were successfully restored retaining their ability to produce monoclonal antibodies specific to protein C.

Characterization of Two Highly Specific Monoclonal Antibodies Targeting the Glycan Loop of the Zika Virus Envelope Protein.

Zika virus (ZIKV) is an emerging flavivirus associated with several neurological diseases such as Guillain-Barré syndrome in adults and microcephaly in newborn children. Its distribution and mode of transmission (via Aedes aegypti and Aedes albopictus mosquitoes) collectively cause ZIKV to be a serious concern for global health. High genetic homology of flaviviruses and shared ecology is a hurdle for accurate detection. Distinguishing infections caused by different viruses based on serological recognition can be misleading as many anti-flavivirus monoclonal antibodies (mAbs) discovered to date are highly cross-reactive, especially those against the envelope (E) protein. To provide more specific research tools, we produced ZIKV E directed hybridoma cell lines and characterized two highly ZIKV-specific mAb clones (mAbs A11 and A42) against several members of the Flavivirus genus. Epitope mapping of mAb A11 revealed glycan loop specificity in Domain I of the ZIKV E protein. The development of two highly specific mAbs targeting the surface fusion protein of ZIKV presents a significant advancement in research capabilities as these can be employed as essential tools to enhance our understanding of ZIKV identification on infected cells ex vivo or in culture.

MONOCLONAL ANTIBODIES AGAINST CTLA-4 AND PD-L1 RECEPTORS OF THE CATTLE IMMUNE SYSTEM

With the progression of bovine leukemia virus (BLV), the concentration of T-cells, as well as CTLA-4 and PD-1 receptors on their cytoplasmic membrane increases. Elevated concentrations of regulatory T-cells lead to increased production of transforming growth factor-β (TGF β), suppression of interferon-γ (IFN-γ) expression, tumor necrosis factor-α (TNF-α), and inhibition of natural killer (NK) cells. Effector and cytotoxic T-lymphocytes, as well as the production of cytokines IFN-γ and TNF-α, play a crucial role in immune response against viral infections. However, at late subclinical stages, T-lymphocyte activity decreases due to the activity of regulatory T-lymphocytes, contributing to infection growth and progression to clinical disease. Blockade of CTLA-4 and PD-1/PD-L1 receptors with specific antibodies restores the immune response against BLV. Monoclonal antibodies (mAbs) against recombinant bovine CTLA-4 and PD-L1 were obtained using hybridoma technology methods. The obtained mAbs were analyzed using enzyme-linked immunosorbent assay (ELISA) and western blotting methods. As a result of the study, hybridoma cell lines producing mAbs to recombinant bovine CTLA-4 and PD-L1 receptors were obtained. The hybridomas produced IgG1 class mAbs that specifically reacted with standard proteins and had a binding constant: 3b3 - 2.9×108 M-1, 4h10 - 1.9×108 M-1. The obtained mAbs effectively blocked the reaction of commercial bovine CTLA 4 and PD-L1 proteins with specific polyclonal antibodies in an indirect fluorescent antibody assay (IFA).

Preparation and interaction mechanism analysis of single-chain fragment variables against phenylethanolamine A

This is the first report on the screening, expression, and recognition mechanism analysis of single-chain fragment variable (scFv) against phenylethanolamine A (PEAA), a newly emerged β-adrenergic agonist illegally used as a feed additive for growth promotion. The PEAA-specific scFv scFv, called scFv-32, was screened from hybridoma cell lines by phage display and was found to be optimally expressed in the E. coli system. The ic-ELISA results revealed an IC50 value of 10.34 μg/L for scFv-32 and no cross-reactivity with other β-adrenergic agonists. Homology modeling and molecular docking revealed the key binding sites VAL178, TYP228, and ASP229. One hydrogen bond, two pi-sigma bonds, and one pi-pi bond maintain the formation of the antibody‒drug complex. Alanine scanning mutagenesis of the three predicted key binding sites showed that the mutants completely lost their recognition activity, which confirmed the accuracy of the theoretical analysis. These results are valuable for the preparation of scFvs and the analysis of the molecular recognition mechanism of antigen-antibodies.Graphical abstract

A Rapid and Sensitive Gold Nanoparticle-Based Lateral Flow Immunoassay for Chlorantraniliprole in Agricultural and Environmental Samples.

Chlorantraniliprole (CAP) is a new type of diamide insecticide that is mainly used to control lepidopteran pests. However, it has been proven to be hazardous to nontarget organisms, and the effects of its residues need to be monitored. In this study, five hybridoma cell lines were developed that produced anti-CAP monoclonal antibodies (mAbs), of which the mAb originating from the cell line 5C5B9 showed the highest sensitivity and was used to develop a gold nanoparticle-based lateral flow immunoassay (AuNP-LFIA) for CAP. The visible limit of detection of the AuNP-LFIA was 1.25 ng/mL, and the detection results were obtained in less than 10 min. The AuNP-LFIA showed no cross-reactivity for CAP analogs, except for tetraniliprole (50%) and cyclaniliprole (5%). In the detection of spiked and blind samples, the accuracy and reliability of the AuNP-LFIA were confirmed by a comparison with spiked concentrations and verified by ultra-performance liquid chromatography-tandem mass spectrometry. Thus, this study provides the core reagents for establishing CAP immunoassays and a AuNP-LFIA for the detection of residual CAP.

Production of Antibodies to Peptide Targets Using Hybridoma Technology.

Hybridoma technology is a well-established and indispensable tool for generating high-quality monoclonal antibodies and has become one of the most common methods for monoclonal antibody production. In this process, antibody-producing B cells are isolated from mice following immunization of mice with a specific immunogen and fused with an immortal myeloma cell line to form antibody-producing hybridoma cell lines. Hybridoma-derived monoclonal antibodies not only serve as powerful research and diagnostic reagents but have also emerged as the most rapidly expanding class of therapeutic biologicals. In spite of the development of new high-throughput monoclonal antibody generation technologies, hybridoma technology still is applied for antibody production due to its ability to preserve innate functions of immune cells and to preserve natural cognate antibody paring information. In this chapter, an overview of hybridoma technology and the laboratory procedures used for hybridoma production and antibody screening of peptide-specific antibodies are presented.

Preparation of colloidal gold test strips for the detection of antibodies to peste des petits ruminants based on monoclonal antibodies to N protein

A simple, fast, and visual method for detecting antibodies against peste des petits ruminants virus (PPRV) using colloidal gold strips was developed. In this study, the pET-32a-N was transformed into Escherichia coli Rosetta (DE3) for expression. Hybridoma cell lines were generated by fusing SP2/0 myeloma cells with splenocytes from immunized mice with the expressed and purified N protein of PPRV. The PPRV N protein was labeled with colloidal gold particles as the gold-labeled antigen. The N protein served as the gold standard antigen and as the test (T) line-coated antigen, while the monoclonal antibody served as the quality control (C) line-coated antibody to assemble the colloidal gold immunochromatographic test strips for detecting antibodies against the N protein of PPRV. Hybridoma cell line designated as 1F1 was able to stably secrete the monoclonal antibody against the N protein of PPRV. The titer of 1F1 monoclonal antibody in ascites was 1:128 000 determined by indirect enzyme-linked immunosorbent assays (ELISA), and the immunoglobulin subtype of the monoclonal antibody was IgG1, with kappa chain. The obtained monoclonal antibody was able to specifically recognize the N protein of PPRV, as shown by Western blotting and indirect immunofluorescent assay (IFA). The developed colloidal gold test strip method was able to detect PPRV antibodies specifically, and there was no difference between different batches of the test strips. Testing of a total of 122 clinical sera showed that the compliance rate of the test strip with ELISA test was 97.6%.The test strip assay developed in this study has good specificity, reproducibility, and sensitivity, and it can be used for the rapid detection of PPRV antibodies.

Structural and functional Analysis of novel 3‐Nitrotyrosine Aβ‐specific Antibodies

AbstractBackgroundPosttranslational modified (PTM) amyloid peptides are increasingly within the spotlight of Alzheimer’s drug research. Some of these modifications, such as the formation of pyroglutamate or isoaspartate, are associated with the deposition and increased toxicity of amyloid peptides. The role of nitration at Tyr10 of Aβ and its abundance in AD brain is less well understood. How 3NY10‐Aβ is formed is controversial, as well as the influence on aggregation behaviour. Hence, we here aimed at generation and characterization of monoclonal antibodies specific for 3NY10‐Aβ as research tool and potential therapeutic molecules.MethodMonoclonal antibodies were developed by immunizing mice with 3NY10‐Aβ(6‐14) and derivation of corresponding hybridoma cell lines. Characterisation of the antibodies was achieved by a variety of biochemical and kinetic analyses: isothermal titration calorimetry (ITC), dot blot analysis and ThioflavinT aggregation studies. X‐ray crystallography was used for structure determination. We used immunohistochemical (IHC) staining of brain tissue from 5xFAD mice and enzyme linked immune sorbent assay (ELISA) to assess the formation of 3NY10‐Aβ in transgenic mice.ResultTwo highly affine 3NY10‐Aβ antibodies 4C3 and 3B3 have been generated, both binding 3NY10‐Aβ(1‐16) in the nanomolar range (KD4C3 = 11 nM, KD3B3 = 88,5 nM) as determined by ITC. The specificity of the antibodies was confirmed by dot blot analysis, using several post‐translationally modified Aβ‐peptides. Aggregation studies of 3NY10‐Aβ(1‐40/42) peptides in presence of 3B3 or 4C3 showed an influence of fibril formation, as could be verified by means of transmission electron microscopy. Co‐crystallisation of 4C3 Fab‐fragment and 3NY10‐Aβ(1‐16) at a resolution of 2.5 Å revealed a binding of 3NY10. To investigate the role of 3NY10‐Aβ in AD, IHC‐staining of brain from mice with AD‐like pathology (5xFAD mice) was performed. The data support presence of 3NY10‐Aβ within amyloid deposits, although staining appeared weak. Likewise, a Sandwich‐ELISA of homogenized murine brain tissue displayed no significant proportion of 3NY10‐modified Aβ in relation to the total Aβ(1‐40/42).ConclusionIt has been demonstrated that the unique monoclonal 3NY10‐Aβ specific antibodies 4C3 and 3B3 are a suitable tool for investigating the role of 3NY10‐Aβ in AD. Future research aims to quantify the level of 3NY10‐Aβ in human brain tissue with AD‐pathology.