Hybridoma Cell Lines Research Articles

Preparation and characterisation of monoclonal antibodies against deoxynivalenol

To prepare broad-spectrum and specific monoclonal antibodies (mAb) against deoxynivalenol (DON), DON-BSA was synthesised by the carbonyl diimidazole method as an artificial antigen to immunise Balb/C mice, and the coated DON-OVA was synthesised by the carbodiimide method to detect anti-DON antibodies. Monoclonal antibodies were screened by indirect ELISA and indirect competitive ELISA. A hybridoma cell line (4F3) capable of stably secreting DON antibody was obtained. The titre of antibody in the culture supernatant was 1:1.28 × 103, and the titre of the ascites antibody was 1:3.2 × 105. The monoclonal antibody homology was IgG1/λ. The half inhibitory concentration (IC50) of DON was 9.84 ng/mL. The cross-reaction rates with 3-Ac-DON and 15-Ac-DON were 60.44% and 52.04%, respectively, but no cross-reaction with other mycotoxins was observed. The results showed that the anti-DON monoclonal antibodies prepared in this experiment could recognise not only DON but also 3-Ac-DON and 15-Ac-DON, which could provide materials for the next step to establish a method to detect DON and its similar compounds. Highlights This study demonstrated that an artificial antigen was synthesised by carbonyl diimidazole method, and a novel broad-spectrum, high affinity monoclonal antibody was developed. The anti-DON mAb developed in this study has better sensitivity, moreover the antibody was sensitive to DON and its derivatives. Therefore, it could be used for simultaneous monitoring of three mycotoxins. Results of this study will further lay the foundation for the establishment of immunological assays for the total amount of similar compounds.

The monofunctional platinum(II) compounds, phenanthriplatin and pyriplatin, modulate apoptosis signaling pathways in HEI-OC1 auditory hybridoma cells

Cisplatin is a platinum(II) chemotherapy drug that can cause the side-effect of ototoxicity and hearing loss. The monofunctional platinum(II) complexes, phenanthriplatin and pyriplatin, have recently been investigated as anti-cancer agents but their side-effects are largely unknown. Here, we used the auditory hybridoma cell line, HEI-OC1, to investigate the ototoxicity of cisplatin, phenanthriplatin and pyriplatin. The effect of these compounds against cellular viability, on reactive oxygen species (ROS) production, mitochondrial membrane polarization, caspase-3/7 activity, DNA integrity and caspase-12 expression were measured using spectrophotometric, flow cytometric and blot analyses. We found that the monofunctional complexes and cisplatin decreased cellular viability. All three compounds increased ROS yield at 24 h, but at 48 h, ROS levels returned to normal. Also, the compounds did not depolarize the mitochondrial membrane. All three compounds reduced caspase-3/7 activity at 24 h; cisplatin increased caspase-3/7 activity and caused apoptosis at 48 h. Caspase-12 expression was associated with all three compounds. In summary, the monofunctional complexes may cause ototoxicity like cisplatin. Phenanthriplatin and pyriplatin may cause ototoxicity initially by inducing ROS production, but they may also signal through distinct apoptotic pathways that do not integrate caspases-3/7, or may act at different time-points in the same pathways.

Undergraduate immunology lab project including opportunities for experimental design, a poster presentation, and the opportunity to partially purify antibodies from hybridoma cells to utilize in a Western blot

Abstract Undergraduate students benefit from lab activities that more closely mirror research conditions. In this 7 week lab project, students maintain the hybridoma cell line MARC 2B7 (ATCC CRL-2509), and design a mini-research project to examine antibody production under a modified growth condition of their choice. Lab groups are responsible for making their own solutions, designing controls, and monitoring and maintaining the cell line over a two week period of time. Antibody levels are measured using a dot blot. For each phase, students are presented with a standard lab protocol accompanied by a list of suggestions and/or modifications so that the students are responsible for writing their own final protocol. For example, the dot blot protocol provided is based on chemiluminescent detection; however, the commercial secondary antibody is HRP conjugated so students are also provided the manufacturer’s information on the secondary antibody and the HRP substrate available in lab. This mirrors more real-world lab experiences, and requires students to think more deeply about the experimental design. Each lab group presents a poster on their mini-project to the class. Subsequently, lab groups use ammonium sulfate precipitation followed by a desalting column to concentrate and partially purify the antibodies from hybridoma supernatants that demonstrated positive results on the dot blots. This product is used as the primary antibody for a Western blot at the end of the experiment arc. In addition to providing a platform to discuss both monoclonal and polyclonal antibodies, and enabling students to practice experimental design, this project requires students to utilize their work from early in the semester in a productive final lab experience.

Development of a Chimeric Antigen-Binding Fragment Directed Against Human Galectin-3 and Validation as an Immuno-Positron Emission Tomography Tracer for the Sensitive In Vivo Imaging of Thyroid Cancer.

Background: The lack of facile methods for the specific characterization of malignant thyroid nodules makes the diagnosis of thyroid cancer (TC) challenging. Due to its restricted expression in such nodules, the cell-associated lectin galectin-3 (Gal3) has emerged as a marker for TC with growing interest for in vivo imaging as well as targeted radionuclide therapy. To accelerate translation into clinical application, we have developed a cognate chimeric human antigen-binding fragment (Fab) derived from the rat anti-Gal3 monoclonal antibody M3/38. Methods: The variable immunoglobulin (Ig) light and heavy chain sequences were cloned from the hybridoma cell line, and the corresponding Fab carrying human IgG1/κ constant genes was functionally produced in the periplasm of Escherichia coli and purified to homogeneity. To moderately prolong its plasma half-life and, thus, increase tumor uptake, the recombinant Fab was fused with a long disordered amino acid chain comprising in total 200 Pro, Ala, and Ser residues (PASylation). This novel tracer was subjected to in vitro characterization and in vivo validation by using two thyroid cancer orthotopic murine models. To this end, the αGal3-Fab-PAS200 was conjugated with deferoxamine (Dfo), labeled with 89Zr under mild conditions and tested for binding on TC cell lines. Athymic nude mice were inoculated either with FRO82-1 or with CAL62 tumor cells into the left thyroid lobe. After intravenous injection with ∼3.0 MBq of 89Zr-Dfo-PAS200-Fab, these mice were subjected to positron emission tomography (PET)/computed tomography imaging followed by quantification of tumor accumulation and immunohistochemical analysis. Results: The αGal3-Fab-PAS200 revealed high affinity toward the recombinant Gal3 antigen, with a dissociation constant ≤1 nM as measured via enzyme-linked immunosorbent assay, surface plasmon resonance spectroscopy, and radioactive cell binding assay. The in vivo Gal3-targeting by the 89Zr(IV)-labeled protein tracer, as investigated by immuno-PET, demonstrated highly selective and fast accumulation in orthotopically implanted tumors, with strong contrast images achieved 24 hours postinjection, and no uptake in the tumor-free thyroid lobe, as also confirmed by biodistribution studies. Conclusions: The chimeric αGal3 89Zr-Dfo-PAS200-Fab tracer exhibits selective accumulation in the tumor-bearing thyroid lobe of xenograft mice. Thus, this novel radioactive probe offers potential to change TC management, in addition to current diagnostic procedures, and to reduce unnecessary thyroidectomies.

Immunostimulating effect of sweet potato fiber extract on IgM production by HB4C5 cells

Sweet Potato (Ipomea batatas) is a local tuber potentially to be developed as functional food. The aims of this research is to evaluate the immunomodulatory effect of the sweet potato fibre extract (SFE) in vitro by animal cell culture techniques and identification the active compounds of SFE. SFE were prepared by autoclaving the sweet potato fiber powder in distilled water with room temperature 25 °C for 2 hours in distillated water and dialyzed with membrane with molecular weight cut off 14 kDa and 30 kDa. The IgM production of SFE against human hybridoma cell line HB4C5 cells was evaluated. The results of this research indicated that SFE stimulated IgM production by HB4C5 cells with heat and dialysis treatments in dose dependent manner. IgM production was increased by SFE with molecular weight more than 14 kDa and less than 30 kDa. The conclusion of this evaluation, SFE facilitated IgM production by HB4C5 cells and revealed that the SFE has positive effects on immunostimulatory activity in vitro.

Effects of L-Serine Against Cisplatin-Mediated Reactive Oxygen Species Generation in Zebrafish Vestibular Tissue Culture and HEI-OC1 Auditory Hybridoma Cells.

Cisplatin is a platinum-based chemotherapy compound effective against a variety of cancers. However, it can cause increased reactive oxygen species (ROS) production in auditory and vestibular tissue leading to permanent hearing and balance loss. The amino acid, L-serine, has been shown to reduce ROS in some tissue types. In this project, we first investigated whether L-serine could reduce cisplatin-mediated ROS generation in zebrafish utricular tissue culture using spectrophotometry and the fluorescent ROS detector dye, H2DCFDA. Then, we examined whether L-serine could prevent the effect of cisplatin against cellular viability in the mouse auditory hybridoma cell line, HEI-OC1, using the spectrophotometric (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay. As a final step, we used H2DCFDA dye and flow cytometry analysis to determine if L-serine could counteract the effect of cisplatin on ROS production in this cell line. We found that cisplatin and L-serine treatment may influence ROS production in utricular tissue. Further, although L-serine did not counteract the effect of cisplatin against HEI-OC1 cellular viability, the amino acid did prevent the platinum compound's effect to increase ROS in these cells. These results suggest that L-serine may act in auditory and vestibular tissues as an effective protectant against cisplatin-mediated toxicity.

Development of an Enzyme-Linked Immunosorbent Assay Method for the Detection of Rhein in Rheum officinale.

Rhein is an important quality-control marker of Rheum officinale. The aim of this study was to develop an indirect competitive enzyme-linked immunosorbent assay (icELISA) for rhein detection, which acts as a powerful tool for quality control and proper usage of Rheum officinale. First, a specific and sensitive monoclonal antibody (mAb) against rhein was produced from a stable hybridoma cell line, 1F8, generated by the fusion of mouse myeloma sp2/0 with spleen cells obtained from a Bal b/c mouse immunized with rhein-BSA. Then, an icELISA method was developed with an IC50 value and working range of 0.05 μg L−1 and 0.02–0.11 μg L−1, respectively. The icELISA revealed high assay specificity, since it only had a relatively high cross reactivity with aloe-emodin (27%) and almost no cross reactivity with any other anthraquinones (<1%). When spiked with 0.2–2 mg kg−1 of rhein, the recoveries ranged from 84.19% to 102.90%. Finally, icELISA was used to detect rhein contents of Rheum officinale collected from different regions, and the results corresponded well with those of HPLC. Overall, the developed icELISA with high specificity and sensitivity provided a rapid and simple method for rhein detection, and it may be a powerful tool for quality control and proper usage of Rheum officinale.

Production and identification of monoclonal antibodies and development of a sandwich ELISA for detection of the H3-subtype avian influenza virus antigen

The H3 subtype of avian influenza virus (AIV) is widespread in avian species and is frequently isolated in surveillance projects; thus, we have developed a more effective diagnostic approach of a monoclonal antibody (mAb)-based sandwich ELISA for the H3 AIV detection. First, we have produced the essential reagent of mAb against AIV H3 strains with the development of an mAb-Mouse immunization with a purified H3-subtype AIV strain and cell fusion to generate hybridoma cells. These cells were screened with hemagglutination inhibition (HI) tests, and optimal cells were subcloned. We chose a hybridoma cell line that steadily secreted a specific H3-subtype AIV mAb, designated 9F12, that belongs to the IgG1 subclass and has a K-type light chain. 9F12 was shown to bind specifically to the H3-subtype AIV antigen by both immunofluorescence assay and Western blot analysis. Finally, a 9F12-based sandwich ELISA was successfully developed and used to specifically test for this antigen. The sandwich ELISA conditions were optimized, and the specificity and sensitivity were validated. The results for clinical sample detection were consistent with viral isolation. Consequently, the 9F12-based sandwich ELISA is a specific, sensitive, robust, rapid and versatile diagnostic tool for H3-subtype AIV and provides a promising strategy for effective influenza virus prevention and control.

Development of ELISA and Lateral Flow Immunoassays for Ochratoxins (OTA and OTB) Detection Based on Monoclonal Antibody.

Ochratoxins were important secondary metabolites secreted by fungi, and OTA and OTB are mainly significant mycotoxin, having toxic effects on humans and animals. Therefore, it is important to establish a rapid, sensitive, and precise method for ochratoxins detection and quantification in real samples. In this study, a stable monoclonal antibody (mAb) that recognizing both OTA and OTB toxins was employed for the establishment of indirect competitive ELISA (ic-ELISA), colloidal gold nanoparticles (CGNs), and nanoflowers gold strips (AuNFs) for detection of ochratoxins in real samples. A 6E5 hybridoma cell line stable secreting mAb against both OTA and OTB toxins was obtained by fusion of splenocytes with myeloma SP2/0 cells. The 6E5 mAb had a high affinity (3.7 × 108 L/mol) to OTA, and also showed similar binding activity to OTB. The optimized ic-ELISA resulted in a linear range of 0.06–0.6 ng/mL for ochratoxins (OTA and OTB) detection. The IC50 was 0.2 ng/mL and the limit of detection (LOD) was 0.03 ng/mL. The mean recovery rate from the spiked samples was 89.315 ± 2.257%, with a coefficient variation of 2.182%. The result from lateral flow immunoassays indicated that the LOD of CGNs and AuNFs were 5 and 1 μg/mL, respectively. All these results indicated that the developed ic-ELISA, CGNs, and AuNFs in this study could be used for the analysis of the residual of ochratoxins (OTA and OTB) in food and agricultural products.

Construction of a new anti-CD123 chimeric antigen receptor T cells and effect of anti-acute myeloid leukemia

目的构建一种新的靶向CD123的嵌合抗原受体T细胞(CAR-T细胞)，为CD123阳性白血病的免疫治疗提供实验参考。方法通过单克隆筛选技术获得能稳定分泌CD123抗体的杂交瘤细胞株6E11，将杂交瘤细胞扩增后腹腔注射至Balb/c小鼠腹腔内，收集腹水并处理、纯化得到单克隆抗体，测定抗体效价并对其特异性进行验证；RT-PCR法获得轻链和重链可变区序列，并以此为基础利用分子克隆技术构建一种新的靶向CD123嵌合抗原受体，包装病毒后感染T细胞制备CD123CAR-T细胞，通过功能实验初步探讨6E11CAR-T细胞体外抗白血病的能力。结果①获得1株稳定分泌抗人CD123抗体的杂交瘤细胞株6E11并获得其可变区序列。②6E11单克隆抗体对CD123蛋白亲和性高，解离常数(Kd值)为2.10nmol/L，特异性识别CD123阳性细胞且与CD123阴性细胞无交叉反应。 ③成功构建了CD123CAR慢病毒载体，感染T细胞后获得了靶向CD123的CAR-T细胞(6E11CAR-T)，感染效率大于60％。 ④6E11CAR-T能明显杀伤CD123阳性靶细胞MV4-11，效靶比1∶1时6E11CAR-T细胞对MV4-11细胞的杀伤比例明显高于 Vecor-T细胞[(98.60±1.20)％对(20.28±6.74)％，P<0.001]，但对CD123阴性靶细胞K562没有明显杀伤作用。 ⑤MV4-11细胞可以显著激活6E11CAR-T，但对Vecor-T细胞无明显激活作用[(26.33±3.30)％对(1.17±0.06)％，P<0.001]。⑥6E11CAR-T与MV4-11细胞共培养上清中细胞因子水平均显著高于Vecor-T组[IL-2：(92.90±1.51)pg/ml对(6.05±3.41)pg/ml，P<0.001；TNF-α：(1 407.20±91.95)pg/ml对(7.86±0.85)pg/ml，P<0.001；IFN-γ：(5 614.60±170.17)pg/ml对(8.42±2.70)pg/ml，P<0.001]但与 K562细胞共培养后，两组各细胞因子水平差异无统计学意义。 ⑦6E11CAR-T在与CD123阳性髓系白血病(AML)原代细胞共培养过程中被显著激活，且能有效杀伤原代 AML细胞。结论杂交瘤细胞株6E11能稳定分泌高效特异的抗人CD123单克隆抗体，可用于检测表达人CD123的细胞，也能应用在靶向人CD123蛋白的肿瘤免疫治疗中，以 6E11Ig可变区序列为抗原识别区的CD123CAR-T细胞，具有明确的体外抗白血病活性，为进一步的临床研究奠定了基础。

Development of monoclonal antibodies against melon necrotic spot virus and their use for virus detection

Melon necrotic spot virus (MNSV) is endemic in cucurbit crops worldwide, causing epidemic outbreaks from time to time. MNSV is transmitted in nature by a soil-inhabiting fungus and also through seeds, making its detection in seed certification programs a necessity. Polyclonal antisera and RT-PCR-based detection assays have been developed for MNSV, but up to now no monoclonal antibodies (mAbs) have been described for this virus. In this study, we have produced mAbs in BALB/c mice against the MNSV over-expressed coat protein (CP). Titers of the antibodies produced against the recombinant MNSV CP ranged around 10−3–10−4 and the IgG yields for each mAb from ascitic fluids ranged from 1.51 to 6 mg/mL. Supernatants from ten hybridoma cell lines were evaluated in Western blot analysis and seven of them efficiently recognized the MNSV CP in crude extracts of MNSV-infected leaf material; the 2D4H4 hybridoma cell line was selected for further purification and characterization. The isotype of the 2D4H4 immunoglobulin class was identified as IgG2a and kappa light-chain. Western-blot analyses showed that mAb 2D4H4 provided sensitive and specific detection of MNSV. A TAS-ELISA protocol was developed for mAb 2D4H4. Using this protocol, limits of detection of 1:20,480 and 1:10,240 (g/mL, w/v) were attained for the homologous isolate and a heterologous MNSV isolate, respectively. Moreover, mAb 2D4H4 was used successfully to localize the MNSV CP in infected cells by immunocytochemistry/transmission electron microscopy, illustrating the usefulness of this mAb for advanced cellular studies.

Recombinant Factor VIII Fc Inhibits B Cell Activation via Engagement of the FcγRIIB Receptor.

The development of neutralizing antibodies (inhibitors) against factor VIII (FVIII) is a major complication of hemophilia A treatment. The sole clinical therapy to restore FVIII tolerance in patients with inhibitors remains immune tolerance induction (ITI) which is expensive, difficult to administer and not always successful. Although not fully understood, the mechanism of ITI is thought to rely on inhibition of FVIII-specific B cells (1). Its efficacy might therefore be improved through more aggressive B cell suppression. FcγRIIB is an inhibitory Fc receptor that down-regulates B cell signaling when cross-linked with the B cell receptor (BCR). We sought to investigate if recombinant FVIII Fc (rFVIIIFc), an Fc fusion molecule composed of FVIII and the Fc region of immunoglobulin G1 (IgG1) (2), is able to inhibit B cell activation more readily than FVIII. rFVIIIFc was able to bind FVIII-exposed and naïve B cells from hemophilia A mice as well as a FVIII-specific murine B cell hybridoma line (413 cells). An anti-FcγRIIB antibody and FVIII inhibited binding, suggesting that rFVIIIFc is able to interact with both FcγRIIB and the BCR. Furthermore, incubation of B cells from FVIII-exposed mice and 413 cells with rFVIIIFc resulted in increased phosphorylation of SH-2 containing inositol 5-phosphatase (SHIP) when compared to FVIII. B cells from FVIII-exposed hemophilia A mice also exhibited decreased extracellular signal-regulated kinase (ERK) phosphorylation when exposed to rFVIIIFc. These differences were absent in B cells from naïve, non-FVIII exposed hemophilic mice suggesting an antigen-dependent effect. Finally, rFVIIIFc was able to inhibit B cell calcium flux induced by anti-Ig F(ab)2. Our results therefore indicate that rFVIIIFc is able to crosslink FcγRIIB and the BCR of FVIII-specific B cells, causing inhibitory signaling in these cells.

Development of Indirect Competitive Enzyme-Linked Immunosorbent Assay and Lateral-Flow Immunochromatographic Strip for the Detection of Digoxin in Human Blood.

Mouse–mouse hybridoma cell lines producing stable, highly specific monoclonal antibodies with good affinity for the cardiac glycoside digoxin (DIG) were established to construct an indirect enzyme-linked immunosorbent assay and lateral-flow immunochromatographic strip to detect DIG in human blood. The hapten DIG was coupled to bovine serum albumin or chicken ovalbumin by sodium periodate oxidation. The highest sensitivity and specificity antibody had a median inhibitory concentration (IC50) of 0.45 ng/mL, a linear range of detection of 0.293–0.7 ng/mL, and low cross-reactivity with several DIG analogues. The cut-off value of the lateral-flow immunochromatographic strip was 5 ng/mL when the strip was tested with human blood. The immunochromatographic lateral flow strip test provides a quick and convenient method for determining DIG in plasma which can be visually observed in only 5 min to promote rational drug use.

PRESCIENT: platform for the rapid evaluation of antibody success using integrated microfluidics enabled technology.

Identifying antibodies (Abs) that neutralize infectious agents is the first step for developing therapeutics, vaccines, and diagnostic tools for these infectious agents. However, current approaches for identifying neutralizing Abs (nAbs) typically rely on dilution-based assays that are costly, inefficient, and only survey a small subset of the entire repertoire. There are also intrinsic biases in many steps of conventional nAb identification processes. More importantly, conventional assays rely on simple Ab-antigen binding assays, which may not result in identifying the most potent nAbs, as the strongest binder may not be the most potent nAb. Droplet microfluidic systems have the capability to overcome such limitations by conducting complex multi-step assays with high reliability, resolution, and throughput in a pico-liter volume water-in-oil emulsion droplet format. Here, we describe the development of PRESCIENT (Platform for the Rapid Evaluation of antibody SucCess using Integrated microfluidics ENabled Technology), a droplet microfluidic system that can enable high-throughput single-cell resolution identification of nAb repertoires elicited in response to viral infection. We demonstrate PRESCIENT's ability to identify Abs that neutralize a model viral agent, Murine coronavirus (murine hepatitis virus), which causes high mortality rates in experimentally infected mice. In-droplet infection of host cells by the virus was first demonstrated, followed by demonstration of in-droplet neutralization by nAbs produced from a single Ab-producing hybridoma cell. Finally, fluorescence intensity analyses of two populations of hybridoma cell lines (nAb-producing and non-nAb-producing hybridoma cell lines) successfully discriminated between the two populations. The presented strategy and platform have the potential to identify and investigate neutralizing activities against a broad range of potential infectious agents for which nAbs have yet to be discovered, significantly advancing the nAb identification process as well as reinvigorating the field of Ab discovery, characterization, and development.

Development, characterization of monoclonal antibodies specific for the ORF25 membrane protein of Cyprinid herpesvirus 2 and their applications in immunodiagnosis and neutralization of virus infection

Cyprinid herpesvirus 2 (CyHV-2) is the causative agent of haematopoietic necrosis disease in farmed gibel carp (Carassius auratus gibelio). It appears to be spreading cross the world, and has resulted in massive mortality and great economic losses. Although many sensitive nucleic acid-based diagnostic methods have been developed, effective immunodiagnosis and neutralization approaches based on monoclonal antibodies (MAbs) against CyHV-2 membrane protein are still scarce to study. In this study, ORF25 gene was chemically synthesized and cloned into a pET-28a expression vector to produce rORF25 (recombinant ORF25) in E. coli BL21 (DE3). The purified rORF25 was served as an immunogen to prepare monoclonal antibodies (MAbs), and five efficient hybridoma cell lines were selected by enzyme-linked immunosorbent assay (ELISA). Among them, MAbs 7H10-1B6 and 1F8-1B6 belonged to the IgG2b isotype, while the other three MAbs designed as 8D9-1H6, 3H2-1G5 and 2C3-1E6 belonged to the IgG1 isotype. Western blotting results indicated that MAbs could specifically identify the rORF25. MAb 2C3-1E6 could specifically detect ORF25 in CyHV-2 infected Fathead Minnow cells and gible carp tissues by ELISA, indirect immunofluorescence and immunohistochemistry. In vitro neutralization assay showed that MAb 2C3-1E6 exhibited a titer-dependent neutralization effect on CyHV-2 infection in FHM cells. The results indicated that MAbs against rORF25 could serve as effective detection probes and potential neutralizing antibody for haematopoietic necrosis disease in gibel carp.

Toolkit for the study of yellow fever virus

Yellow fever virus (YFV) is a dangerous re-emerging mosquito borne flavivirus with a high lethality, which causes untreatable haemorrhagic fever in humans. Important disadvantages regarding safety and limited vaccine supply have been identified in the existing live attenuated vaccine. Alternative safer therapeutic strategies are needed to reduce the lethality of infections and protect a wider group of people. To this point, we created an array of essential tools for the study of YFV. We designed a novel YFV reporter virus to develop a highly sensitive high throughput neutralisation assay (Z’=0.65), which can screen virus inhibitors in only 48 hours. Moreover, we designed a site-specific biotinylated soluble YF envelope (E) protein by a cloning strategy involving the use of a biotin acceptor peptide and E. coli Biotin-protein birA ligase. The biotinylated E protein was used to develop a highly sensitive ELISA to screen antibodies that bind to the YFV E protein. Furthermore, we immunised mice with a soluble YFV E protein and generated anti-YFV E IgG secreting hybridoma cell lines. Monoclonal antibodies secreted by hybridomas were purified by affinity chromatography and are being characterised. The developed antibodies against YF E protein are a crucial tool for the molecular study of YF. Additionally, neutralising antibodies against YFV could potentially be developed into the first therapeutic treatment against YF infection. The toolkit developed in this project includes a neutralisation assay, a binding ELISA, and anti YFV E antibodies. These are essential instruments to expand the knowledge of YFV.

Rapid Detection of Staphylococcal Enterotoxin-B by Lateral Flow Assay.

A cohort of monoclonal antibodies (mAbs) were generated against Staphylococcal enterotoxin-B (SEB) and selected by double sandwich enzyme-linked immunosorbent assay (ELISA) for solution capture of the toxin. Clonal hybridoma cell lines were established and a pair of anti-SEB mAbs selected for the development of a sandwich ELISA. Immobilized 3D6 mAb (IgG1, kappa) when paired with 4C9 mAb (IgG1, kappa) conjugated to horseradish peroxidase generates a typical dose–response curve with an EC50 of 24.8 ng/mL for purified SEB using chemiluminescent detection. These mAbs bind SEB by Western blot and ELISA binding to classical enterotoxin serotypes show that the 3D6 mAb binds both SEB and the SEC1 serotypes, whereas 4C9 binds only SEB. These mAbs effectively port onto lateral flow test strips with a visual detection sensitivity for SEB of 5 ng/mL in <10 minutes using a 4C9 conjugated to a 40 nm gold reporter.

Serum low replacement medium versus serum rich replacement medium for production of Anti-Schistosoma Monoclonal antibody: A comparative study

Objectiveto assess the production of Anti-Schistosoma Monoclonal Antibodies by growing IgM producing hybridoma cell line in serum low medium and to evaluate their diagnostic potential to replace the old conventional method in animal ascetic fluid. MethodsHighly reactive and specific IgM MAb secreting hybridoma cell line to S. mansoni SEA was cultured in three different media; serum rich replacement medium (conventional), serum low replacement medium (SLRM) and serum free replacement medium (SFRM). ResultsCell count and viability were better in conventional medium and SLRM than SFRM. The lower detection limit of the assay Mab of SLRM culture was 1.1 ng/ml of S. mansoni SEA. SFRM was excluded because of the low concentration and reactivity. MAb was tested against 34 human samples. Sensitivity and specificity of the assay for each were 92% and specificity 90% for SLRM respectively. The diagnostic efficacy of the assay was 94%. Conclusionsthe production and diagnostic efficacy of antischistosomal MAbs was comparable on usage of either in vitro cell culture supernate in SLRM or conventional media.

Preparation of monoclonal antibodies against human interleukin-35 and their application in a quantitative ELISA for interleukin-35 detection

To establish a quantitative ELISA for human interleukin-35 (IL-35) detection, we cloned cDNAs encoding the 2 subunits IL-27EBI3 and IL-12p35 of IL-35 by RT-PCR and transformed the cDNAs into Escherichia coli BL21 star (DE3) by recombinant DNA technology. IL-27EBI3 and IL-12p35 were expressed as recombinant proteins and used as immunogen to immunize Balb/c mice. Spleen cells from the positive serum mice were isolated and fused with SP-2/0 myeloma cells. We obtained the hybridoma cell lines stably secreting target antibodies by indirect ELISA screening of the cell supernatants with recombinant IL-27EBI3 and IL-12p35 as antigen and consecutive subcloning of the cells in the well with positive supernatant. Following further measurement of supernatant titers of the antibodies and identification of their antigen specificity, we obtained a hybridoma cell line 3B11 that stably secrets antibody against IL-27EBI3 and a hybridoma cell line 3A10 that secrets antibody against IL-12p35. Both monoclonal antibodies (mAbs) were identified as the subtype of IgG1. Finally, using the anti-IL-27EBI3 mAb from 3B11 as the capture antibody and the anti-IL-12p35 mAb from 3A10 as the secondary antibody, we established a quantitative double-antibodies sandwich ELISA for IL-35 detection with streptavidin-biotin amplification system. Results demonstrated that the quantitative assay had a detection range of 3.12-200 pg/mL, a detectability of 1.26 pg/mL, and a crossing-reactive rate of 0.1%. The intra-batch RSD and the inter-batch RSD of the quantitative assay were 5.1%-5.6% and 5.6%-7.2%, respectively, and the fortified recovery was 89%-103%. Therefore, the sandwich ELISA assay for IL-35 meets the qualification of quantitative analysis and laid a stable foundation for the development of quantitative ELISA kit for IL-35 detection.

Monoclonal Antibody Production: A Project-Based Laboratory Program for Final Year Biotechnology Undergraduate Students

A semester-long, project-based series of laboratory exercises is described that provides practical experience of the upstream and downstream processes involved in monoclonal antibody production. This 6 week project is aimed at final year undergraduate students undertaking primary degrees in biotechnology, biochemistry, or related disciplines. Students develop subject matter knowledge of biologics production and practical skills related to cell culture, column chromatography, quality control, and information literacy. Students are assigned a vial containing a hybridoma cell line stored in liquid nitrogen. They thaw the vial and culture the hybridoma over the course of 4 weeks. They perform daily counts of live/dead cells and quantify glucose levels in the media. During the 4 weeks they will expand their cells from a single 60 mm dish into two large T-175 flasks, one for antibody production and one for generating stocks. They harvest the medium from the production flask and purify the antibody via Protein G affinity chromatography on an FPLC instrument. The purity of the antibody is tested by SDS-PAGE, and the concentration is determined on a Nanodrop instrument. Students present their results in the form of a project thesis, a product datasheet, and a public presentation. Learning was assessed relative to six learning objectives using a student survey administered at the beginning (presurvey) and end (postsurvey). The students who have undertaken this module demonstrated substantial learning gains. This laboratory project is highly adaptable with scope to adapt it for longer or shorter study periods.