Development of Monoclonal Antibody Based IgG and IgM ELISA for Diagnosis of Severe Fever with Thrombocytopenia Syndrome Virus Infection

Background: Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerged virus that possesses a great threat to human health because of the high fatality rate. Method: To develop sensitive and specific sero-diagnosis systems for SFTSV infections, monoclonal antibodies (MAbs) against recombinant SFTSV nucleocapsid (rSFTSV-N) protein were developed by immunizing BALB/C mice with rSFTSV-N protein and fusing the spleen cells with SP2/0 myeloma cells. Three hybridoma cell lines secreting MAbs against rSFTSV-N were obtained. MAb based IgG sandwich enzyme linked immunosorbent assay (ELISA) and IgM capture ELISA systems were established by using the newly developed MAbs. One hundred fifteen clinical suspected SFTS patient serum samples were used to evaluate the newly established systems by comparing with the total antibody detecting sandwich ELISA system and indirect ELISA systems. Results: The MAb based sandwich IgG ELISA was perfectly matched with that of the total antibody sandwich ELISA and the indirect IgG ELISA with a sensitivity and specificity of 100%. IgM capture ELISA results perfectly matched with that of the total antibody sandwich ELISA while was more sensitive comparing with the indirect IgM ELISA. Conclusions: The MAbs against rSFTSV-N protein offer new tools for SFTSV studies and our newly developed MAb-based IgG and IgM capture ELISA systems would offer safe and useful tools for diagnosis of SFTS virus infections and epidemiological investigations.

Growing Hybridomas

This introduction discusses the techniques used to grow and maintain myeloma and hybridoma cell lines, the production and collection of monoclonal antibodies, and methods for drug selection used in hybridoma work.

Preparation and Characterization of Monoclonal Antibodies with High Affinity and Broad Class Specificity against Zearalenone and Its Major Metabolites

This study aimed to detect and monitor total Zearalenone (ZEN) and its five homologs (ZENs) in cereals and feed. The monoclonal antibodies (mAbs) with a high affinity and broad class specificity against ZENs were prepared, and the conditions of a heterologous indirect competitive ELISA (icELISA) were preliminarily optimized based on the ZEN mAbs. The immunogen ZEN-BSA was synthesized using the oxime active ester method (OAE) and identified using infrared (IR) and ultraviolet (UV). The coating antigen ZEN-OVA was obtained via the 1,4-butanediol diglycidyl ether method (BDE). Balb/c mice were immunized using a high ZEN-BSA dose with long intervals and at multiple sites. A heterologous indirect non-competitive ELISA (inELISA) and an icELISA were used to screen the suitable cell fusion mice and positive hybridoma cell lines. The ZEN mAbs were prepared by inducing ascites in vivo. The standard curve was established, and the sensitivity and specificity of the ZEN mAbs were determined under the optimized icELISA conditions. ZEN-BSA was successfully synthesized at a conjugation ratio of 17.2:1 (ZEN: BSA). Three hybridoma cell lines, 2D7, 3C2, and 4A10, were filtered, and their mAbs corresponded to an IgG1 isotype with a κ light chain. The mAbs titers were between (2.56 to 5.12) × 102 in supernatants and (1.28 to 5.12) × 105 in the ascites. Besides, the 50% inhibitive concentration (IC50) values were from 18.65 to 31.92 μg/L in the supernatants and 18.12 to 31.46 μg/L in the ascites. The affinity constant (Ka) of all of the mAbs was between 4.15 × 109 and 6.54 × 109 L/mol. The IC50 values of mAb 2D7 for ZEN, α-ZEL, β-ZEL, α-ZAL, β-ZAL and ZAN were 17.23, 16.71, 18.27, 16.39, 20.36 and 15.01 μg/L, and their cross-reactivities (CRs, %) were 100%, 103.11%, 94.31%, 105.13%, 84.63%, and 114.79%, respectively, under the optimized icELISA conditions. The limit of detection (LOD) for ZEN was 0.64 μg/L, and its linear working range was between 1.03 and 288.55 μg/L. The mAbs preparation and the optimization of icELISA conditions promote the potential development of a rapid test ELISA kit, providing an alternative method for detecting ZEN and its homologs in cereals and feed.

Preparation of highly sensitive monoclonal antibody against α-zearalanol based on the similar antigen determinant structure to zearalanone

In this study, we report a new method to prepare highly sensitive monoclonal antibody against α-zearalanol (ZAL) based on a similar antigen determinant structure. Zearalanone (ZAN), structural analogs of ZAL, was modified by oximation to obtain ZAN-O. ZAN-O was then coupled with bovine serum albumin using 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) to synthesise the artificial complete antigen ZAN-O-BSA. ZAN-O-BSA was used to immunise the BALB/c mice. The splenocytes of the immunised mice were fused with myeloma NS0 cells. During the process of cell fusion, ZAL was used as an inhibitor instead of ZAN to screen the hybridoma cell lines that can secrete monoclonal antibodies against ZAL. The sensitivity (half inhibitory concentration, IC50) of the prepared monoclonal antibody was 0.475 ng/ml, the limit of detection (LOD) was 0.050 ng/ml, the linear range of detection was 0.066-3.399 ng/ml, the affinity constant Kaff was 6.18×107 l/mol, the cross-reactivity rate with structural analogues, such as β-zearalanol, α-zearalenol, β-zearalenol, ZAN and zearalenone were 28.07, 13.16, 15.83, 60.28 and 7.95% respectively. The cross-reactivity with other mycotoxin and carrier proteins were all less than 0.05%. The prepared monoclonal antibody can be used to establish a highly sensitive immunoassay for the detection of ZAL.

Three Highly Sensitive and High-Throughput Serological Approaches for Detecting Dickeya dadantii in Sweet Potato

Sweet potato stem and root rot is an important bacterial disease and often causes serious economic losses to sweet potato. Development of rapid and sensitive detection methods is crucial for diagnosis and management of this disease in field. Here, we report the production of four hybridoma cell lines (25C4, 16C10, 9B1, and 9H10) using Dickeya dadantii strain FY1710 as an immunogen. Monoclonal antibodies (MAbs) produced by these four hybridoma cell lines were highly specific and sensitive for D. dadantii detection. Indirect enzyme-linked immunosorbent assay (indirect-ELISA) results showed that the four MAbs 25C4, 16C10, 9B1, and 9H10 could detect D. dadantii in suspensions diluted to 4.89 × 104, 4.89 × 104, 9.78 × 104, and 9.78 × 104 CFU/ml, respectively. Furthermore, all four MAbs can react strongly and specifically with all four D. dadantii strains used in this study, not with the other seven tested bacterial strains. Using these four MAbs, three different serological approaches, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA), dot-ELISA, and tissue-print-ELISA, were developed for detection of D. dadantii in crude extracts prepared from field-collected sweet potato plants. Among these three methods, TAS-ELISA and dot-ELISA were used to detect D. dadantii in suspensions diluted up to 1.23 × 104 and 1.17 × 106 CFU/ml, respectively, or in sweet potato crude extracts diluted up to 1:3,840 and 1:1,920 (wt/vol, grams per milliliter), respectively. Surprisingly, both TAS-ELISA and dot-ELISA serological approaches were more sensitive than the conventional PCR. Analyses using field-collected sweet potato samples showed that the newly developed TAS-ELISA, dot-ELISA, or tissue-print-ELISA were reliable in detecting D. dadantii in sweet potato tissues. Thus, the three serological approaches were highly valuable for diagnosis of stem and root rot in sweet potato production.

Generation of Rat Monoclonal Antibody to Detect Hydrogen Sulfide and Polysulfides in Biological Samples

Hydrogen sulfide (H2S) is endogenously produced by enzymes and via reactive persulfide/polysulfide degradation; it participates in a variety of biological processes under physiological and pathological conditions. H2S levels in biological fluids, such as plasma and serum, are correlated with the severity of various diseases. Therefore, development of a simple and selective H2S measurement method would be advantageous. This study aimed to generate antibodies specifically recognizing H2S derivatives and develop a colorimetric immunoassay for measuring H2S in biological samples. We used N-ethylmaleimide (NEM) as an H2S detection agent that forms a stable bis-S-adduct (NEM-S-NEM). We also prepared bis-S-heteroadduct with 3-maleimidopropionic acid, which, in conjugation with bovine serum albumin, was to immunize Japanese white rabbits and Wistar rats to enable generation of polyclonal and monoclonal antibodies, respectively. The generated antibodies were evaluated by competitive enzyme-linked immunosorbent assay. We could obtain two stable hybridoma cell lines producing monoclonal antibodies specific for NEM-S-NEM. By immunoassay with the monoclonal antibody, the H2S level in mouse plasma was determined as 0.2 μM, which was identical to the level detected by mass spectrometry. Taken together, these monoclonal antibodies can be a useful tool for a simple and highly selective immunoassay to detect H2S in biological samples.

Production of monoclonal antibody to quantify serum CA125 concentration in breast and ovarian cancer patients

Ovarian carcinoma (OC) and breast cancer (BC) are mainly caused by alterations in genes such as BRCA1 and BRCA2, which are involved in differentiation and survival of cancer cells. The protein CA125 (MUC16) is released by cancer cells in most OC and a few BC patients. The cut-off point of CA125 level for tumor growth and metastasis is 35 U/mL. Production of CA125 monoclonal antidody (mAb) to determine expression level of this antigen by ELISA has been well known. In this study, we aimed to generate CA125-specific mAb for developing a new in-house ELISA kit. To this end, BALB/c mice were immunized with CA125 protein and splenocytes of immunized mice were fused with myeloma Sp2/0 cells. Efficiency of mAbs secreted from the hybridoma clones was examined by ELISA and flow cytometry analysis. As a result, among 3 stable hybridoma cell lines identified, A1 clone attained about 90% positive for anti-CA125 mAb, whereas H1 and H3 clones were about 40% and 50% positive for anti-CA125 mAb, respectively. By flow cytometry analysis, anti-CA125 mAb from A1 clone was more specific to CA125 antigen present in OVCAR -3 cells than those from H1 or H3 clone. In addition, the isotype of the obtained mAb was specific IgG1 and Kappa light chain. In conclusion, the mouse anti-human CA125 mAb generated in our lab was specifically binding to CA125 antigen and used as the capture antibody in sandwich ELISA system for early diagnosis as well as monitoring therapeutic response in OC patients.

Development of monoclonal antibodies and simplified sandwich ELISA for canine neutrophil gelatinase-associated lipocalin A detection

Background Acute kidney injury (AKI) is one of the most common canine diseases with a high mortality rate. Neutrophil gelatinase-associated lipocalin (NGAL) is the novel biomarker for early diagnosis of renal injury. Only few sandwich ELISA kits are commercially available, all of which require the use of expensive enzyme-conjugated secondary or biotinylated antibody. The aims of this study were to develop high affinity monoclonal antibodies (mAbs) and simplified sandwich ELISA for canine NGAL detection. Results Recombinant canine NGAL was expressed in E. coli and purified to a high purity. Six hybridoma cell lines were generated by immunization of mice with the purified protein, all of which secreted high titers of specific mAbs. By screening 36 different antibody combinations, a pair of mAbs with high additivity and P/N ratio was selected as the capture and detection antibodies. By conjugation of the detection mAb with horse radish peroxidase, a simplified sandwich ELISA was developed with a correlation coefficient of 0.9939, detection limit of 8.28 ng/mL. The parallel test of 42 samples from healthy and AKI dogs showed the good agreement in NGAL concentrations detected by the simplified sandwich ELISA and commercial ELISA kit. Conclusions We developed canine NGAL-specific monoclonal antibodies and simplified sandwich ELISA. The simplified sandwich ELISA could replace the commercial ELISA kits for canine NGAL detection with the advantages of reduced cost and detection time.