Monoclonal Antibody-based Assays Research Articles

Concordance between two monoclonal antibody-based antigen detection enzyme-linked immunosorbent assays for measuring cysticercal antigen levels in sera from pigs experimentally infected with Taenia solium and Taenia hydatigena

BackgroundAntigen detection in Taenia solium cysticercosis confirms viable infection in the intermediate host (either pig or human). The reference B158/B60 monoclonal antibody (mAb)-based Ag-enzyme-linked immunosorbent assay (ELISA) has acceptable levels of sensitivity and specificity in human neurocysticercosis with multiple brain cysts, although its sensitivity is lower in cases with single brain cysts, whereas in porcine cysticercosis the assay specificity is affected by its frequent cross-reaction with Taenia hydatigena, another common cestode found in pigs. Our group has produced 21 anti-T. solium mAbs reacting against antigens of the whole cyst, vesicular fluid, and secretory/excretory products, identifying TsW8/TsW5 as the most promising pair of mAbs for an Ag-ELISA.MethodsWe report the use of the TsW8/TsW5 Ag-ELISA to measure cysticercus antigen levels [expressed as optical density (OD) values] in two panels of sera collected from day 0 (baseline) to day 90 postinfection (PI) from pigs experimentally infected with T. solium (n = 26) and T. hydatigena (n = 12). At baseline and on days 28 and 90 PI, we used Bland–Altman (BA) analysis and Lin’s concordance correlation coefficients (CCC) to determine the concordance between the TsW8/TsW5 and the B158/B60 Ag-ELISA.ResultsThe TsW8/TsW5 Ag-ELISA was able to efficiently measure circulating antigen levels in T. solium-infected pigs, similar to that obtained with the B158/B60 Ag-ELISA. Almost all paired log-OD differences between assays were within the limits of agreement (LoA) in the BA analysis at baseline and on days 28 and 90 PI (92.3%, 100%, and 100%, respectively), and a high concordance of log-ODs between assays was also found (Lin’s CCC: 0.69, 0.92, and 0.96, respectively, all P < 0.001). In pigs infected with T. hydatigena, almost all paired log-OD differences were within the LoA in the BA analysis, whereas the concordance of log-ODs between assays was low at baseline (Lin’s CCC: 0.24) but increased on days 28 and 90 PI (Lins’ CCC: 0.88 and 0.98, P < 0.001).Conclusions/significanceThe TsW8/TsW5 Ag-ELISA recognizes antigens in pigs with T. solium cysticercosis and is highly concordant with the B158/B60 Ag-ELISA. However, its diagnostic use is hampered by cross-reactions with T. hydatigena, as in other mAb-based Ag-ELISAs.Graphical

Evaluation of protection and immunity induced by infectious bronchitis vaccines administered by oculonasal, spray or gel routes in commercial broiler chicks

Broiler chicks’ responses following combined IBV live attenuated Massachusetts and 793B strains through gel, spray or oculonasal (ON) vaccination routes were cross-compared. Subsequently, the responses following IBV M41 challenge of the unvaccinated and vaccinated groups were also assessed. Post-vaccination humoral and mucosal immune responses, alongside viral load kinetics in swabs and tissues, were determined using commercial ELISA assays, monoclonal antibody-based IgG and IgA ELISA assays and qRT-PCR respectively. After challenged with IBV-M41 strain, humoral and mucosal immune responses, ciliary protection, viral load kinetics, and immune gene mRNA transcriptions between the three vaccination methods were examined and compared. Findings showed that post-vaccinal humoral and mucosal immune responses were similar in all three vaccination methods. Post vaccinal viral load kinetics is influenced by method of administration. The viral load peaked in the ON group within the tissues and the OP/CL swabs in the first and third weeks respectively. Following M41 challenge, ciliary protection and mucosal immune responses were not influenced by vaccination methods as all three methods offered equal ciliary protection. Immune gene mRNA transcriptions varied by vaccination methods. Significant up-regulation of MDA5, TLR3, IL-6, IFN-α and IFN-β genes were recorded for ON method. For both spray and gel methods, significant up-regulation of only MDA5 and IL-6 genes were noted. The spray and gel-based vaccination methods gave equivalent levels of ciliary protection and mucosal immunity to M41 virulent challenge comparable to those provided by the ON vaccination. Analysis of viral load and patterns of immune gene transcription of the vaccinated-challenged groups revealed high similarity between turbinate and choanal cleft tissues compared to HG and trachea. With regards to immune gene mRNA transcription, for all the vaccinated-challenged groups, similar results were found except for IFN-α, IFN-β and TLR3, which were up-regulated only in ON compared to gel and spray vaccination methods.

Significant Improvement in Digoxin Immunoassays Over Four Decades: Newer Assays are Less Affected by Interferences.

Digitalis glycosides derived from foxglove plants have been used for medicinal purposes since the 16th century. Currently, digoxin derived from foxgloves is used clinically. Owing to the narrow therapeutic range, therapeutic drug monitoring is essential; however, digoxin immunoassays suffer from interference. The issue of interference was reviewed for both older polyclonal antibody-based digoxin assays and newer monoclonal antibody-based digoxin assays. A literature search was conducted using PubMed, ScienceDirect, Scopus, Web of Science, and ResearchGate for studies on digoxin immunoassays published in the English language from 1969 to the present. Radioimmunoassays for digoxin in the 1970s and, later, first-generation nonradioimmunoassay methods were liable to several interferences, including digoxin-like immunoreactive substances, spironolactone, potassium canrenoate, and various digoxin metabolites. However, for the last 10-15 years, next next-generation digoxin immunoassays have been virtually free from such interferences. Nevertheless, certain herbal supplements, as well as both Digibind and DigiFab, interfere with serum digoxin measurement, even with the more recently developed digoxin assays. More recently introduced monoclonal antibody-based digoxin assays are superior to the older polyclonal antibody-based digoxin assays.

COVID-19-Associated Mucormycosis: A Matter of Concern Amid the SARS-CoV-2 Pandemic.

Mucormycosis is an invasive fungal infection caused by fungi belonging to order Mucorales. Recently, with the increase in COVID-19 infections, mucormycosis infections have become a matter of concern globally, because of the high morbidity and mortality rates associated with them. Due to the association of mucormycosis with COVID-19 disease, it has been termed COVID-19-associated mucormycosis (CAM). In the present review, we focus on mucormycosis incidence, pathophysiology, risk factors, immune dysfunction, interactions of Mucorales with endothelial cells, and the possible role of iron in Mucorales growth. We review the limitations associated with current diagnostic procedures and the requirement for more specific, cost-effective, convenient, and sensitive assays, such as PCR-based assays and monoclonal antibody-based assays for the effective diagnosis of mucormycosis. We discuss the current treatment options involving antifungal drug therapies, adjunctive therapy, surgical treatment, and their limitations. We also review the importance of nutraceuticals-based therapy for the prevention as well as treatment of mucormycosis. Our review also highlights the need to explore the potential of novel immunotherapeutics, which include antibody-based therapy, cytokine-based therapy, and combination/synergistic antifungal therapy, as treatment options for mucormycosis. In summary, this review provides a complete overview of COVID-19-associated mucormycosis, addressing the current research gaps and future developments required in the field.

New Diagnostic Cutoffs for Adrenal Insufficiency After Cosyntropin Stimulation Using Abbott Architect Cortisol Immunoassay

IntroductionThe accurate interpretation of the cosyntropin (adrenocorticotropic hormone [ACTH]) stimulation test requires method- and assay-specific cutoffs of the level of cortisol. Compared with a historical cutoff (18 μg/dL) for polyclonal antibody-based immunoassays, lower thresholds were proposed for the Roche Elecsys II assay, which uses a monoclonal antibody. However, cutoffs for other commonly adopted, monoclonal antibody-based cortisol assays were not yet available. Here, we established the thresholds for the level of cortisol specific to the Abbott Architect immunoassay by comparing the measurements of the level of cortisol using 3 immunoassays. MethodsThe ACTH stimulation test was performed in patients with suspected adrenal insufficiency (n = 50). The serum cortisol level was measured using the Abbott Architect, Roche Elecsys II, and Siemens Centaur assays. The results of the Abbott assay were also compared with those of liquid chromatography-tandem mass spectrometry. The receiver operating characteristic analysis was performed to derive new diagnostic thresholds for the Abbott assay using the polyclonal antibody-based Siemens assay as the reference method. ResultsThe concentrations of cortisol measured using the Abbott assay were similar to those measured using liquid chromatography-tandem mass spectrometry and the Roche Elecsys II assay but significantly lower than those measured using the Siemens assay. The optimized threshold for cortisol using the Abbott assay was 14.6 μg/dL at 60 minutes after stimulation (sensitivity, 92%; specificity, 96%) and 13.2 μg/dL at 30 minutes after stimulation (sensitivity, 100%; specificity, 89%). ConclusionWe recommend a threshold of 14.6 μg/dL for the level of cortisol at 60 minutes after ACTH stimulation for the Abbott assay. In comparison with the historical threshold of 18 μg/dL, the application of the new cutoff may significantly decrease false-positive results due to ACTH stimulation testing. The use of assay-specific cutoffs will be essential for reducing misclassification and overtreatment in patients with suspected adrenal insufficiency.

Three highly sensitive monoclonal antibody-based serological assays for the detection of tomato mottle mosaic virus

In recent years, tomato mottle mosaic virus (ToMMV) has become one of the most important viral pathogens affecting solanaceous crop production in Yunnan, Hainan, and Shandong provinces of China, often causing huge yield reductions. To provide farmers and vegetable industry with reliable and easy-to-use ToMMV detection methods, we immunized BALB/c mice with purified ToMMV and obtained six hybridoma cell lines (i.e., 2D6, 9C12, 26A10, 3A4, 23A4 and 17B11) that secrete anti-ToMMV monoclonal antibodies (MAbs) through the hybridoma technology. Using these MAbs as the detection antibody, we developed three serological assays: antigen-coated-plate enzyme-linked immunosorbent assay (ACP-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and tissue print enzyme-linked immunosorbent assay (tissue print-ELISA) for ToMMV detection. Our test results showed that these three newly developed serological methods can be used to specifically detect ToMMV infection in plant samples, but not tobacco mosaic virus, tomato mosaic virus, cucumber green mottle mosaic virus and cucumber mosaic virus. Sensitivity analyses further showed that ACP-ELISA and dot-ELISA can be used to detect ToMMV infection in plant crude extracts diluted at 1:81,920 and 1:40,960 (weight/volume, g/mL), respectively. Surprisingly, the detection limit of the developed dot-ELISA was 26 times higher than that of traditional RT-PCR. Using field-collected plant samples, we have demonstrated that these three new serological methods are accurate and easy-to-use for large-scale detection of ToMMV in fields.

Clinical Comparison of a New Serum Free Light Chain (FLC) Assay Based on the Seralite Monoclonal Anti-FLC Antibodies with the Freelite and N-Latex FLC Assays in the Diagnosis of AL Amyloidosis

Aims: Accurate quantitation of FLC is essential for the diagnosis and monitoring of patients with AL amyloidosis. Current assays, however, have their limitations. Recently a new assay based on monoclonal antibodies (Seralite, Abingdon Health Ltd, UK) has been developed which appears to have some analytical advantages over existing assays and has been clinically validated in patients with myeloma (BJH 2017;178:220). We compared the antibodies used in this novel assay for FLC quantitation to the established monoclonal (N-Latex) and polyclonal (Freelite) antibody-based assays in AL amyloidosis.Methods: 61 diagnostic samples from patients with AL amyloidosis were analysed. Serum FLC concentration was measured using Seralite monoclonal anti-FLC antibodies on an in-house ELISA platform in the Clinical Immunology Service Birmingham, and Freelite (The Binding Site, UK) and N-Latex (Siemens, Germany) immunoassays on a BNII nephelometer in the Pathology Queensland laboratory, Brisbane. Serum and urine immunofixation electrophoresis (IFE) were performed on Hydrasys gel systems (Sebia, France).Results: For AL of kappa type (n=18), the median involved FLC (iFLC) was non-significantly lower by the Seralite assay than by the N-Latex or Freelite assay (220 vs 289 vs 667mg/L, p=0.64 and 0.27 for Seralite vs N-Latex and Freelite, respectively). In AL of lambda type (n=43) the values were significantly different (37 vs 144 vs 156mg/L, p=0.005 and 0.006 for Seralite vs N-Latex and Freelite, respectively). Measurable disease is currently defined as a difference between the involved and uninvolved FLC (dFLC) of > 50mg/L. By these criteria, 54% of AL would be measurable by the Seralite FLC assay compared to 82% by the N-Latex assay and 89% by the Freelite assay indicating that different cut-offs are likely to be required for different FLC assays.In terms of the diagnostic sensitivity, the κ:λ FLC ratio was normal in 30%, 21% and 15% of patients by the Seralite, N- Latex and Freelite assays, respectively. All patients with AL amyloidosis of kappa type had an abnormal κ:λ ratio by all 3 assays. This was not the case for the 43 patients with lambda AL amyloidosis where the Seralite determined κ:λ FLC ratio was normal in 18 (42%) of cases compared with 30% and 21% for the N-Latex and Freelite assays, respectively. Providing greater detail of these 18 cases with a normal κ:λ ratio by the Seralite reagents: in 5 cases the κ:λ ratio was normal by all 3 assays; in 9 cases the κ:λ ratio was abnormal by both Freelite and N Latex assays; 1 case had a normal κ:λ ratio by Freelite and abnormal by N Latex; and 3 cases had an abnormal κ:λ ratio by Freelite and normal by N latex assays. For many of these cases the Seralite reagent appeared not to be reacting well with a lambda light chain clearly identifiable by one of the alternate assays. The combination of serum and urine IFE with either FLC assay, however, allowed identification of the amyloidogenic clone in 95%, 98% and 98% producing comparable overall diagnostic sensitivity for the methods. An attempt by the Birmingham laboratory to modify the assay to improve lambda FLC reactivity failed to improve the results.Conclusions: There are significant differences between iFLC as measured by this in-house ELISA assay using Seralite monoclonal antibodies as compared to the existing N Latex and Freelite assays. In particular, the Seralite assay appears to react poorly with amyloidogenic lambda light chains and the current in-house ELISA assay is not suitable for diagnosis or monitoring of AL amyloidosis. It may be that a combination of anti-FLC monoclonal antibodies is required to improve detection of lambda clones by the assay. This data also highlights that different cut-offs and response criteria will be required depending on the assay used in patients with AL amyloidosis which should be a focus of further research. DisclosuresDrayson:Abingdon Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mapping the probability of detecting Burkholderia pseudomallei in rural rice paddy soil based on indicator kriging and spatial soil factor analysis.

Melioidosis is an infectious disease commonly found in Thailand. This infectious disease is caused by Burkholderia pseudomallei in soil. This study aims to analyze the association between spatial soil factors and B. pseudomallei detection, as well as to map the probability of B. pseudomallei contamination based on indicator kriging in paddy soil. Seventy-eight soil samples were collected randomly on 22 April 2018 in various paddy fields. Oxidase, Gram staining and monoclonal antibody-based latex agglutination assays were performed to confirm the presence of B. pseudomallei in soil samples. The association between B. pseudomallei detection and spatial soil factors including soil temperature, soil pH, soil texture and soil drainage were analyzed by the Mann-Whitney U test and χ2 test. Subsequently, a semivariogram model and indicator kriging were used to map the probability of B. pseudomallei contamination. Of the 78 samples, B. pseudomallei was detected in 32 (41.03%). The presence or absence of B. pseudomallei was not significantly associated with spatial soil factors. The semivariogram model showed that the lag distance between positive B. pseudomallei samples was 90.51m. The empirical semivariogram and indicator kriging are an alternative option for predicting the spatial distribution of B. pseudomallei in soil.

Monoclonal antibody-based diagnostic assays for pepper mild mottle virus

A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) for pepper mild mottle virus (PMMoV) detection were developed based on monoclonal and polyclonal antibodies produced in this study. Production of anti-PMMoV monoclonal antibodies (MAbs) was achieved using the hybridoma technique and a recombinant protein of the PMMoV coat protein as the antigen. Production of a polyclonal antibody (PAb) against the recombinant coat protein of PMMoV was carried out in a New Zealand White rabbit. The PAb was specific to five tobamoviruses consisting of PMMoV, tobacco mosaic virus (TMV), odontoglossum ringspot virus (ORSV), tomato mosaic virus (ToMV) and ribgrass mosaic virus (RMV). Two TAS-ELISA protocols were developed based on the characteristics of the antibodies produced. One protocol, based on MAb TOBA1, could detect five tobamoviruses (TMV, PMMoV, ORSV, ToMV, and RMV), four of which infect peppers. In the other protocol, TOBA10 was used to specifically detect PMMoV. We also successfully developed an IC-RT-PCR procedure using TOBA1. The sensitivity of the IC-RT-PCR (1:2,000,000) was much higher than TAS-ELISA (1:5120) for detection of the virus in plant sap. We anticipate these assays to facilitate the screening process of tobamovirus-resistant pepper cultivars in breeding programs.

Monoclonal antibody-based serological assays for detection of Potato virus S in potato plants.

Potato virus S (PVS) often causes significant losses in potato production in potato-growing countries. In this study, the ordinary strain of PVS (PVSO) was purified from PVS-infected potato plants and used as the immunogen to produce hybridomas secreting monoclonal antibodies (MAbs). Five highly specific and sensitive murine MAbs (1A3, 16C10, 18A9, 20B12, and 22H4) against PVS were prepared using conventional hybridoma technology. Using these MAbs, tissue print-enzyme-linked immunosorbent assay (ELISA), dot-ELISA, and double-antibody sandwich (DAS)-ELISA were developed for sensitive and specific detection of PVS infection in potato plants. The results of sensitivity assays revealed that PVS could be reliably detected in PVS-infected leaf crude extracts diluted at 1:10 240 and 1:163 840 (w/v, g/ml) in phosphate buffer saline (PBS) by dot-ELISA and DAS-ELISA, respectively. Twenty-two samples collected from potato fields in Yunnan Province, China were tested for PVS infection using the serological assays we had developed, and 14 of them were found to be positive. This indicates that PVS is now prevalent in potato fields in Yunnan Province.

Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc.

Analytical issues of serum free light chain assays and the relative performance of polyclonal and monoclonal based reagents.

Serum free light chain (FLC) assays have been incorporated into routine clinical practice and their use is recommended in international guidelines for the management of monoclonal gammopathies. Given that FLCs are not simple analytes, laboratories should be aware of potential analytical issues when using FLC assays, including antigen excess, lot-to-lot variation and non-linearity. Whilst manufacturers of monoclonal antibody-based assays claim that they overcome such issues, the evidence available to date does not support this. Here we review and compare the technical performance of both polyclonal and monoclonal antibody-based assays. The evidence suggests that the Freelite assay, based on polyclonal antisera, gives a broader recognition of monoclonal FLCs than the N Latex assay, based on monoclonal antisera, and despite being cited as a technical concern, we show that lot-to-lot variation of the Freelite assay is good. Both non-linearity and antigen excess are characteristic of FLC analysis and laboratories should be aware of these phenomena regardless of the assay system they use. Comparisons of the absolute values of sFLCs determined using monoclonal and polyclonal antibody-based assays show poor quantitative agreement and, because current guidelines have been established using the polyclonal antibody-based Freelite assay, it should not be assumed that assays utilizing monoclonal antibodies will give compliance with these guidelines.

Development of ELISA Assays for Measurement of Can f 1 and Can f 3 in Dog Allergen Extracts

RationaleDog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3.MethodsMouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts.ResultsScreening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays.ConclusionssELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts. RationaleDog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. Dog allergen extract is a non-standardized extract with no potency measures. Understanding dog allergen extract potency will enhance the safety and efficacy of this product. Our goal is to develop monoclonal antibody-based sandwich ELISA assays for the measurement of Can f 1 and Can f 3. MethodsMouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. Mouse monoclonal antibodies were generated against natural Can f 1 and dog serum albumin (Can f 3). Anti-Can f 1 and anti-Can f 3 hybridoma supernatants were screened against dog hair and dog epithelium extracts. Purified IgG antibodies were biotinylated for further pairwise screening and optimization. The defined concentrations of capture (non-biotinylated) and primary (biotinylated) antibodies were used for initial testing of various commercially available dog allergen extracts. ResultsScreening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. Screening of Can f 1 antibodies revealed consistent strong responses with natural protein and dog extracts during indirect ELISA. Three pairs were selected for Can f 1 measurement: 6G1 as a capture antibody (2.5 μg/mL) and 4C3, 7D12, or 9A9 as biotinylated primary antibody (each at 1.0 μg/mL). Anti Can f 3 antibodies exhibit greater variability and less affinity: 10-20 μg/mL of each capture and primary antibody are required for consistent measurement of Can f 3. Further studies are needed to optimize both the assays. ConclusionssELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts. sELISA assays have been developed for determining Can f 1 and Can f 3 contents of non-standardized dog allergen extracts.

Toward the development of monoclonal antibody-based assays to probe virion-like epitopes in hepatitis B vaccine antigen

Prophylactic vaccines against hepatitis B Virus (HBV) infection were produced in different expression systems under different processing conditions. Since the recombinant HBV surface antigen (HBsAg) in these vaccines is a cysteine-rich protein with 14 cysteines among a total of 226 amino acids, the epitopes are dependent on the formation of intra- and intermolecular disulfide bonds. A panel of 22 monoclonal antibodies (mAbs) were developed and evaluated with respect to their sensitivity to disulfide reduction treatment of recombinant HBsAg. Not surprisingly, different mAbs showed different degree of sensitivity to controlled HBsAg disulfide reduction. With a view to exploring the functionality of anti-HBsAg mAbs to be used in HBsAg quality analysis, in vitro neutralization activity for the mAbs was assessed. One of the mAbs tested, 5F11, which showed high sensitivity to the disulfide integrity in HBsAg, was shown also to be highly effective in neutralizing HBV in vitro. Conversely, 42B6, while exhibiting similar neutralization activity, showed comparable binding HBsAg with or without reduction treatment. Based on these mAb characteristics, a sandwich ELISA with 42B6 being the capture Ab and detection Ab was developed to quantify HBsAg (like a “mass” assay) during antigen bioprocessing or in vaccine products. In parallel, when 5F11 was used as the detection Ab (with the same capture Ab), the assay can be used to probe disulfide-dependent and virion-like epitopes in intermediates or final products of hepatitis B vaccine, serving as a surrogate marker for vaccine efficacy to elicit neutralizing antibodies. This approach enables the comparative epitope specific antigenicity analysis of HBsAg antigen preparations from different sources.

Monoclonal antibody-based serological assays and immunocapture-RT-PCR for detecting Rice dwarf virus in field rice plants and leafhopper vectors

Rice dwarf virus (RDV) causes Rice dwarf disease, which leads to considerable losses in rice production in Asia. Purified RDV virions were used as the immunogen to prepare monoclonal antibodies (mAbs). Three murine mAbs against RDV were prepared. Plate-trapped antigen enzyme-linked immunosorbent assay (PTA-ELISA), dot enzyme-linked immunosorbent assay (dot-ELISA) and immunocapture-RT-PCR (IC-RT-PCR) were then developed for sensitive, specific, and rapid detection of RDV in rice and leafhopper samples obtained in the field using the mAbs. The PTA-ELISA, dot-ELISA and IC-RT-PCR detected the virus in infected tissue crude extracts diluted at 1:81,920, 1:10,240 and 1:655,360 (w/v, gmL−1), in individual viruliferous rice green leafhopper crude extracts diluted at 1:25,600, 1:6400 and 1:3,276,800 (individual leafhopper/μL), respectively. 878 rice field samples and 531 leafhopper field samples from ten provinces of China were screened for the presence of RDV using the two serological assays and the IC-RT-PCR and the results indicated that 37 of the 878 rice samples and 22 of the 531 leafhopper samples were infected by RDV. All positive samples were from Yunnan Province, indicating that RDV is prevalent in this province, but not in the other nine provinces. The dot-ELISA is suitable for routine detection of large-scale rice and leafhopper samples in field surveys.

Development of monoclonal antibody-based assays for the detection of Vibrio tubiashii zinc-metalloprotease (VtpA)

Vibrio tubiashii has been linked to disease outbreaks in molluscan species, including oysters, geoducks, and clams. In particular, oyster hatcheries in the Pacific Northwest have been plagued by intermittent vibriosis since 2006. Accurate detection of vibrios, including V. tubiashii, is critical to the hatcheries in order to allow for rapid remediation efforts. The current methods for detection of Vibrio spp. are not ideal for use at the hatchery. Plating samples require time and is not sensitive to extracelluar pathogenic products, such as the secreted zinc-metalloprotease, VtpA. Other sensitive methods to detect bacteria, such as qPCR, require a high level of laboratory skills and expensive supplies that are prohibitive for use at hatchery sites. Thus, hatcheries would benefit from a sensitive, simple method to detect V. tubiashii and its secreted toxin. Here, we describe the development of two inexpensive and highly specific tests for the shellfish—toxic zinc-metalloprotease secreted by V. tubiashii: enzyme-linked immunoassays (ELISA) and a lateral flow immunoassay (dipstick assay). Both technologies rely on a set of monoclonal antibodies used in a sandwich format, with the capture antibody recognizing a different epitope than the detection antibody on the mature VtpA protein. Both assays are quantitative and give colorimetric readouts. The sandwich ELISA was sensitive when VtpA was diluted into PBS, but was markedly less sensitive in conditions that correlate with the environment of hatchery-derived samples, such as in the presence of seawater, algae, or oyster larvae. In contrast, the dipstick assay remained very sensitive in the presence of these contaminants, is less work-intensive, and much more rapid, making this format the preferred assay method for detecting VtpA on site in a hatchery or environmental setting.

Clinical comparison of new monoclonal antibody-based nephelometric assays for free light chain κ and λ to polyclonal antibody-based assays and immunofixation electrophoresis

Clinical comparison of new monoclonal antibody-based nephelometric assays for free light chain κ and λ to polyclonal antibody-based assays and immunofixation electrophoresis

Clinical comparison of new monoclonal antibody-based nephelometric assays for free light chain kappa and lambda to polyclonal antibody-based assays and immunofixation electrophoresis

New monoclonal antibody-based assays for serum-free light chains (FLC) have become available. In a clinical study with 541 patients, the new N Latex FLC assays were compared with the Freelite FLC assays and immunofixation electrophoresis (IF). Comparison of the different FLC kappa (κ) assays showed a slope of 0.99 with a deviation of 5.0%, rs=0.92, for FLC lambda (λ) a slope of 1.22, deviation 13.8%, rs=0.90 and for the κ/λ ratio a slope of 0.72, deviation -4.6%, rs=0.72. The concordance for the FLC κ assays was 91%, for FLC λ 85% and κ/λ ratio 95%. The clinical sensitivity and specificity of the κ/λ ratios in the study were comparable: 60% and 99% for the N Latex FLC assay and 61% and 97% for the Freelite assay. In IF-FLC positive samples, the N Latex FLC κ/λ ratio scored 20/23 (87%) samples outside the reference range and Freelite 21/23 (91%). For IF-FLC negative samples, N Latex FLC assay κ/λ ratio scored 338/350 (97%) within the reference range and Freelite scored 332/350 (95%). The concordance scores and the clinical sensitivity and specificity of the new N Latex FLC assays and Freelite assays appeared comparable, but there are some differences in measurement of concentrations between the methods.

Proinsulin-to-C-peptide ratio versus proinsulin-to-insulin ratio in the prediction of incident diabetes: the Insulin Resistance Atherosclerosis Study (IRAS)

Associations of proinsulin-to-insulin ratios with incident type 2 diabetes have been inconsistent. The use of C-peptide as the denominator in the ratio may allow for better prediction because C-peptide concentration is not affected by hepatic insulin clearance. The objective of this paper was to compare fasting intact and split proinsulin-to-insulin ratios (PI/I, SPI/I) with intact and split proinsulin-to-C-peptide ratios (PI/C-pep, SPI/C-pep) in the prediction of type 2 diabetes. Prospective data on 818 multi-ethnic adults without diabetes at baseline from the Insulin Resistance Atherosclerosis Study (IRAS) were used. Insulin sensitivity (S(I)) and acute insulin response (AIR) were determined from frequently sampled intravenous glucose tolerance tests, and fasting intact and split proinsulin were measured using specific two-site monoclonal antibody-based immunoradiometric assays. Associations of proinsulin ratios with type 2 diabetes were determined using logistic regression and differences in prediction were assessed by comparing areas under the receiver operating characteristic curve (AROCs). In logistic regression analyses, PI/C-pep and SPI/C-pep were more strongly associated with incident type 2 diabetes (n = 128) than PI/I and SPI/I, and were significantly better predictors of diabetes in AROC analyses (PI/C-pep = 0.662 vs PI/I = 0.603, p = 0.02; SPI/C-pep = 0.690 vs SPI/I = 0.631, p = 0.01). Both PI/C-pep and SPI/C-pep were associated with type 2 diabetes after adjustment for age, sex, ethnicity, waist circumference, impaired glucose tolerance, lipids and S(I). Both PI/C-pep and SPI/C-pep were significantly associated with incident type 2 diabetes in models that included AIR. Proinsulin-to-C-peptide ratios were stronger predictors of diabetes in comparison with proinsulin-to-insulin ratios. These findings support the use of C-peptide as the denominator for proinsulin ratios, to more accurately reflect the degree of disproportional hyperproinsulinaemia.

Development of an Automated and Multiplexed Serotyping Assay for Streptococcus pneumoniae

Streptococcus pneumoniae expresses more than 90 capsule types, and currently available pneumococcal vaccines are designed to provide serotype-specific protection. Consequently, serotyping of pneumococcal isolates is important for determining the serotypes to be included in pneumococcal vaccines and to monitor their efficacy. Yet serotyping of pneumococcal isolates has remained a significant technical challenge. By multiplexing many assays, we have now developed a simple yet comprehensive serotyping assay system that can not only identify all known pneumococcal serotypes but also subdivide nontypeable (NT) isolates into those with or without the conventional capsule locus. We have developed this assay system to require only six key reagents: two are used in one multiplex inhibition-type immunoassay, and four are required in two multiplex PCR-based assays. The assay system is largely automated by a seamless combination of monoclonal antibody-based and PCR-based multiplex assays using the flow cytometric bead array technology from Luminex. The assay system has been validated with a panel of pneumococci expressing all known pneumococcal serotypes and was found to be easily transferable to another laboratory.