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The 2018 12 th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis , issues 22 and 24 (2018), respectively.

The 2018 12 th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis , issues 22 and 23 (2018), respectively.

A novel ultra-low-cost biochemical analysis platform to quantify protein dissociation binding constants and kinetics using paper microfluidics is reported. This approach marries video imaging with one of humankind's oldest materials: paper, requiring no large, expensive laboratory equipment, complex microfluidics or external power. Temporal measurements of nanoparticle-antibody conjugates binding on paper is found to follow the Langmuir Adsorption Model. This is exploited to measure a series of antibody-antigen dissociation constants on paper, showing excellent agreement with a gold-standard benchtop interferometer. The concept is demonstrated with a camera and low-end smartphone, 500-fold cheaper than the reference method, and can be multiplexed to measure ten reactions in parallel. These findings will help to widen access to quantitative analytical biochemistry, for diverse applications spanning disease diagnostics, drug discovery, and environmental analysis in resource-limited settings., (© 2018 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760μM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system., (Copyright © 2017 Elsevier B.V. All rights reserved.)

BackgroundBrucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable.Methodology/principal findingsIn this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen.ConclusionsB cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.

Roach (Rutilus rutilus), from a unpolluted lake, were caged in a lake receiving treated waste waters from a pulp and paper mill 16 km upstream, and simultaneously a group of fish were held in cages in the unpolluted reference lake. The level of serum immunoglobulin of the fish kept in cages in these lakes was quantified. Serum immunoglobulin levels decreased when the fish were transferred to the contaminated lake and remained significantly reduced for the 8-week period fish were monitored. In another experiment fish were immunized with bovine gamma-globulin (BGG) after 5 weeks caging in the lakes. The kinetics of specific antibody synthesis were determined by assaying the number of antibody secreting cells in the spleen using enzyme-linked immunospot assay and by measuring the concentration of specific antibody in the serum. The peak response of the effluent exposed fish against BGG was lower than that for fish kept in the unpolluted reference lake, and the antibody titers, due to immunization, increased more slowly in the exposed fish. These results demonstrate that subchronic exposure of fish in a lake receiving treated waste waters from a bleached kraft mill affects the antibody-mediated immunity resulting in, besides lower levels of Ig, weak responsiveness against antigen.

The enzyme-linked immunospot (ELISpot) assay is a highly useful and sensitive method to detect total immunoglobulin and antigen-specific antibody-secreting cells. In addition, this method can measure biological activity and immunological secretions from immune cells. In general, membrane-bound antigen allows binding of antibody secreted by B cells, or a membrane-bound analyte-specific antibody binds to the specific analyte (e.g., cytokines) elicited from cells added to the well containing the bound antibody. The response from added cells is then detected by using an anti-Ig antibody and a colorimetric substrate, while in the case of non-B cells, the elicited antigen is detected with appropriate antibodies and enzyme-conjugated antibodies. Specificity of antibodies binding the protein of interest is necessary to achieve correct results. Western blotting can be used for this with/without siRNA knockdown of proteins of interest or with the use of peptide inhibitors to inhibit the binding of specific antibodies to the target protein. Despite its general simplicity, western blotting is a powerful technique for immunodetection of proteins (notably low abundance proteins) as it provides simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Now, we have plethora of immunoblotting methods to validate antibodies for ELISpot., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Agglutinated blood transports differently onto paper than stable blood with well dispersed red cells. This difference was investigated to develop instantaneous blood typing tests using specific antibody-antigen interactions to trigger blood agglutination. Two series of experiments were performed. The first related the level of agglutination and the fluidic properties of blood on its transport in paper. Blood samples were mixed at different ratios with specific and nonspecific antibodies; a droplet of each mixture was deposited onto a filter paper strip, and the kinetics of wicking and red cell separation were measured. Agglutinated blood phase separated, with the red blood cells (RBC) forming a distinct spot upon contact with paper while the plasma wicked; in contrast, stable blood suspensions wicked uniformly. The second study analyzed the wicking and the chromatographic separation of droplets of blood deposited onto paper strips pretreated with specific and nonspecific antibodies. Drastic differences in transport occurred. Blood agglutinated by interaction with one of its specific antibodies phase separated, causing a chromatographic separation. The red cells wicked very little while the plasma wicked at a faster rate than the original blood sample. Blood agglutination and wicking in paper followed the concepts of colloids chemistry. The immunoglobin M antibodies agglutinated the red blood cells by polymer bridging, upon selective adsorption on the specific antigen at their surface. The transport kinetics was viscosity controlled, with the viscosity of red cells drastically increasing upon blood agglutination. Three arm prototypes were investigated for single-step blood typing.

Antigen binding activities of 25 kinds of filter papers, including nitrocellulose (NC), nylon or polyvinylidine difluoride (PVDF), in the binding of 5 viruses, 3 bacteria, 2 mycoplasmas and 1 chicken serum protein antigens in dot immunoassay were compared. Immobilon affinity membrane type D (IAM-D) was found best in binding viral antigens, followed by Ultrabind SV-450 (SV-450). SV-450 was found best in binding bacterial antigen, followed by Ultrabind US-450 (US-450), IAM-D and NC 0.45 micron. IAM-D was the best in binding mycoplasma antigen, followed by Ultrabind HP, US-450, NC 0.2 micron. Overall, IAM-D had the best capability in the binding of the antigens.

Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 μl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 μl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests. [Display omitted] [ABSTRACT FROM AUTHOR]

We have developed a novel histamine release test (HR) using whole blood and antigen-coupled RAST paper discs, for the screening of allergens rather in a short time with a small amount of blood. Histamine was determined by a RIA kit. In order to evaluate the diagnostic value, the results of the test were compared with those of RAST, intracutaneous tests, and eye tests in 45 allergic patients. HR correlates better with RAST than intracutaneous test in almost all antigens. The closest positive correlation with the other tests was seen in mite allergen, followed by pollen, foods and mould spores in that order. When the HR was positive for house dust, 100% of the patients were also positive for the eye test, which is reported to closely correlate with bronchial provocation tests. HR seemed to be a useful test not only for the screening but also for the determination of pathogenic allergens.

A method is described which allows antigens electrophoretically 'blotted' onto nitrocellulose to be probed using small volumes (less than 0.3 ml) of antibody solutions. Reagent volumes required for this method are more than 10-fold lower than the minimum volumes attainable using either custom made or commercial incubation trays. The procedure is simple and economical and should be applicable to any situation where it is desirable to either conserve or reduce the amount of screening reagent used.

In 2022, the outbreak of the monkeypox virus occurred in many non-endemic countries, and the World Health Organization (WHO) assessed that this outbreak was "atypical". The establishment of a rapid and effective assay that can be used for the early diagnosis of monkeypox virus infection is crucial for outbreak prevention and control. In this study, the monkeypox virus A29 protein and the homologous vaccinia virus A27 protein and cowpox virus 162 protein were expressed in Escherichia coli BL21 for screening. We synthesized the monkeypox virus A2917–49 peptide as the immunogen and obtained 25 monoclonal antibodies (mAbs) against the A29 protein using mouse hybridoma techniques. Then an immunochromatographic test strip method for detecting A29 was established. The strips utilizing mAb-7C5 and 5D8 showed the best sensitivity and lowest limit of detection: 50 pg mL−1 for purified A29 and specificity tests showed that the strips did not cross-react with other orthopox viruses (vaccinia virus or cowpox virus) as well as common respiratory pathogens (SARS-CoV-2, influenza A and influenza B). Therefore, this method can be used for early and rapid diagnosis of monkeypox virus infection by antigen detection. [ABSTRACT FROM AUTHOR]

Many studies have been conducted on measuring avian influenza viruses and their hemagglutinin (HA) antigens via electrochemical principles; most of these studies have used gold electrodes on ceramic, glass, or silicon substrates, and/or labeling for signal enhancement. Herein, we present a paper-based immunosensor for label-free measurement of multiple avian influenza virus (H5N1, H7N9, and H9N2) antigens using flexible screen-printed carbon nanotube-polydimethylsiloxane electrodes. These flexible electrodes on a paper substrate can complement the physical weakness of the paper-based sensors when wetted, without affecting flexibility. The relative standard deviation of the peak currents was 1.88% when the electrodes were repeatedly bent and unfolded twenty times with deionized water provided each cycle, showing the stability of the electrodes. For the detection of HA antigens, approximately 10-μl samples (concentration: 100 pg/ml–100 ng/ml) were needed to form the antigen–antibody complexes during 20–30 min incubation, and the immune responses were measured via differential pulse voltammetry. The limits of detections were 55.7 pg/ml (0.95 pM) for H5N1 HA, 99.6 pg/ml (1.69 pM) for H7N9 HA, and 54.0 pg/ml (0.72 pM) for H9N2 HA antigens in phosphate buffered saline, and the sensors showed good selectivity and reproducibility. Such paper-based sensors are economical, flexible, robust, and easy-to-manufacture, with the ability to detect several avian influenza viruses. [ABSTRACT FROM AUTHOR]

[Display omitted] • A µPAD was introduced for quantitative detection of the SARS-CoV-2 antigen employing electrocatalytic activity of the Ni Fe layered double hydroxide. • TO evaluate the applicability of the proposed sensor, nasopharyngeal swabs samples in COVID-19 patient samples collected. • The µPES displayed the attributes of low cost , reliability, selectivity, and high sensitivity for low-level detection of the target. In the offered study, the proposed sensor combined the innovative disposable microfluidic paper-based immunosensor and cost-effective plant-based anti-SARS-CoV-2 monoclonal antibody CR3022, expressed in Nicotiana benthamiana. An electrochemical microfluidic paper-based analytical device (µPAD) was introduced for quantitative detection of the SARS-CoV-2 antigen employing electrocatalytic activity of the Ni Fe layered double hydroxide (NiFe LDH). Its electrocatalytic activity asthe platform of proposed the analytical device enhanced the signal output via redox-recycling of the species the ferri/ferrocyanide [Fe(CN) 6 ]3-/4- in solution and is applied to µPAD response signal. Layered NiFe double hydroxide (LDH) nanosheets with a suitable redox potential were prepared by incorporating Fe into the Ni-based LDH. With the help of Fe, the charge-transfer kinetics for the reduction of Ni3+ to Ni2+ was improved and the formation of unwanted Ni components with higher oxidation state was suppressed. The incorporated Fe as the electron transfer mediator enhanced the process of Ni(OH) 2 /NiOOH redox cycle. Under the optimum conditions, the dependency of impedance signal and SARS-CoV-2 concentration showed a linear region from 10 fg/mL-5 ng/mL with a detection limit of 0.23 fg/mL. This Paper-based immunosensor device also showed long stability and good reproducibility, which can be used for the quantitative assay of the SARS-CoV-2. The results of nasal samples analysis revealed the applicability of the resulting biosensor. [ABSTRACT FROM AUTHOR]

A very sensitive method for the detection of antigen-antibody complexes on nitrocellulose paper immunoblots is described. The protein antigens are separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by their electrophoretic transfer onto a nitrocellulose sheet ("Western blot"). The protein antigens bound to the nitrocellulose paper are exposed to the monoclonal antibody and the antibody-antigen complexes are detected on the paper by an immunoenzymatic reaction. The improved sensitivity of this method is the result of (i) the use of the detergent Tween 20 in blocking the nonspecific binding of the antibodies to the nitrocellulose paper, (ii) the use of a peroxidase-antiperoxidase (PAP) reaction, and (iii) the intensification of the diaminobenzidine reaction product with nickel and cobalt ions in phosphate buffer.

Freeze-thawed extracts of mature schizonts of Plasmodium knowlesi (strain W1) were electrophoresed in sodium dodecylsulphate (SDS) polyacrylamide gels, then transferred electrophoretically to diazophenylthioether paper. The transferred antigen was probed using a purified polyvalent, polyclonal immune serum pool isolated from rhesus monkeys (Macaca mulatta). This method for identifying parasite antigens was compared with immunoabsorption, using parasites which had been metabolically labelled in vitro with 35S-methionine. The methods showed general correspondence but some antigens failed to react after transfer because of loss of antigenicity during SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Some antigens were observed preferentially after the transfer procedure, possibly because of their failure to elute from the immunoabsorbant or because they may have a low methionine content. The transfer method failed to reveal the antigen reactivity of three out of four monoclonal antibodies tested, apparently because the epitopes involved were inactivated by SDS. During P. knowlesi infection in the resistant kra monkey (Macaca fascicularis) the transfer method showed the presence by day 10 of antibodies against two components having an apparent molecular weight by SDS-PAGE similar to those of putative protective antigens described in other studies using monoclonal antibodies.

The application of immunogold for the detection of antigen-antibody complexes on nitrocellulose paper is described. Tobacco mosaic virus (TMV) protein, either purified or in leaf extract, was bound to the nitrocellulose paper and then exposed to rabbit anti-TMV serum. The antigen-antibody complexes were detected by gold-labeled goat anti-rabbit immunoglobulin G. The gold-labeled antibody is directly visible because of its pink color. This method can detect 1-5 pg of TMV protein, either in purified form or in the unpurified plant extract, with high specificity.

An immunoblotting procedure using a peroxidase-anti-peroxidase (PAP) complex was developed for the detection of autoantigens in crude mixtures by human autoimmune sera. Thymus proteins were transferred to a nitrocellulose sheet after electrophoresis in polyacrylamide gels and probed with a 1:100 dilution of serum. The location and extent of immunoglobulin G (IgG) binding was determined by sequential reaction with: rabbit anti-human IgG, goat anti-rabbit IgG and rabbit peroxidase-anti-peroxidase complex. The peroxidase was allowed to react with chloronaphthol and low levels of autoantigen/autoantibody complex were detectable with virtual absence of background colour. The inclusion of human IgG and its pepsin-generated fragment provided a means of controlling and calibrating the blotting procedure.

Normal human diploid cells, such as WE-38, transformed by an oncogenic virus, SV40, are greatly depleted of a cathodal esterase isozyme using both immunological and enzymatic criteria. This cathodal esterase is primate-specific and associated with lysosomes of many different tissue cells. It provides a clear example of antigenic loss associated with malignant change.

Two novel immunization methods designed for immunization with small quantities of antigen, immobilized on a solid matrix and without the use of adjuvant, are presented. The major test antigen used in order to evaluate these methods was bovine serum albumin (BSA). It was deposited in the spleen of mice and rabbits, either attached to Sepharose beads (Pharmacia Sepharose 4B) or to nitrocellulose (NC) paper strips (Millipore). BSA was attached to NC by direct application or by electroblotting after SDS-polyacrylamide gel electrophoresis. The antibody response in mouse and rabbit serum, after intrasplenic immunizations with various quantities of antigen, was analyzed in an ELISA standard procedure. In mice, an antibody response in serum was detected after three intrasplenic immunizations with a total quantity of 73.6 ng BSA bound to Sepharose beads and after two immunizations with a total quantity of 800 ng BSA attached to NC. Determination of the antigen-binding to NC and the clearance rate of antigen attached to NC deposited in the spleen of mice was performed with 125I-labeled BSA. In rabbits, an antibody response in serum was detected after a single intrasplenic immunization with 2.6 micrograms BSA attached to NC. When testing human insulin and sheep prolactin, attached to NC, as antigens in intrasplenic immunization of rabbits, an antibody response was found after a total quantity of 3.2 micrograms insulin and 10.5 micrograms prolactin, respectively.