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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]

Abstract Antibody-based diagnostics and therapeutics have huge commercial value. However, applications of antibodies are often limited by instability, particularly for recombinant antibody formats. This paper describes the conversion of a single-chain variable fragment (scFv) antibody to a single-chain antibody fragment (scAb) with notably improved stability characteristics. This scAb retains antigen-binding activity (i) at high temperature (up to 60 °C), (ii) in guanidine hydrochloride (GdnHCl, up to 1 M), and (iii) when stored at 37 °C for 6 months. However, limited improvement was observed when the original scFv was converted to a larger fragment antigen-binding (Fab) format. Certain Cys-to-Ala mutations in the third complementarity determining region of the antibody heavy chain (CDR H3) also led to stability improvements. Our findings indicate that the stability of an antibody derivative depends on its format and on the positions of cysteines in the CDRs. Graphical abstract Unlabelled Image Highlights • Changing 4G5 scFv antibody to scAb improved stability to heat (≤60 °C) and guanidine hydrochloride (≤1M), with little change in binding properties • The 4G5 scAb showed much better stability when stored at 37 °C for 6 months • Paired Cys in the 4G5 CDR-H3 region (SSYQCSGDYC) can influence the antibody's stability, with minimal effects on its affinity (antigen binding) [ABSTRACT FROM AUTHOR]

Photonic ring resonance is a property of light where in certain circumstances specific wavelengths are trapped in a ring resonator. Sensors based on silicon photonic ring resonators function by detecting the interaction between light circulating inside the sensor and matter deposited on the sensor surface. Binding of biological material results in a localized change in refractive index on the sensor surface, which affects the circulating optical field extending beyond the sensor boundary. That is, the resonant wavelength will change when the refractive index of the medium around the ring resonator changes. Ring resonators can be fabricated onto small silicon chips, allowing development of a miniature multiplex array of ring based biosensors. This paper describes the properties of such a system when responding to the refractive index changed in a simple and precise way by changing the ionic strength of the surrounding media, and in a more useful way by the binding of macromolecules to the surface above the resonators. Specifically, a capture immunoassay is described that measures the change of resonant wavelength as a patient serum sample with anti-SS-A autoantibodies is flowed over a chip spotted with SS-A antigen and amplified with anti-IgG. The technology has been miniaturized and etched into a 4 × 6 mm silicon chip that can measure 32 different reactions in quadruplicate simultaneously. The variability between 128 rings on a chip as measured by 2 M salt assays averaged 0.6% CV. The output of the assays is the average shift per cluster of 4 rings, and the assays averaged 0.5% CV between clusters. The variability between chips averaged 1.8%. Running the same array on multiple instruments showed that after some improvements to the wavelength referencing system, the upper boundary of variation was 3% between 13 different instruments. The immunoassay displayed about 2% higher variability than the salt assays. There are several outstanding features of this system. The amount of antigen used on the chip for each test is around 200 picograms, only a few microliters of sample is necessary, and the assays take < 10 min. [ABSTRACT FROM AUTHOR]

Taenia solium oncosphere protein TSOL18 is the host-protective antigen against porcine cysticercosis. Little attention has been given to use it as target molecule in immunodiagnostic tests. The objective of this paper is to describe the immunodiagnostic potential of monoclonal antibodies (MoAbs) raised against conformational epitopes of TSOL18. Three murine IgG1 MoAbs (25D12C1, 21C2D2, 10H1F2) against three different conformational epitopes of TSOL18 were produced and evaluated with an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. Serum samples from pigs immunized with TSOL18 inhibited the binding of the three MoAbs to TSOL18 antigen in i-ELISA. The highest inhibition of anti-TSOL18 antibodies in immunized pigs was observed with MoAb 25D12C1. Ten field sera (12.19%) from 82 non-vaccinated and non-infected pigs showed anti-oncosphere antibodies inhibiting the binding of MoAb 25D12C1. Anti-oncosphere antibodies in pigs experimentally infected with T. solium eggs inhibited the binding of MoAb 25D12C1 from 2 to 8 week-post infection. It is concluded that MoAb 25D12C1 has excellent immunodiagnostic potentials to detect anti-oncosphere antibodies in the intermediate hosts at early exposure to T. solium eggs. Further investigations on potential use of MoAb 25D12C1 in a capture antigen ELISA for the detection of post-oncospheral antigens in infected pigs cannot be overemphasized. [Display omitted] • We describe the potential usefulness of the monoclonal antibody 25D12C1 to TSOL18 protein. • Serum samples from pigs immunized with TSOL18 inhibited the binding of 25D12C1 in an inhibition enzyme-linked immunosorbent assay (i-ELISA) for the detection of anti-TSOL18 and anti-oncosphere antibodies. • Anti-oncosphere antibodies in pigs infected with T. solium eggs inhibited also the binding of 25D12C1. • It is concluded that MoAb i-ELISA using 25D12C1 has the potential to identify animals exposed to T. solium infection. [ABSTRACT FROM AUTHOR]

Several diagnostic tools have been developed for clinical and epidemiological assays. RT-PCR and antigen detection tests are more useful for diagnosis of acute disease, while antibody tests allow the estimation of exposure in the population. Currently, there is an urgent need for the development of diagnostic tests for COVID-19 that can be used for large-scale epidemiological sampling. Through a comprehensive strategy, potential 16 mer antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified. A systematic scan of the three structural proteins (S,N and M) and the non-structural proteins (ORFs) present in the SARS-CoV-2 virus was conducted through the combination of immunoinformatic methods, peptide SPOT synthesis and an immunoassay with cellulose-bound peptides (Pepscan). The Pepscan filter paper sheets with synthetic peptides were tested against pools of sera of COVID-19 patients. Antibody recognition showed a strong signal for peptides corresponding to the S, N and M proteins of SARS-CoV-2 virus, but not for the ORFs proteins. The peptides exhibiting higher signal intensity were found in the C -terminal region of the N protein. Several peptides of this region showed strong recognition with all three immunoglobulins in the pools of sera. The differential reactivity observed between the different immunoglobulin isotypes (IgA, IgM and IgG) within different regions of the S and N proteins, can be advantageous for ensuring accurate diagnosis of all infected patients, with different times of exposure to infection. Few peptides of the M protein showed antibody recognition and no recognition was observed for peptides of the ORFs proteins. • Potential antigenic peptides suited for antibody-based SARS-CoV-2 diagnosis were identified • Selected peptides were synthesized using the SPOT technique and exposed to pools of COVID-19 patients' sera • Antibody recognition showed a strong signal for peptides of the S, N and M proteins, but not for the ORFs proteins • Peptides of the N protein C- terminal region showed strong recognition with all three immunoglobulins in the pools of sera [ABSTRACT FROM AUTHOR]

The SARS-CoV-2 pandemic has provided the stimulus for the rapid development of a variety of diagnostic testing methods. Initially these were deployed as screening tools to evidence spread of the virus within populations. The recent availability of vaccines against the virus and the need to better understand the parameters of post-infection protective immunity requires development of methods, suitable for use in the routine diagnostic laboratory, capable of characterising the viral immune response in greater detail. Such methods need to consider both cellular and humoral immunity. Toward this aim we have investigated use of a commercial multiplex assay (COVID Plus Assay, One Lambda), providing assessment of the SARS-CoV-2 response at structural level, and developed an in-house cell stimulation assay using commercially available viral peptides (Miltenyi). This paper reports our experience in use of these methods in extended investigation of a cohort of healthcare workers with prior screening results indicative of viral infection. The antibody response generated is shown to be both qualitatively and quantitatively different in different individuals. Similarly a recall response to SARS-CoV-2 antigen involving the T cell compartment can be readily demonstrated in recovered individuals but is of variable magnitude. [ABSTRACT FROM AUTHOR]

Dried blood spots (DBS) are routinely used in screening newborns for treatable disorders. Immunoglobulin extraction from DBS, serum or other biological fluids loaded on filter paper cards could represent a valuable method of specimen preservation in monitoring immune response against pathogens as well as vaccination efficiency. In this study using different sources including serum, and monoclonal antibodies we established parameters for antibody extraction from the filter cards to assess antibody reactivity against Helicobacter pylori , measles virus (MV) and the novel coronavirus SARS-CoV-2 antigens. We demonstrated that DBS and dried undiluted serum result in completely preserved antibody activity for immunoassays, including in virus neutralization assays against MV. Extraction efficiency was determined by IgG concentration measurements. The plaque-reduction neutralization titer 50% of dried human serum spots remained stable after more than 10-day storage – 1:359 vs. 1:345 for the corresponding frozen sample. DBSs could be used to monitor immune response to bacterial and viral antigens following natural exposure or immunization. Mice immunized with recombinant spike protein receptor-binding domain of SARS-CoV-2 developed a strong antibody response by day 14 and reached titers above 1:64,000 on day 21 following the secondary boost immunization as measured on DBS samples in antigen-mediated ELISA. Variability in IgG concentration of eluted DBS could be influenced by factors involved in sample application, extraction process and sample characteristics. Adjustment of antibody specific activity to the eluted IgG concentration can increase accuracy of the result interpretation, including in SARS-CoV-2 serological diagnostics. • Immunoglobulins could be efficiently extracted from dried filter cards and used in testing immune response against pathogens. • Antibody specific activity is preserved in serum and blood spot extracted samples run in ELISA and virus neutralization test. • Extraction conditions and dilution factor are critical for running the dried spot samples in virus neutralization test. • Measurement of IgG concentration and calculation of specific IgG activity is the most accurate way of result interpretation. [ABSTRACT FROM AUTHOR]