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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]

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.

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.

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.

A device is described which is used to measure the concentration of antigen in solution. A wedge-shaped gap containing the antigen to be detected is bounded on one side by a reservoir containing specific antiserum and on the opposite side by a detection slide which has been previously coated with a layer of adsorbed antigen. As the antibodies diffuse through the gap, they complex with the soluble antigen in the wedge until these become saturated leaving additional antibody free to complex with antigen on the detection slide. By allowing diffusion to continue for an appropriate time, it is possible to relate the distance along the gap where this has occurred to the concentration of antigen within the gap. Using human IgG as a model antigen, we have been able to measure concentrations down to approximately 10 micrograms/ml with this test.

Utilizing the principles of counterimmunoelectrophoresis, a technique was devised to immunologically identify non-denatured protein antigens resolved by polyacrylamide gel electrophoresis. Proteins were electrophoretically transferred from polyacrylamide gel to antibody-containing agarose gel in a commercially available blotting apparatus. Detection of the antigen/antibody complex by Coomassie blue staining (in the ng range for unlabeled antigen) or autoradiography (in the pg range for radiolabeled antigen) established the sensitivity and specificity of this method. Similar sensitivity could be obtained following conventional sodium dodecyl sulfate polyacrylamide gel electrophoresis after partial removal of the detergent and possible renaturation of proteins. Placement of a nitrocellulose sheet on the anodal side of the agarose gel produced a 'negative replica' (proteins/polypeptides not reacting with specific antibodies in the agarose gel). Counterimmunoblotting provides an additional method for molecular weight determination of protein antigens with sensitivity and specificity comparable to established nitrocellulose blotting (unlabeled proteins) or immunoprecipitation (labeled proteins) procedures.

This paper describes the development of an enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of autoantibodies against the nuclear protein Scl-70. The isolation of Scl-70 from rat livers and the conditions for the ELISA are described. Compared with the already established double diffusion in gel for detection of anti-Scl-70 antibodies this ELISA has advantages.

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.

This paper describes the evaluation and optimisation of boric acid antigen retrieval (AR) in rat joint tissue immunohistochemistry (IHC), with reference to two sample IHC targets, CD31 (PECAM-1) and Proliferating Cell Nuclear Antigen (PCNA). Sections of buffered formalin-fixed arthritic tibial/talus joints, decalcified with EDTA, EDTA/formalin or Surgipath(R) Decalcifier I(R), were subjected to one of a number of pre-treatments (none, 0.1% trypsin, 0.2 M acetic acid pH 7.0 or 0.2 M boric acid pH 7.0) and then immunostained for CD31 or PCNA. Of the pre-treatment AR regimens, boric acid gave the most consistent and specific immunostaining of both antigens in joints from the three different decalcification protocols. Satisfactory CD31 and PCNA staining was also achieved in EDTA decalcified joints with no pre-treatment. Likewise, PCNA could be demonstrated in Surgipath(R) decalcified tissue without pre-treatment, albeit at slightly lower staining intensity than achieved following boric acid. The remaining decalcification/pre-treatment conditions were unsatisfactory for IHC of the two antigens investigated because of lack of staining, non-specific staining or consistent loss of sections from the slides. Boric acid pre-treatment provides a valuable alternative low temperature AR method where conventional heat-mediated AR methods are normally required but cannot be used due to tissue type.

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]

The production of murine monoclonal antibodies against specific antigens by hybridomas is a well utilised technique. The production of hybridomas secreting specific human antibodies would have many advantages in therapeutic applications of monoclonal antibodies. The immortalised human lymphocytes themselves would also provide valuable tools in research on lymphocyte development. Preparation of human-human hybridomas has been limited by a lack of suitable fusion partners. This protocol paper describes the production of human-mouse heterohybridomas by two independent laboratories. The purpose of this protocol is to provide a basis for the development of heterohybridoma technology in laboratories with limited hybridoma experience.

Many enzyme-linked immunosorbent assays take advantage of immobilized antigens for the identification of antibody binding sites. Generally, the analysis of cellulose membrane-bound B-cell epitopes is currently considered of high utility. We adapted this methodology for the stimulation of a T helper cell hybridoma with known specificity. Forty overlapping peptides corresponding to the entire rabies virus nucleoprotein were synthesized in duplicates on a single sheet of 90x130 mm size amino-modified paper. The efficacy of the peptide assembly was monitored by color staining of the unreacted amino groups. After completion of the synthesis, the side-chain protecting groups were removed, and the membrane was thoroughly cleaned of all organic and inorganic contaminants. The membrane was cut into pieces, and a standard lymphokine release assay was performed directly from the paper-bound antigens. From all the 40 peptide spots only peptide 31D stimulated the proliferation of the 9C5.D8-H T-cell hybridoma, known to react to this peptide. By using this protocol, as little as 0.4 microgram (approximately 200 pmole) of peptide could be detected. According to mass spectrometry the T-cell stimulation proceeded as a true solid-phase assay. The peptide neither leached from the membrane nor was cleaved by the medium-splenocyte mixture. Additionally, tryptic digestion of the cellulose membrane released the expected peptide fragments.

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10(5) of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.

A method is described for producing 1-3 microns sized particles of nitrocellulose (NC) which are able to absorb protein. Protein is absorbed onto preformed particles made by first dissolving a sheet of nitrocellulose paper in DMSO, and then precipitating it with sodium carbonate/bicarbonate buffer. The efficiency of binding is the same as that of an equivalent sheet of non-processed NC filter paper. Antibodies absorbed onto preformed particles are not exposed to DMSO and carbonate buffer and therefore retain a high antigen binding capacity. Antigen and antibody-absorbed NC preformed particles were used to capture antibody and antigen, respectively. Using lysis buffer, the captured antibodies and antigens were readily released from the NC particles. This makes the latter an appropriate matrix for immunoprecipitation assays either for an antigen or for specific antibody. Antigen-coated NC particles were specifically aggregated ('agglutinated') by specific antibodies and thus can be used in semi-quantitative tests.

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]

A simple chemical method is described which creates cyanate groups on the saccharide core of bacterial lipopolysaccharide (LPS). The activated LPS molecule is stable at pH 3.5 and can be kept for months at -20 degrees C without loss of properties. The strong reactivity of the cyanate groups permits efficient covalent coupling of various molecules to LPS. This is done under non-denaturing conditions. This paper also describes the coupling of microquantities of antigen (5-10 micrograms) to LPS. The 'LPS-antigen' conjugates are easily purified upon centrifugation at 15,000 X g. These compounds are mitogenic for B cells and trigger in vivo or in vitro production of antibodies directed against the coupled antigen. In vitro, a concentration of conjugate as low as 10(-2) micrograms/ml triggers specific antibody synthesis. Injection of 5 micrograms of conjugate in mice induces a humoral response detectable 7 days after immunization. B cells cultured for 5 days with an adequate dose of the LPS-beta-galactosidase conjugate were fused with the Sp2-0-Ag cell line to give anti-beta-galactosidase hybridomas.

This paper describes a radioimmunoassay (RIA) for the quantitation of a major protein antigen (L1) released from human granulocytes. A protein A-carrying strain of Staphylococcus aureus was used as a solid phase IgG-binding reagent for the separation of antigen-antibody complexes from free antigen in the assay. A two-step version of the RIA may be completed in 2.5 h and has a detection limit of about 5 ng/ml. The sensitivity may be increased by minor modifications. A single step version takes 1.5 h and has adequate sensitivity to cover clinically important plasma levels. Selection and optimal use of reagents for the two-step procedure is described and their storage discussed. The simplicity of the procedure combined with high sensitivity and satisfactory accuracy, precision and reproducibility makes this assay suitable for quantitation of L1 in various body fluids.

This paper describes the use of phenyl-Sepharose CL-4B as a solid-phase hydrophobic adsorbent in the purification of S-antigen from protein extracts of bovine, porcine and human retina. Chromatographic conditions were ascertained whereby the majority of contaminating proteins were bound to the adsorbent leaving S-antigen in the liquid phase. In combination with size fractionation on Ultrogel AcA, the method conveniently yielded porcine and bovine S-antigen preparations up to 100% purity. Immunogenicity of purified S-antigens was verified by induction of experimental autoimmune uveoretinitis in albino Lewis rats. The method is preparative in scale, fast in performance and yields S-antigen in high purity and antigenic potency.

Immunoadsorption is a simple and effective method for purifying antigens and antibodies. The major interfering effect is non-biospecific binding, especially when crude preparations of antibodies or antigens are coupled to the matrix. In this paper various possibilities for eliminating these effects are compared and analysed. It was found that for antigen (AFP) purification pre-elution with a pH 10.0 buffer of high ionic strength (1.0 M) is effective. A major problem in purifying antibodies in the non-biospecific binding of IgG. This can be eliminated by using a non-biospecific immunoadsorbent column, where specific antibodies are not, or only weakly bound and IgG with irrelevant specificity but exhibiting high non-biospecific interaction is retained. This has been applied to preparation of sheep anti-pepsinogen II group protease antibodies.

A method is described for detection of antibodies by means of nitrocellulose or diazobenzyloxymethyl (DBM) paper on which various antigens have been spotted. The sensitivity of this antigen spot test (AST) is comparable with that of RIA and ELISA. The method requires only nanogram amounts of antigen. Since a variety of antigens can be spotted on a single piece of nitrocellulose or DBM paper, this antigen spot test is especially useful for specificity controls on antibodies.

The use of Sepharose beads in the defined antigen substrate sphere (DASS) immunofluorescent system has been restricted by the need for sophisticated equipment for fluorescence quantitation since reliable subjective reading is not possible. This paper describes a method for visual assessment of results in this system. Antigen is coupled to sodium metaperiodate activated. Cross-linked Sepharose 4B beads and blocked with rhodamine-BSA prior to reduction. This procedure eliminates the use of the hazardous cyanogen bromide activation process and is potentially suitable for use in both direct systems for evaluation of specificity and potency of fluorescent conjugates and in indirect systems for serological screening of sera for specific antibody.

The amount of antigen on the solid phase was studied during various steps of an enzyme-linked immunosorbent assay (ELISA) for chicken anti-bovine serum albumin (BSA) antibodies. Three materials for the solid phase were used: polystyrene, nylon and cyanogen bromide activated paper. There was noticeable leakage of antigen during the assay from both polystyrene (30%) and nylon (60%). This occurred during all steps of the assay: during incubation with the sample serum, incubation with the enzyme conjugated antibodies and during the washing procedures. During incubation with the sample serum detachment of antigen was quite rapid and could result in competition between bound and free antigen for antibodies. The presence of antigen-antibody complexes in reaction mixtures could also lead to erroneous results in the antibody assay. With covalent coupling of antigen to solid phase (cyanogen bromide activated paper), batch-to-batch variation in the surface concentration of antigen was strikingly less than in the assay using plastics. Detachment of antigen from activated paper during the assay was less than with polystyrene or nylon. ELISA with activated paper as the solid phase thus seems suitable as a reference method for standardisation of the antigen phase in ELISA systems.

This paper describes a sequential staining procedure for double immunoenzymatic staining of pairs of antigens in frozen tissue sections and cell smears using monoclonal antibodies. This technique involves performance of an indirect immunoperoxidase sandwich (including development of the enzyme reaction) followed by an unlabelled immuno-alkaline phosphatase sandwich (the APAAP method). The two enzyme labels are revealed using DAB/H2O2 for peroxidase and naphthol AS-MX plus fast blue or fast red for alkaline phosphatase. When compared with a hapten-sandwich/biotin-avidin system, the sequential staining procedure proved to be simpler and more sensitive and was also more suitable for double immunoenzymatic staining when monoclonal antibodies were only available in small amounts. The sequential staining procedure is particularly useful for the identification of antigens distributed in different cell populations or in different sites (e.g., nucleus and cytoplasm or cell surface) of the same cell. In contrast, this method does not appear to be very suitable for demonstrating two antigens located in the same site (e.g., surface membrane) of the same cell for which purpose double immunofluorescence remains the first choice.

Abstract: Detection of circulating antibodies to bovine herpes virus 1 (BHV-1), parainfluenza 3 virus (PI3V), bovine viral diarrhoea virus (BVDV) and bovine respiratory syncytial virus (BRSV) using ELISA is widely used for veterinary diagnostics and surveillance. In this paper, the potential of a multiplex serology test based on Luminex technology, where all antibodies are simultaneously detected in a single assay was investigated. The performance of “in-house” separate ELISAs which use relatively crude lysates of cultured virus as capture antigens, was compared to the multiplex assay where the same antigens were covalently bound to the fluorescent beads used in the Luminex platform. A panel of field serum samples was tested by the multiplex assay in parallel with the separate routine ELISAs to provide a comparison between tests. The BHV-1 and PI3V components of the multiplex test showed similar sensitivities and specificities to the separate “in-house” ELISAs. The performance of the BVDV and BRSV components was less successful and was attributed to relatively low signal strength for these antigens, leading to higher assay variability and a reduced ability to distinguish positive and negative samples compared to the “in-house” ELISAs. The results illustrated that antigens commonly used successfully in ELISAs cannot always be transferred for use in alternative assay systems. The use of recombinant BVDV E2 protein was investigated and was shown to lead to an appreciable increase in signal strength compared to the use of crude BVDV antigen in the Luminex system. [Copyright &y& Elsevier]

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]

Abstract: Autoimmune (Hashimoto''s) thyroiditis is a chronic inflammatory disease which affects >3% of the population and shows an increasing prevalence with increasing age. Anti-thyroid autoantibodies, particularly against thyroperoxidase (also known as thyroid peroxidase or TPO), occur commonly in humans with autoimmune thyroid disease, and assays for anti-TPO autoantibodies are used in clinical diagnosis. In contrast anti-TPO autoantibodies have not been observed in classical mouse models of autoimmune thyroiditis, except in cases where mice were deliberately immunized with TPO. In the past, detection of anti-TPO autoantibodies in mice has relied on an indirect immunofluorescence assay (iIFA) which screens for thyroid follicle membrane staining in frozen sections of mouse thyroid glands. Since recent transgenic mouse models of autoimmune thyroiditis spontaneously develop anti-TPO autoantibodies, an assay other than serial dilution and iIFA would be useful to detect and quantify these autoantibodies. In this paper we describe such an assay, based on the capacity of autoimmune mouse sera to bind to the extracellular domain of mouse TPO which was produced in a radioactively labeled form using a coupled in vitro transcription/translation system. The same approach, using human TPO, could provide a highly sensitive method to detect anti-TPO autoantibodies in humans. [Copyright &y& Elsevier]

Abstract: Time-resolved fluorescence has been developed for immunoassay to obtain higher sensitivity than usual immunoassays. In this paper, a simple, sensitive and specific method was developed for immunoassay of serum carcinoembryonic antigen (CEA) by combining time-resolved fluoroimmunoassay (TRFIA) and flow injection analysis. Based on a sandwich immunoassay format, a monoclonal antibody immobilized immunoaffinity column inserted in a flow system was used for immunoreactions. The cleaved solution was collected after the reaction between the immunocomplex in the immunoaffinity column and the enhancement solution that was used to cleave the Eu-labels from the immunocomplex, and then detected by time-resolved fluorescence. Serum CEA could be detected in the linear range from 2.5 to 100 ng/ml with a correlation coefficient of 0.997 and the detection limit of 1.0 ng/ml. Twenty human serum samples detected by this method were in good agreement with the results obtained by the electrochemiluminescence immunoassay. This method could be further developed for fast practical clinical detection of serum CEA levels. [Copyright &y& Elsevier]

Abstract: This study presents an improved method for the complete transfer of proteins separated by two-dimensional gel electrophoresis to a membrane, specifically designed for the screening and identification of antigens recognized by autoantibodies in patients with breast cancer (BCP) and healthy volunteers. This paper reports the evaluation of this technique using proteins from MCF7 as a source of antigens following 2-DE separation. The appropriate quantity of protein to be loaded on gels (150 μg) has been determined, the aim being a complete and reproducible recovery of all separated proteins onto the polyvinylidene fluoride membrane (2D-blot) after a semi-dry electrotransfer. Several different transfer methods were tested in parallel, resulting in the selection and optimisation of one using a discontinuous buffer system, based on the isotachophoresis theory. To facilitate the comparative analysis of the different sets of 2D-blots probed with individual sera from BCP and healthy volunteers, the 2D-blots were stained with colloidal gold following the immunodetection step. The gels and 2D-blots were scanned and analysed by imaging software. The matching permitted exact localisation of particular relevant protein spots hybridised by antibodies on the 2D-blots. These spots were subsequently located on preparative gels for identification by mass spectrometry. A set of 40 2D-blots was probed with 20 sera from patients with breast cancer and 20 sera from healthy volunteers. In the protein profiles submitted to immunodetection, 15 proteins were repeatedly immunodetected by both BCP and sera from healthy people. Those proteins were identified by mass spectrometry. Conversely, some protein isoforms were preferentially immunodetected by BCP sera and may reflect the presence of this cancer. The improved isotachophoretic method described in this study is suitable for comparing the overall profile of autoimmunity between different populations and for subsequent identification of relevant antigens. [Copyright &y& Elsevier]

Different single-cell analyses for the detection of antigen-specific T cells based on antigen-triggered induction of cytokine production (elispot, intracellular cytokine staining, cytokine secretion assay, etc.) have been analyzed. In this paper we present the data of a thorough validation of the IFNγ Secretion Assay (ISA, Miltenyi Biotec, Bergisch Gladbach, Germany). In this assay the secreted IFNγ is bound to the cell surface and is then stained as an artificial surface molecule and analyzed by flow-cytometry. The introduction of five quality criteria markedly improved the reproducibility of this assay and made it very reliable (intra-assay variability<5%; inter-assay variability<20%). Recovery experiments further demonstrated that almost 100% of IFNγ+ labeled cells could be detected by this technology. In order to analyze which cell subsets contribute to IFNγ-production, we compared the results obtained in different individuals after VZAg-stimulation. Three different IFNγ-secretion patterns could be discerned. In Pattern 1 there is a predominant and almost equal contribution of T cells and NK cells with a minor contribution of CD3+CD56+ and B cells. Pattern 2, which is most abundant, is characterized by a predominance of NK cells (60–70%). Pattern 3 differs from the previous one in its minor contribution of NK cells. Here T cells predominate the IFNγ secretion. These results clearly demonstrate that the IFNγ+ subset distribution after VZAg-stimulation is not uniform and differs individually. Furthermore, the ISA-technology proves to be very useful in vaccine research. This was demonstrated by testing the IFNγ+ secretion pattern after HBsAg-stimulation in PBMC from HBsAg-vaccinated individuals. [Copyright &y& Elsevier]

Given the key role 4-1BB plays in the stimulation of T cells, humanization of agonistic anti-human 4-1BB monoclonal antibody (mAb) may have important clinical applications. In this paper, we present the humanization of agonistic anti-human 4-1BB mAb, BBK-4, using a phage display library. We first prepared the combinatorial library by incorporating murine and human alternative at positions representing buried residues that might affect the structural integrity of the antigen binding site. Six humanized single chain Fv (scFv) fragments were selected from the combinatorial library expressing phage-displayed humanized scFv. They were found to retain the epitope specificity of the original mAb but had affinities of lower than 1/10 of the original. In spite of the lower affinity, the humanized scFv coated on the surface expanded human peripheral blood mononuclear cells (PBMCs) in MLR similarly to the original mAb in the presence of anti-CD3 mAb. These results suggest that humanized anti-human 4-1BB scFvs can be used as a valuable reagent for clinical application. [Copyright &y& Elsevier]

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]