Search

The level of T-cell receptor excision circles (TREC), which decline with advancing age in normal individuals, has recently gained interest as a reference marker for studies on premature or early immunosenescence under particular health conditions. In order to facilitate translational studies at population and clinical levels, essential for the understanding of how changes in TREC levels are associated with responsiveness of the immune system, we have developed and optimized a real-time polymerase chain reaction (qPCR) assay which quantifies the TREC ratio from dried blood spot (DBS) samples. The present study considers a fully automated procedure to purify DNA and amplify sequences of interests by means of qPCR from DBS samples collected in healthy adults. Both TREC:PBMC (peripheral blood mononuclear cell) and TREC:T-cell ratios were compared for intra- and inter individual reproducibility. Furthermore, the impact of the length of storage on the quality of the DNA generated was also analyzed. In conclusion we describe a fully automated procedure for extracting DNA and qPCR set up, which offers a high-precision, robust qPCR assay for the quantification of both TREC:T-cell ratio and TREC:PBMC from DBS samples.

Epitope mapping with synthetic peptides bound to derivatized paper discs has been investigated using the discs both as a solid-phase matrix for peptide synthesis as well as the solid phase in immunologic testing procedures without detachment of the peptides from the paper discs. Using the T bag (tea bag) method the simultaneous synthesis and subsequent immunologic testing of large numbers of peptides was demonstrated for the feline major allergen Fel d I. A total of 15,000 paper disc-bound peptides, comprising the 146 nonapeptides overlapping by eight amino acid residues on both chains, were synthesized simultaneously with 100 paper discs per T bag. Using these paper disc-bound peptides as the solid phase in radioimmunoassays the binding sites found coincided with those detected in the PEP-SCAN with the commercially available epitope mapping kit and with the binding sites that had been found with Sepharose-coupled peptides. The signal to background-ratio in the paper disc-RIA was comparable to that in the PEPSCAN and the reproducibility was good. The bound antibodies could be eluted from the paper disc-bound peptides, permitting regeneration and repeated use of the paper discs for immunologic testing. This method was shown to be a useful alternative to the PEPSCAN and to have the major advantage that large numbers of antibodies could be tested with large numbers of peptides simultaneously.

The desire to produce specific antibodies to substances available only in m inute quantities and increased concern for laboratory animal welfare have each contributed to heightened interest in alternative immunization methods. In this report, we describe the production of polyclonal antibodies against microgram quantities of a weakly immunogenic tumor-associated protein of canine origin. Our technique employs the subcutaneous implantation of nitrocellulose electroblot strips without the use of adjuvant. The method is simple, appears reliable, can be used to improve antigen purity, and is applicable for either polyclonal or monoclonal antibody production in different host species. In addition, because traditional adjuvants are not required with this system, severe inflammatory responses and associated animal discomfort are reduced.

A simple method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed ‘Key paper’ is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins.

An immunobinding assay is described that tests antigens attached to nitrocellulose paper against antisera contained in microtiter plates. In comparison to a conventional microtiter plate radioimmunoassay, the nitrocellulose paper radioimmunoassay is clearly superior in both antigen attachment and antibody binding. Studies using bovine serum albumin and human IgG demonstrated superior antigen attachment extending from 5-fold in a physiological solution, to 50-fold in 50% fetal calf serum, to over 1000-fold in detergent solutions. With titrations using a rabbit anti-human IgG serum, the antibody binding in the nitrocellulose paper radioimmunoassay averaged over 5 times the binding in the microtiter plate radioimmunoassay. The nitrocellulose paper radioimmunoassay was also modified to quantitate human IgG. With this assay, 15 pg of human IgG inhibited the antibody binding by 50%. The nitrocellulose paper radioimmunoassay is easy to perform, and, since it combines the antigen-binding properties of the nitrocellulose paper with the convenience of assaying samples in microtiter plates, this assay should prove useful for investigating the many antigens that attach to nitrocellulose paper.

Horse anti-alpha-fetoprotein was coupled to CM-cellulose discs by a modified carbodiimide reaction. The resulting coupling CM-discs were used in solid-phase radioimmunoassay of human alpha-fetoprotein. The sensitivity of these discs and conventional BrCN activated filter paper discs coupled anti-alpha-fetoprotein was approximately the same. A fair correlation between the alpha-fetoprotein levels determined by both methods was observed. The coupling procedure with carbodiimide is simple and the use of hazardous BrCN is eliminated.

A novel technique for establishing short term clones of antigen- or mitogen-activated splenic B lymphocytes is described. Spleen cells are plated onto the surface of filter paper discs and subsequently stimulated by antigen or mitogen in situ; activated B cells proliferate and differentiate into pure colonies of cells analogous to bacterial colonies growing on agar. These colonies of lymphocytes may be characterized in a series of replica hemolytic-plaque, autoradiographic, or immunoenzyme assays making possible a full characterization of the frequency of secreted idiotopes and paratopes and of the cells that produce them. Coloby induction by either antigen or mitogen occurs under idnentical conditions, thus a rigorous comparison between the mitoge-selected and antigen-selected antibody repertoires may be made.

A sensitive and universal assay for the detection of monoclonal antibodies is described. One microliter of each hybrid cell supernatant is transferred to activated diazobenzyloxymethyl paper and, after coating the uncovered activated sites by incubation with bovine serum albumin, the paper is dipped in a solution of 125 I-labeled antigen. After incubation to allow for fixation of antigen to antibody present in the supernatants, the paper is rinsed and autoradiographed. As many as 96 wells can be tested on one paper and more than 20 papers can be treated in 1 day (∼ 2000 wells). The sensitivity and reliability of the method were tested with monoclonal antibodies against a subunit of scorpion ( Androctonus australis ) hemocyanin. Unlike other immunobinding assays the test depends only on antigen-antibody interaction and not on class or subclass of immunoglobulin. It is suitable for a variety of hybridomas.

A paper radioimmunosorbent test (PRIST) was shown to be sensitive and reproducible when used with excretory/secretory antigen of Toxocara canis second stage larvae. Whatman No. 50 filter paper (5 mm discs) gave the most consistent and clear results with antigen at a concentration of 100 micrograms/ml, and could be stored for up to 3 weeks in vacuo at -70 degrees C. Antigen coated discs were incubated with test sera at 1:10 dilution for 3 h at room temperature (21 degrees C), reacted with [125I]anti-human IgG for 1 h and counts determined in a gamma counter. Sera from patients with fascioliasis, taeniasis, schistosomiasis, oxyuriasis, trichinellosis and ancyclostomiasis gave counts similar to cord serum controls. Sera from patients with ascariasis gave counts of up to twice as great as controls, but sera from patients with toxicariasis produced counts of 7,000-13,000, a 4-6-fold increase.

A new method has been developed by which human myeloid progenitor cells can be grown on nitrocellulose paper. The method uses human placental conditioned medium for colony-stimulating activity in an agar layer while the cells grow on the overlying nitrocellulose paper in compact colonies containing granulocytic and macrophage cells. The importance of this method is discussed in the light of its usefulness in carrying out molecular studies on differentiating hematopoietic cells.

A synthetic peptide corresponding to Cys-thymopoietin 28-39 was synthesized and coupled by diazo linkages to aminophenyl thioether-derivatized paper disks. Disks were implanted in the peritoneal cavities of mice, initially after soaking in complete Freund's adjuvant and subsequently, at 3 week intervals, without further treatment. After four implantations, 6/6 mice developed antibodies reacting with the synthetic peptide and with native thymopoietin. In contrast, mice conventionally immunized with peptide alone (six mice) or with peptide complexed with thyroglobulin (six mice) all failed to develop antibodies. Mice immunized with disks derivatized with Cys-thymopoietin 9-20, corresponding to the other hydrophilic region of thymopoietin, also failed to develop antibodies. Thymopoietin 28-39 corresponds to an antigenic hydrophilic region of thymopoietin that contains the pentapeptide active site (thymopoietin 32-36, Arg-Lys-Asp-Val-Tyr).

A simple method to increase the overall sensitivity of immunoblot assays is described. The method is based on fixation of the primary monoclonal antibody-antigen complex on nitrocellulose paper by a divalent crosslinker, glutaraldehyde. The fixation of the antibody-antigen complex significantly increases the sensitivity of the assay. Unexpectedly, the glutaraldehyde treatment also improves the signal/noise ratio by decreasing non-specific binding of secondary antibodies and/or tertiary reagents. Thus, a significant overall improvement of immunoblot assays is achieved by this method.

A method is described for the silver staining of polyacrylamide gels and transfer to nitrocellulose sheets which couples the advantages of silver stain detection sensitivity with efficient transfer to nitrocellulose sheets. Using this procedure, a detection sensitivity of 10 ng was achieved while retaining a transfer efficiency for most proteins that is equivalent or better than methodology that does not permit prior visualization of protein. When coupled with the use of an acrylamide template, this procedure permits protein detection, simultaneous transfer of a large number of cored protein products from many one- or two-dimensional polyacrylamide gels to a single sheet of nitrocellulose, and retention of immunoreactivity. Prior detection, simultaneous transfer, and retention of immunoreactivity provide a substantial number of biochemical and technical advantages.

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.

Monitoring the burden and spread of infection with the new coronavirus SARS-CoV-2, whether within small communities or in large geographical settings, is of paramount importance for public health purposes. Serology, which detects the host antibody response to the infection, is the most appropriate tool for this task, since virus-derived markers are most reliably detected during the acute phase of infection. Here we show that our ELISA protocol, which is based on antibody binding to the Receptor Binding Domain (RBD) of the S1 subunit of the viral Spike protein expressed as a novel fusion protein, detects antibody responses to SARS-CoV-2 infection and COVID-19 vaccination. We also show that our ELISA is accurate and versatile. It compares favorably with commercial assays widely used in clinical practice to determine exposure to SARS-CoV-2. Moreover, our protocol accommodates use of various blood- and non-blood-derived biospecimens, such as breast milk, as well as dried blood obtained with microsampling cartridges that are appropriate for remote collection. As a result, our RBD-based ELISA protocols are well suited for seroepidemiology and other large-scale studies requiring parsimonious sample collection outside of healthcare settings.

In response to the SARS-CoV-2 pandemic many vaccines have been developed and evaluated in human clinical trials. The humoral immune response magnitude, composition and efficacy of neutralizing SARS-CoV-2 are essential endpoints for these trials. Robust assays that are reproducibly precise, linear, and specific for SARS-CoV-2 antigens would be beneficial for the vaccine pipeline. In this work we describe the methodologies and clinical qualification of three SARS-CoV-2 endpoint assays. We developed and qualified Endpoint titer ELISAs for total IgG, IgG1, IgG3, IgG4, IgM and IgA to evaluate the magnitude of specific responses to the trimeric spike (S) antigen and total IgG specific to the spike receptor binding domain (RBD) of SARS-CoV-2. We also qualified a pseudovirus neutralization assay which evaluates functional antibody titers capable of inhibiting the entry and replication of a lentivirus containing the Spike antigen of SARS-CoV-2. To complete the suite of assays we qualified a plaque reduction neutralization test (PRNT) methodology using the 2019-nCoV/USA-WA1/2020 isolate of SARS-CoV-2 to assess neutralizing titers of antibodies in plasma from normal healthy donors and convalescent COVID-19 individuals., Graphical abstract Image 1

Many studies seek to explore the impact of extrinsic soluble factors present in serum, interstitial fluids or cell-conditioned media on cells in vitro. A convenient approach to elucidate the effects of a particular factor is its selective neutralization. However, intrinsic production of soluble factors such as cytokines by the cultured cells is common and can have an impact via autocrine mechanisms. The addition of cytokine-specific neutralizing antibodies leads to neutralization of the targeted factors irrespective of their source and affects paracrine and autocrine effects alike. Thus, neutralization assays are not suitable to irrevocably demonstrate that the examined factors exert their effect via a paracrine mechanism. We were interested in investigating the impact of immunosuppressive factors present in ovarian carcinoma-associated ascites by dissecting paracrine versus autocrine effects of interleukin 10 (IL-10) and prostaglandin E2 (PGE2) on the activation of monocyte-derived dendritic cells (DC). We explored several methods of depletion based on introduction of the neutralizing antibodies bound to beads. Here we describe the pitfalls of the investigated depletion approaches and show the importance of monitoring the presence of residual neutralizing antibodies in the sample upon depletion, which impacts on the suitability of the approach to distinguish paracrine from autocrine effects. Only one of three investigated approaches showed no dislocation of neutralizing antibody from the beads into the sample. This method, which is based on covalently linking antibody to magnetic beads harbouring a reactive group allowed for the complete removal of the investigated factors from ascites and represents an elegant tool to elucidate immunoregulatory or -stimulatory cytokine networks in considerably more depth than the use of neutralizing antibodies in cell cultures alone can contribute.

Compliance and acceptance for the finger-prick method of blood collection is generally better than for venipuncture. A finger-prick method of blood collection with quantitative antibody recovery is even more important for seroepidemiological surveys. Finger-prick blood collected and dried onto filter paper has been used; but, unfortunately, this method has several disadvantages, including loss of antibody activity, possible contact contamination from blood spots on adjacent filter papers, and difficulties in extracting antibodies, justifying the search for other methods of collecting and transporting blood samples. We report on a simple method of collecting a measured amount of finger-prick blood onto a sample pad, which is immediately transferred to storage/extraction buffer. The diluted blood sample is never dried, and because of the storage buffer, can be transported and stored without refrigeration. Furthermore, the diluted blood samples can then be tested directly without further preparation. We systematically compared several storage/extraction buffers and commercially available filter papers. We showed that antibody recovery was not significantly affected by the type of filter papers used but was significantly affected by the storage/extraction buffer used. The best such buffer is StabilZyme Select.

Parenterally administered rotavirus vaccines may overcome the low efficacy observed in resource-poor regions that use live oral formulations. We have reported work on a trivalent nonreplicating rotavirus vaccine (NRRV) for parenteral administration consisting of the recombinant tetanus toxoid P2 CD4 epitope fused to a truncated VP8* fragment (P2-VP8*) for the P[4], P[6], and P[8] serotypes of rotavirus adjuvanted with aluminum. An essential part of developing this vaccine candidate was devising quantification methods for each antigen in the trivalent NRRV in the presence of aluminum adjuvant. This report describes the development of quantitative inhibition enzyme-linked immunosorbent assays (ELISAs) for in vitro antigenicity determination of the adjuvanted trivalent NRRV using serotype-specific monoclonal antibodies (mAbs) against each of the P2-VP8* antigens. Adjuvanted trivalent vaccine samples are titrated and incubated with a constant concentration of specific mAbs against each NRRV P2-VP8* antigen variant. Unbound mAbs are measured by ELISA to indirectly quantify the amount of each antigen present in the trivalent vaccine. Sensitive, specific, and reproducible inhibition ELISAs were developed and qualified for each antigen and used for final product quantification and release testing without desorption of the vaccine antigen.

Several lines of evidence indicate that antibody-dependent cellular cytotoxicity (Wren et al., 2013) is important in the pathogenesis of HIV-1 infection. Namely, ADCC is induced during natural HIV-1 infection or in HIV-1 vaccine studies, the latter demonstrated by the RV144 vaccine trial. To expedite the assessment of ADCC in studies of HIV, we have developed a high throughput assay. We have optimized the rapid fluorometric antibody-mediated cytotoxicity assay (RFADCC) by transfecting the EGFP-CEM-NKr cell line to constitutively express SNAP-tagged CCR5. This cell line can then serve as a source of HIV-specific targets when coated with monomeric gp120, spinoculated with inactivated intact virions, infected by cell-free viral diffusion or infected by cell-to-cell transmission of virus. The optimized strategy has two significant advantages over the original RFADCC method: First, the preparation of detectable target cells is less labor intensive and faster as it does not rely on multiple staining and washing steps for target cells. Second, because the target cell markers GFP and SNAP are constitutively expressed, the assay provides highly reproducible data. These strengths make the optimized RFADCC assay suitable not only for studies of HIV-1 specific cytotoxicity but also for studies of cell–cell transmission of virus. In conclusion, this assay provides a new generation T cell line that can expedite large clinical studies as well as research studies in humans or non-human primates.

A method is described for producing 1–3 μm 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.

Sensitive measurement of multiple cytokine profiles from small mucosal tissue biopsies, for example human gastric biopsies obtained through an endoscope, is technically challenging. Multiplex methods such as Luminex assays offer an attractive solution but standard protocols are not available for tissue samples. We assessed the utility of three commercial Luminex kits (VersaMAP, Bio-Plex and MILLIPLEX) to measure interleukin-17A (IL-17) and interferon-gamma (IFNγ) concentrations in human gastric biopsies and we optimised preparation of mucosal samples for this application. First, we assessed the technical performance, limits of sensitivity and linear dynamic ranges for each kit. Next we spiked human gastric biopsies with recombinant IL-17 and IFNγ at a range of concentrations (1.5 to 1000 pg/mL) and assessed kit accuracy for spiked cytokine recovery and intra-assay precision. We also evaluated the impact of different tissue processing methods and extraction buffers on our results. Finally we assessed recovery of endogenous cytokines in unspiked samples. In terms of sensitivity, all of the kits performed well within the manufacturers' recommended standard curve ranges but the MILLIPLEX kit provided most consistent sensitivity for low cytokine concentrations. In the spiking experiments, the MILLIPLEX kit performed most consistently over the widest range of concentrations. For tissue processing, manual disruption provided significantly improved cytokine recovery over automated methods. Our selected kit and optimised protocol were further validated by measurement of relative cytokine levels in inflamed and uninflamed gastric mucosa using Luminex and real-time polymerase chain reaction. In summary, with proper optimisation Luminex kits (and for IL-17 and IFNγ the MILLIPLEX kit in particular) can be used for the sensitive detection of cytokines in mucosal biopsies. Our results should help other researchers seeking to quantify multiple low concentration cytokines in small tissue samples., Highlights • Challenge: To evaluate in vivo cytokine profiles using small mucosal tissue samples • Solution: Low volume multiplex assays and optimised sample preparation • We compared low concentration IL-17 and IFNγ quantification by three Luminex kits. • MILLIPLEX was most consistent for sensitivity and accurate spiked cytokine recovery. • Manual tissue processing and PBS-based extraction buffers were superior.

Cervical cytobrushing is a useful and non-invasive method for obtaining mucosal mononuclear cells from the female genital tract, but yields few cells. The aim of this study was to compare in vitro expansion protocols (anti-CD3, anti-CD3/CD28 or Dynal anti-CD3/CD28 beads) and cytokine combinations (IL-2, IL-7 and IL-15) to improve cervical T cell yields and viability. Eighteen HIV-infected women were included in this study to compare methods for polyclonal expansion of T cells from the female genital tract and blood. Comparison of T cell yields, viability and maturational status (by differential staining with CD45RO, CCR7 and CD27) was determined following 7days of in vitro expansion. Anti-CD3 and IL-2 resulted in a 4.5-fold (range 3.7–5.3) expansion of cervical CD3+ T cells in 7days compared to day 0. Inclusion of anti-CD28 or addition of IL-7 and IL-15 to this combination did not improve expansion. Culturing cells with Dynal beads (1:1) and IL-2, IL-7 and IL-15 gave rise to the highest yields after 7days in both blood (7.1-fold) and cervix (5.6-fold). While expansion with anti-CD3 led to the accumulation of effector memory T cells (CD45RO+CCR7−CD27−), expansion with Dynabeads selected for accumulation of central memory T cells (CD45RO+CCR7+CD27+). We conclude that in vitro expansion with Dynabeads (1:1) in the presence of IL-2, IL-7 and IL-15 resulted in the greatest increase in viable T cells from both blood and cytobrush. Irrespective of the expansion method used, the T cell memory profile was altered following expansion.

The high-affinity IgE receptor (FcepsilonRI)-beta gene is one of the atopy-associated genes, but its biological significance is largely unknown. In this study, we generated the anti-FcepsilonRI-beta chain antibody to clarify beta-chain protein expression in human mast cells. The FcepsilonRI-beta antibody showed specific binding to a 27 kDa protein with Western blotting and membrane bound immunostaining using cultured mast cells. Monomeric IgE sensitization increased beta-chain expression as well as mature alpha-chain expression in mast cells. Upregulation of beta-chain expression with monomeric IgE treatment suggests possible roles of FcepsilonRI-beta protein as an atopy-related molecule.

Here, we report an ultra-rapid method for the transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In this procedure, the electro-transfer was performed with heated (70-75 degrees C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight proteins (a purified protein, molecular weight protein standards and proteins from a human tissue extract) could be carried out in 10 min for a 0.75-mm, 7% SDS-PAGE gel. For 10% and 12.5% gels (0.75 mm), the corresponding time was 15 min. In the case of 1.5-mm gels, a complete transfer could be carried out in 20 min for 7%, 10% and 12.5% gels. The permeability of the gel is increased by heat, such that the proteins trapped in the polyacrylamide gel matrix can be easily transferred to the membrane. When the heat-mediated transfer method was compared with a conventional transfer protocol, under similar conditions, we found that the latter method transferred minimal low molecular weight proteins while retaining most of the high molecular weight proteins in the gel. In summary, this procedure is very rapid, avoids the use of methanol and is particularly useful for the transfer of high molecular weight proteins.

A method for coating native, non-derivatized, polysaccharide (PS) onto nitrocellulose (NC) for identifying PS-specific antibodies has been developed. The new feature of this method is that PS molecules are vacuum filtered onto NC in their native state by devices that can accomodate NC of different sizes and shapes. PS-coated NC disks were used to localize antibody secreting hybridoma cells cultured on filter paper disk. These were analyzed by blotting with size-matched PS-coated NC disks and specific antibodies secreted by individual colonies were detected by enzyme-linked immunoblot. In another application of this method, immune sera were separated by isoelectric focusing and the gels were blotted with PS-coated NC sheets. The spectrotype and isotype of antibodies that bound to the NC were examined using isotype specific enzyme-linked antibody. These immunoblots showed high resolution and specificity. The advantages of this method are that the PS used for coating does not need to be derivatized in order to bind the NC, and that smaller quantities of PS may be utilized by this coating method when compared to other techniques. This provides a useful tool to ask many questions regarding the immune response to PS.

Peroxidase-bound protein A and lectins in combination with precipitable substrates were used to detect viral proteins and glycoproteins on ‘Western blots’. The sensitivity of various methods was compared, including radioiodinated protein A, enzyme immunosorbent assay and peroxidase staining with DAP. The sensitivity of the peroxidase reaction, its rapidity and low cost compared with other methods make this a useful choice for developing protein bands transferred to nitrocellulose paper. In addition, a procedure was devised to clarify and fix nitrocellulose sheets for direct quantification of the precipitated substrate by densitometric analysis.

International audience; A library of Plasmodium falciparum genomic DNA on the lambda gt11 phage vector was screened for clones positive to a rabbit serum raised against a purified fraction of P. falciparum proteins and a pool of sera from malaria patients. The positive clones were characterized with antibodies purified by the plaque antibody selection technique. This technique consist of purifying specific antibodies on a nitrocellulose filter blotted directly on a lawn of plaques of an antigen-producing phage clone. The purified antibodies are then used as a probe in a Western blot of parasite protein extract, for preliminary characterization of the clones. Using this method, two different clones coding for P. falciparum antigens were identified with the rabbit serum and about 20 with the human sera. This method can be of general use, i.e. it is not limited to parasite systems, and facilitates the immunological analysis and identification of a large number of clones.

A simple antigen overlay technique for line immunoelectrophoresis and crossed-line immunoelectrophoresis is described. A blind gel is overlaid with antigen-impregnated filter paper instead to mix antigens with molten agarose and to pour them in a uniform sample gel. This makes it easier in line and crossed-line immunoelectrophoresis comparison of different antigen samples, simplifying technique as compared with the conventional method. Moreover, this technique is designated to obtain reproducible patterns from thermolabile proteins since they are unaffected by being heated.

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.

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 μm/ml with this test.

Immunoprecipitates formed in conventional electroimmunoassays were solubilized by brief immersion of the agarose gels in sodium dodecylsulfate. The proteins were then transferred to nitrocellulose sheets by electroblotting. The blotted proteins were readily amenable to analyses by non-precipitating monoclonal antibodies and the immunoblots were developed with second antibody/biotin-streptavidin-peroxidase staining. The electroimmunoassay-immunoblot (EIA-IB) method is of value in (1) specificity assays of monoclonal antibodies in crossed immunoelectrophoresis; (2) analysis of specific molecular interactions between proteins; (3) rapid screening and simple identification of monoclonal antibodies by line immunoelectrophoresis immunoblotting.

Antigenic competition is known to be a widespread phenomenon when using crude extracts of antigens (e.g., Escherichia coli cytoplasmic proteins) for immunization. This non-specific form of immune suppression can be partially overcome by passive immunization with antibodies against dominant antigens (which are the suppressive molecules) before injection of the antigenic mixture. Blocking these immunodominant antigens or antigenic determinants by a passively administered antibody permits antibody responses against hitherto weakly or non-immunogenic molecules.

Conventional cleavage of linked polypeptide chains by heating in SDS can so alter molecular structure as to interfere with antibody binding, on which both immunoprecipitation and 'western blotting' depend. As an alternative, gentle treatment with acid at room temperature or at 0 degrees C was effective in separating the alpha and beta chains of human MHC Class II glycoprotein dimers and proved superior in terms of preservation of at least one labile epitope on the beta chain.

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.

A miniaturization of the immunoblot technique permitting the use of as little as 50 μl of antibody is described. This method does not require any special equipment and the whole experimental procedure (incubation, washing, detection) can be performed in the same chamber, with economy of reagents at all steps and no prenumbering of blotted strips. This method has been applied for screening the presence of antibodies in hybridoma culture supernatants and is of special interest for testing any scarce material (immune sera or antigens).

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.

There is much debate in the pharmaceutical industry on how to translate the current guidelines on immunogenicity testing for biotherapeutics into a testing strategy that suits the specific requirements of individual drug candidates. In this paper, member companies from the European immunogenicity platform (EIP) present a consensus view on the essential requirements for immunogenicity testing of a biotherapeutic throughout the various phases of drug development, to ensure patient safety and to enable successful market entry. Our aim is to open the debate and provoke discussion on this important topic which is unique to biotherapeutic drug development. The scope of this paper is limited to aspects relevant to biotherapeutic drug development and does not include fundamental academic studies of immunogenicity. Here, we propose two pre-defined testing strategies for the detection and characterization of anti-drug antibody (ADA) responses where the different strategies are based on the phase of development for a biotherapeutic, a. without (category 1) and b. with (category 2) the expected potential to elicit ADA mediated severe clinical consequences. The harm of a potential ADA response determines which of the two testing strategies is adopted. Rather than replacing the overall risk assessment which is known to be challenging and multi-factorial, the testing strategy selection is a starting point for immunogenicity testing which adapts throughout drug development as more information becomes available. The scientific rationale on which the "case-by-case" approach advocated in white papers and guidance documents may be translated for each individual drug development program is provided and, underpins the recommendations made here.

Nasal allergen challenge of patients with allergic rhinitis results in increased numbers of inflammatory cells and increased production of pro-inflammatory cytokines including interleukin 5 (IL-5). We report a sensitive, noninvasive method to measure changes in the amount of mRNA for IL-5 in nasal epithelium and have used this method to detect alterations of IL-5 mRNA from patients undergoing a nasal allergen challenge. Ten grass or ragweed allergic adults were challenged out of season with appropriate pollen extracts at sufficient dose to give a rhinitis total symptom score of 5 on a scale of 12. After allergen exposure, symptoms were recorded hourly. At 0, 3, and 6 h after allergen exposure, secreted proteins were collected on filter paper strips and two superficial scrapings of nasal epithelium were obtained. The scrapings of epithelium were immediately immersed in 100 microl of RNAlater (Ambion, Austin, TX) and stored at 4 degrees C for up to 1 month without loss of RNA quality. Total RNA was isolated and RT-PCR was performed. cDNA for IL-5 was then measured by real-time fluorescence quantitative PCR with Pre-Developed TaqMan Assay Reagents (PE Biosystems, Foster City, CA). Sufficient RNA was isolated from eight subjects to measure IL-5 mRNA. Data were normalized for content of ribosomal RNA. The relative amount of cDNA for IL-5 was calculated by comparison with internal standards prepared from phytohemagluttinin-stimulated peripheral blood mononuclear cells. Messenger RNA for IL-5 was increased 8.7+/-2.7-fold at 3 h (p0.01) and 39.5+/-20.9-fold at 6 h (p0.01). Increased IL-5 mRNA levels at 6 h closely correlate with total symptom scores at 6 h (r=0.88; p=0.007). IL-5 protein was measured by ELISA in eluates from the filter papers. At 6 h, there was increased IL-5 protein (7.7+/-2.8 ng/ml) compared with time zero (1.8+/-0.5 ng/ml) (p=0.02). The levels of IL-5 protein did not correlate significantly with the symptoms score or with changes in the levels of IL-5 protein with IL-5 mRNA. These data show that changes in IL-5 mRNA in patients with allergic rhinitis undergoing an allergen challenge correlate with total symptom scores better than changes in IL-5 protein eluted from filter paper. Furthermore, these changes can be measured quantitatively in very small amounts of tissue.

We show in this paper that the survival of antigen-loaded dendritic cells in vivo may be used as a sensitive readout of CTL activity. We have previously shown that dendritic cells labeled with the fluorescent dye CFSE and injected sub-cutaneously into mice migrate spontaneously to the draining lymph node where they persist for several days. In the presence of effector CTL responses, dendritic cells loaded with specific antigen rapidly disappear from the draining lymph node. In this paper we extend the above observations and set up a simple and sensitive method to reveal CTL activity in individual mice in vivo. Dendritic cells were labeled with two different fluorochromes, loaded with antigen or left untreated, and mixed together before injection into mice. We show that only the dendritic cells loaded with specific antigen were cleared from the draining lymph node, while dendritic cells not loaded with antigen remained unaffected. Cytotoxic responses generated by immunization with peptide-loaded dendritic cells, or by infection with influenza virus, could be revealed using this method. Comparison of the differential survival of dendritic cells populations mixed together also allowed us to accurately evaluate the disappearance of dendritic cells, irrespective of variability in the injection site and other parameters. Given the ability of dendritic cells to efficiently take up and present complex antigens, nucleic acids and apoptotic bodies, this method may also allow the evaluation of cytotoxic activity against antigens that are not characterized in terms of peptide epitopes.

Penicilloylated (BPO) and penicillanylated (BPA) poly-L-lysine (PLL) and human serum albumin (HSA) were prepared and characterized by penamaldate assay and proton NMR spectroscopy. The conjugates were coupled to nitrocellulose (NC) discs and cyanogen bromide activated paper discs and their in vitro reactivities with serum IgE antibodies were examined. Results showed that on paper discs, 55.3 and 83% of the sera reacted with PLL conjugates of BPO and BPA, respectively, while 41.5 and 58.1% reacted with HSA conjugates. On NC discs, HSA conjugates gave better results, 75.6 and 70.7%, respectively for BPO and BPA, compared with 38.6 and 50%, respectively for the PLL conjugates. Overall, the BPA-PLL conjugate on paper discs proved to be the most reactive preparation. Addition of the BPO-PLL paper disc preparation detected more positive sera (85.1%) and we believe that the combined use of these two specificities offers the best test for the detection of penicillin-reactive IgE antibodies.

Subcellular fractionation has been an important tool in the investigation of neutrophil structural organization including granule heterogeneity, composition and mobilization. The resolution of organelles obtained by subcellular fractionation was improved considerably after the introduction of nitrogen cavitation as an efficient but gentle means of disrupting neutrophils and with Percoll as a density medium. This paper describes in detail the methodology of subcellular fractionation of nitrogen cavitated neutrophils on one-, two-, and three-layer Percoll density gradients. Appropriate marker proteins are presented for neutrophil organelles including azurophil, specific and gelatinase granules, in addition to secretory vesicles and plasma membranes. The dynamics of granule and secretory vesicle exocytosis is demonstrated by subcellular fractionation of resting and activated human neutrophils. Finally, the paper describes the applications of subcellular fractionation in the investigation of the localization of neutrophil constituents, in protein purification schemes and in the study of translocation of cytosolic proteins to isolated neutrophil organelles.

The conglutinin-binding assay (KgBa) has gained widespread use for the detection of circulating immune complexes. A recent paper questioned the interpretation of the results obtained by this method and the validity of the assay (Holmskov et al. (1992) J. Immunol. Methods 148, 225). We now present hitherto unnoted differences between controls and patients with either rheumatoid arthritis or systemic lupus erythematosus. For this we use simple, but unconventional, graphic representations of the data, based on difference plots and ratio plots. Differences between patients with Burkitt's lymphoma and systemic lupus erythematosus from another previously published study (Macanovic, M. and Lachmann, P.J. (1979) Clin. Exp. Immunol. 38, 274) are also represented using ratio plots. Our observations indicate that analysis by regression analysis may often be misleading. 11111onglutinin-binding assay (KgBa) has gained widespread use for the detection of circulating immune complexes. A recent paper questioned the interpretation of the results obtained by this method and the validity of the assay (Holmskov et al. (1992) J. Immunol. Methods 148, 225). We now present hitherto unnoted differences between controls and patients with either rheumatoid arthritis or systemic lupus erythematosus. For this we use simple, but unconventional, graphic representations of the data, based on difference plots and ratio plots. Differences between patients with Burkitt's lymphoma and systemic lupus erythematosus from another previously published study (Macanovic, M. and Lachmann, P.J. (1979) Clin. Exp. Immunol. 38, 274) are also represented using ratio plots. Our observations indicate that analysis by regression analysis may often be misleading.

This paper describes a method for the detachment of immunomagnetic beads from positively selected human B lymphocytes. After rosetting of B cells using anti-CD19 coated magnetic beads (Dynabeads M-450 Pan B, Dynal), the Dynabeads were rapidly detached (efficiency 80%) from the cells using goat anti-mouse-Fab antiserum (DETACHaBEAD, Dynal) at ambient temperature. Isolated B cells did not show significant differences in the expression of a number of B cell antigens when compared to B cells stained in fresh whole blood. In contrast, positively selected B cells that had detached from the beads following overnight incubation, demonstrated a significantly reduced expression of certain of the antigens examined (CD19, CD20 and CD23). It was further demonstrated that neither anti-CD19 nor anti-Fab resided on the surface of the cells after detachment. The cells were still in G0 phase (greater than 90%) at the end of the isolation procedure. Moreover, anti-IgM antibodies stimulated the vast majority of the cells to leave the G0 phase, and to progress through S phase in the presence of growth factors. The cells could also be stimulated to differentiate, further confirming the normal functional capacity of the isolated cells. The method described in this paper can also be used for the detachment of other positively selected cells, such as CD4+ T cells, CD8+ T cells and CD34+ stem cells.

One of the important steps for antigen presentation by MHC class II molecules involves binding of a peptide fragment of the antigen to the class II molecule followed by recognition of the resulting complex by T cells. The most commonly used methods for studying binding of peptide to MHC II are: equilibrium dialysis, gel filtration chromatography, HPLC and polyacrylamide gel electrophoresis. Each of these methods has some limitations and is time consuming. In addition, each requires a considerable amount of native MHC class II, which is always difficult to obtain. In this report, we describe three different sensitive methods using radiolabeled peptide to study peptide binding to murine MHC class II molecules. These are: nitrocellulose filter binding, thin-layer chromatography (TLC) using plate-supported silica gel or PEI cellulose, and paper electrophoresis using Sepraphor cellulose polyacetate paper. All three methods are rapid, highly sensitive and require only ng quantities of affinity pure MHC class II molecules and peptides. These methods can be used to calculate the peptide occupancy of MHC class II molecules.

A paper by Manghi et a!. (1994) on the 'Devel­ opment of an ELISA for measuring the activity of tetanus toxoid in vaccines and comparison with the toxin neutralization test in mice' prompts this response. A major problem with the paper is that the authors have confused methods of testing the potency of tetanus toxoid (IT) as per the British Pharmacopoeia (1973), the British Pharma­ copoeia (1980) and the United States Minimum Requirements (1952). The three methods are very different. The British Pharmacopoeia (1973) method, which is no longer employed in Great Britain, is based on two sequential injections of IT into guinea pigs. This method has been fol­ lowed by Manghi et al. (1994) who refer to it as the British Pharmacopoeia (1980) method. The British Pharmacopoeia (1980) method of potency testing of adsorbed TT, which is currently used in most European countries including Great Britain and which is recommended by the World Health Organization (1990), does not involve titration of sera from immunized guinea pigs. This method requires a single injection of several dilutions of test and reference vaccines into groups of guinea pigs or mice and challenging the immunized ani­ mals after 28 days with tetanus toxin. The po­ tency of the test vaccine is determined against the reference vaccine by calculating 50% immunizing doses of both test and reference vaccines. The British Pharmacopoeia (1973) method for ad­ sorbed IT requires two injections of nine guinea pigs with 1/10 of a single human dose with a 4 week interval and bleeding of guinea pigs not later than 2 weeks after the second injection. The vaccine passes the test if at least 113 of guinea pigs show tetanus antitoxin levels in their sera not less than 0.5 IUIml or at least 2/3 of guinea pigs show tetanus antitoxin levels not less than 0.05 IU1m!. Manghi et a!. (1994) injected guinea pigs by the British Pharmacopoeia (1973) method and followed the requirements of the US potency method which uses a different immunization schedule. The US method requires injection of guinea pigs with a single dose of half of the total human dose (1.5 times a single human dose) and bleeding the guinea pigs not later than 6 weeks afterwards. The vaccine passes the US minimum requirements if the pooled sera of at least four guinea pigs show no less than 2 IUIml of tetanus antitoxin. It has been shown that the affinity of antibod­ ies elicited in guinea pigs after injection of diph­ theria toxoid (Gupta et aI., 1994) and tetanus toxoid (Gupta and Siber, 1994b) depends upon the immunization status of animals. In the experi­

Mycotoxin is one of the most important natural pollutants, which poses a global threat to food safety. However, the pollution of mold in food production is inevitable. The detection technology of mycotoxins in food production is an important means to prevent the damage of mycotoxins, so rapid detection and screening to avoid pollution diffusion is essential. The focus of this review is to update the literature on the detection of mycotoxins by surface plasmon resonance (SPR) technology, rather than just traditional chromatographic methods. As a relatively novel and simple analytical method, SPR has been proved to be fast, sensitive and label-free, and has been widely used in real-time qualitative and quantitative analysis of various pollutants. This paper aims to give a broad overview of the sensors for detection and analysis of several common mycotoxins.