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In general, vertical flow assay (VFA) has a disadvantage of requiring a complex analysis process that involves manually injecting various reagents (target analyte, washing buffer, detection conjugate, etc.) sequentially. However, in this study, we have developed an innovative paper-based VFA device that replaces the complex analysis process with one-step and enables the detection of multiple targets. The fabrication process of the multi-target detection VFA device is as follows: preparation and pre-treatment of the strip materials, design of strip cartridge, design of the multiple detection VFA device, optimization experiments for strip sample flow rates, determination of device analysis time, determination of device limit of detection (LOD), multiple target signal uniformity experiment, immunoglobulin G (IgG) and C-reactive protein (CRP) antigen-antibody multiple detection experiment, and data extraction and analysis method. The use of paper-based materials enables the device to be produced at cost-effective, and cartridge production allowed for uniform array formation. IgG and CRP are used to evaluate the performance of the device as common biomarkers. The device proposed in this study is currently under research. To validate multiple target detection capability of the VFA device proposed in this study, two types of antigens-antibodies (Human IgG and Human CRP) were employed. The detection limit is 0.15 μg/mL for IgG and 0.19 μg/mL for CRP in naked eye. Furthermore, it was confirmed that there is no cross-reactivity caused by the device structure through IgG and CRP antigens. In conclusion, the VFA device proposed in this study consists of a one-step analysis process, and it has been confirmed that it can detect multiple targets simultaneously., Competing Interests: Declaration of competing interest There are no conflicts to declare., (Copyright © 2024 Elsevier B.V. All rights reserved.)

A machine has been developed for the rapid washing of the cellulose filter paper discs that are used in a number of radioimmunoassays. The machine is simple in design, easy to use, and is capable of washing 96 filter paper discs simultaneously. The efficiency of the machine is demonstrated by a RAST assay for measuring IgE antibodies to the venom. Time taken to wash the discs was reduced 3-fold without loss of sensitivity or reproducibility.

A rapid method for the analysis of antigen-specific human serum antibodies is described. After isoelectric focusing on thin agarose gels, serum proteins are transferred to nitrocellulose paper by capillary blotting. The kinetics of transfer indicated that ca. 90% of 125I-labelled isolated human immunoglobulins (IgG, IgA and IgM) are transferred to the nitrocellulose paper within 10 min. Probing of the nitrocellulose blot with 125I-labelled tetanus toxoid antigen followed by autoradiography reveals the total serum anti-tetanus toxoid antibody repertoire, including high molecular weight IgM. This procedure is rapid (results can be obtained in less than 12 h), sensitive and should prove very useful for detailed studies and analysis of antibody repertoires in body fluids and extracts

Abstract: Dried blood spot samples (DBSS) on filter paper offer several advantages compared to conventional serum/plasma samples: they do not require any phlebotomy or separation of blood by centrifugation; they are less invasive; they allow sample stability and shipment at room temperature; and they pose a negligible risk of infection with blood-borne viruses, such as HIV, HBV and HCV, to those who handle them. Therefore dried blood spot samples (DBSS) on filter paper can be a quick, convenient and inexpensive means of obtaining blood samples for biomarker discovery, disease screening, diagnosis and treatment monitoring in non-hospitalized, public health settings. In this study, we investigated for the first time the potential application of dried blood spot samples (DBSS) in protein expression profiling using antibody array technology. First, optimal conditions for array assay performance using dried blood spot samples (DBSS) was established, including sample elution buffer, elution time, elution temperature and assay blocking buffer. Second, we analyzed dried blood spot samples (DBSS) using three distinct antibody array platforms, including sandwich-based antibody arrays, quantitative antibody arrays and biotin-label-based antibody arrays. In comparison with paired serum samples, detection of circulating proteins in dried blood spot samples (DBSS) correlated well for both low- and high-abundance proteins on all three antibody array platforms. In conclusion, our study strongly indicates the novel application of multiplex antibody array platforms to analyze dried blood spot samples (DBSS) on filter paper represents a viable, cost-effective method for protein profiling, biomarker discovery and disease screening in a large, population-based survey. [Copyright &y& Elsevier]

A simple method for the comparison and identification of protein epitopes recognized by monoclonal antibodies directly on thin-layer plates and 3MM paper chromatograms is described. Enzyme digests of myelin basic protein were separated on thin-layer plates and 3MM paper, fixed with glutaraldehyde and probed directly with affinity-purified mouse monoclonal antibodies. Detection of the immunoreactive peptides was enhanced using a second rabbit anti-mouse immunoglobulin and finally located using an alkaline phosphatase-conjugated anti-rabbit immunoglobulin. By probing the same enzyme digests of MBP with various monoclonal antibodies raised against MBP, a different binding 'pattern' of reactive peptides is rapidly obtained for monoclonal antibodies of differing specificities. This procedure was extended to the identification of the antigenic determinant using synthetic peptides. The major advantages of this procedure are its simplicity, non-radioactive nature and speed. Furthermore, there is the possibility of sequencing immunoreactive peptides eluted from the 3MM 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.

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 microtiter plates, this assay should prove useful for investigating the many antigens that attach to nitrocellulose paper.

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.

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.

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 PEPSCAN 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.

This report describes a modified dot immunobinding assay (DIA) in microplates using a crude mixture of non-purified antigen. Nitrocellulose filter paper discs exposed to the antigen mixture were inserted into the wells and kept in place by a specially constructed device. To test the efficiency of the modification a set of monoclonal antibodies from a mouse immunized with 58 kDa trpE-Bmyc fusion protein were screened. The advantage of this modified method over conventional ELISA is that it permits the use of non-purified antigen for screening large numbers of monoclonal antibodies.

The desire to produce specific antibodies to substances available only in minute 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 new technique is described for the rapid detection and quantitation of herpes simplex virus (HSV) antigens and antiviral antibodies. It involves immobilization of HSV antigens on filter paper discs and subsequent analysis by 125I-labeled staphylococcal protein A (SPA) radioimmunoassay. A specially designed 96-well filtration device is employed which serves both as an incubation chamber and as a filtration manifold. It is rapid, simple, sensitive and specific, and requires only small volumes of antiserum and few target cells. The results may be readily and objectively quantitated. This technique permits the simultaneous assay of a large number of specimens in less than 1 h. Its sensitivity is considerably greater than that of other currently used immunologic techniques, and it is amenable to automation. These characteristics suggest that this [125I]SPA immunofiltration technique may be applicable to the rapid diagnosis of viral infections.

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.

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 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. Colony induction by either antigen or mitogen occurs under identical conditions, thus a rigorous comparison between the mitogen-selected and antigen-selected antibody repertoires may be made.

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.

Immune chromatography, a radioimmunological binding assay, employs paper chromatography to separate immune complexes from free antigen and antibodies. During chromatography free antigen and antibodies become distributed throughout the paper, while immune complexes remain near the bottoms of the strips. The chromatographic differences can be made quantitative by using either iodinated antigens or antibodies. Under these conditions nanogram quantities of antigen can be detected or antibodies in sera diluted several 1000-fold. The immune chromatography assay can also be performed as an indirect assay, since the paper strips are cut from nitrocellulose paper. In this case the immune components are absorbed by the paper during chromatography. Antigen is then detected with an iodinated second antibody. The indirect immune chromatography assay is particularly useful for identifying different sera that react with the same antigen. Reaction with the first serum before chromatography reduces the amount of antigen available to the second serum following chromatography. In addition to characterizing the immune chromatography procedure, we discuss the possible applications of chromatography assays for the quantitation of other types of molecular binding interactions.

We have developed a rapid and sensitive enzyme-linked immunosorbent assay (ELISA) for thyroxine (T4) in dried blood samples spotted on filter paper. The assay is carried out on microtiter plates without extraction or centrifugation steps. The detection limit of the assay is 5 pg/disc/well, equivalent to 1.25 micrograms/1 of whole blood or 2.5 micrograms/1 of serum. Intra- and inter-assay coefficients of variation for various T4 concentrations are 2.4-9.0% and 5.9-17.5% respectively. Correlation between the proposed ELISA method and the RIA is good (r = 0.900, n = 62, y(RIA) = 0.99x(ELISA) + 9.90). The ELISA method is useful for mass-screening of neonatal congenital hypothyroidism using dried blood samples on filter paper, is very simple and one person can assay more than 300 samples per day.

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).