A highly sensitive and subspecies-specific surface antigen enzyme- linked immunosorbent assay for diagnosis of Johne's disease

Johne's disease (JD), or paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis, is one of the most widespread and economically important diseases of livestock and wild ruminants worldwide. Control of JD could be accomplished by diagnosis and good animal husbandry, but this is currently not feasible because commercially available diagnostic tests have low sensitivity levels and are incapable of diagnosing prepatent infections. In this study, a highly sensitive and subspecies-specific enzyme-linked immunosorbent assay was developed for the diagnosis of JD by using antigens extracted from the surface of M. avium subsp. paratuberculosis. Nine different chemicals and various intervals of agitation by vortex were evaluated for their ability to extract the surface antigens. Various quantities of surface antigens per well in a 96-well microtiter plate were also tested. The greatest differences in distinguishing between JD-positive and JD-negative serum samples by ethanol vortex enzyme-linked immunosorbent assay (EVELISA) were obtained with surface antigens dislodged from 50 microg/well of bacilli treated with 80% ethanol followed by a 30-second interval of agitation by vortex. The diagnostic specificity and sensitivity of the EVELISA were 97.4% and 100%, respectively. EVELISA plates that had been vacuum-sealed and then tested 7 weeks later (the longest interval tested) had diagnostic specificity and sensitivity rates of 96.9 and 100%, respectively. In a comparative study involving serum samples from 64 fecal culture-positive cattle, the EVELISA identified 96.6% of the low-level fecal shedders and 100% of the midlevel and high-level shedders, whereas the Biocor ELISA detected 13.7% of the low-level shedders, 25% of the mid-level shedders, and 96.2% of the high-level shedders. Thus, the EVELISA was substantially superior to the Biocor ELISA, especially in detecting low-level and midlevel shedders. The EVELISA may form the basis for a highly sensitive and subspecies-specific test for the diagnosis of JD.     

Development of a particle agglutination assay system for detecting Japanese encephalitis virus-specific human IgM,using hydroxyapatite-coated nylon beads

Japanese encephalitis virus-specific IgM is a reliable indicator for serodiagnosis of Japanese encephalitis. A particle agglutination (PA) assay system was developed to detect anti-Japanese encephalitis virus IgM in human serum samples. The newly developed PA assay consisted of hydroxyapatite-coated nylon beads and V-bottom 96-well microplates. Hydroxyapatite-coated nylon beads were coated with Japanese encephalitis virus antigens. Japanese encephalitis virus antigen-coated, hydroxyapatite-coated nylon beads agglutinated in the IgM-captured wells when anti-Japanese encephalitis virus IgM-positive serum samples were used. A button pattern was formed at the bottom of the wells when anti-Japanese encephalitis virus IgM-negative serum samples were used. Thirty anti-Japanese encephalitis virus IgM-positive serum samples from Japanese encephalitis-confirmed cases were tested by the PA assay. All these serum samples were determined to be Japanese encephalitis virus IgM-positive. IgM titers determined by the PA assay corresponded to those determined by enzyme-linked immunosorbent assay. The titers were consistent in two independent PA assays. These results indicate that the newly developed PA assay is a reliable method for detecting anti-Japanese encephalitis virus IgM in human serum samples and that this assay will be a suitable diagnostic system especially in rural areas of Asia.     

Hydroxyapatite-coated nylon beads as a new reagent to develop a particle agglutination assay system for detecting Japanese encephalitis virus-specific human antibodies.

BACKGROUND: Detection of Japanese encephalitis virus (JEV)-specific antibodies is done today by hemagglutination-inhibition assay (HIA), neutralization assay (NTA) and enzyme-linked immunosorbent assay (ELISA). These conventional assays are often difficult to perform in diagnostic laboratories with insufficient resources. An alternative antibody detection kit, which is simple, preservable and inexpensive, is needed for extended use in rural areas of Asia. OBJECTIVES: (i) Characterization of a new antigen carrier, hydroxyapatite-coated nylon (Ha-Ny) beads, and (ii) evaluation of the JEV antigen-coated Ha-Ny beads as a reagent to detect anti-JEV antibodies in human serum samples. STUDY DESIGN: We examined the Ha-Ny beads for hydroxyapatite content, precipitation efficiency and protein adsorption ability. We then developed a particle agglutination assay system using the JEV antigen-coated Ha-Ny beads, and tried out the newly developed assay system with reference serum samples. RESULTS: The beads had the ability to adsorb 0.44 mg of lysozyme per gram. Sedimentation speed was 10.2 cm/30 min in phosphate buffered saline (PBS), pH 7.0. Binding of the JEV antigen on Ha-Ny beads was confirmed by scanning electron microscopy (SEM) and ELISA. Eighteen confirmed-human serum samples were tested by the newly developed particle agglutination assay system. The results were consistent with those from HIA, NTA and ELISA. CONCLUSION: The Ha-Ny beads can be applicable to the development of a new JEV antibody-detection kit, which does not require specific laboratory facilities.     

Development and Characterization of Microfluidic Devices and Systems for Magnetic Bead-Based Biochemical Detection

This paper presents the development and characterization of a generic microfluidic system for magnetic bead-based biochemical detection. Microfluidic and electrochemical detection devices such as microvalves, flow sensors, biofilters, and immunosensors have been successfully developed and individually characterized in this work. Magnetically driven microvalves, pulsed-mode microflow sensors, magnetic particle separators as biofilters, and electrochemical immunosensors have been sep-arately fabricated and tested. The fabricated microfluidic components have been surface-mounted on the microfluidic motherboard for fully integrated microfluidic biochemical detection system. A magnetic bio-bead approach has been adopted for both sampling and manipulating target biological molecules. Magnetic beads were used as both substrate of antibodies and carriers of target antigens for magnetic bead-based immunoassay, which was chosen as a proof-of-concept for the generic microfluidic bio-chemical detection system. The microfluidic and electrochemical immunosensing experiment results obtained from this work have shown that the biochemical sensing capability of the complete microfluidic subsystem is suitable for portable biochemical detection of bio-molecules. The methodology and system, which has been developed in this work, can be extended to generic bio-molecule detection and analysis systems by replacing antibody/antigen with appropriate bio receptors/reagents such as DNA fragments or oligonucleotides for application towards DNA analysis and/or high throughput protein analysis.     

Avian antigen binding cells: enrichment methods.

Data are presented comparing different methods for the fractionation and enrichment, respectively, of specific antigen binding lymphoid cells from immunized chickens. The bovine serum albumin (BSA) anti-BSA system was chosen as a model. To enrich avian antigen binding cells (ABC) from a mixture of chicken peripheral blood and spleen lymphocytes 3 different methods were used: (1) separation of cells forming rosettes with antigen-coated sheep red blood cells (SRBC) from non-rosetting cells by density centrifugation; (2) isolation of ABC by their specific adherence to antigen bound to immunoadsorptive surfaces (gelatin, plastics); (3) column affinity chromatography with antigen-coated agarose, cross-linked dextran for plastic beads. The most efficient method was column affinity chromatography with antigen-coated polyacrylamide beads which affords up to 12-fold enrichment of ABC. Both the other methods are also suitable for separation and enrichment of specific ABC but can only with difficulty be adapted for processing the large numbers of cells which would be necessary, e.g., for in vivo transfer studies.     

A novel enzyme-linked immunosorbent assay for diagnosis of Mycobacterium avium subsp. paratuberculosis infections (Johne's Disease) in cattle

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.     

Enzyme and radioimmunoassays for specific murine IgE and IgG with different solid-phase immunosorbents

Enzyme and radioimmunoassays of specific murine IgE and IgG were done using antigen-coated flex vinyl plates, antigen-coupled paper discs or Sepharose 4B beads as solid-phase immunosorbents. These 3 immunosorbents differ in their antibody binding capacities. Only Sepharose beads have a high capacity so that they can be used to assay specific IgE in serum samples without interference from specific IgG which can be present in concentrations of several thousand times greater than those of specific IgE. All 3 types of immunosorbents are equally useful for assaying specific IgG, but the flex vinyl plate is the immunosorbent of choice since it requires less time and gives lower blanks than do the other two. Under the conditions tried, the lowest detectable concentration of specific IgE or IgG was about 1 ng/ml on enzyme or radioimmunoassay.The immunoassays described in this paper are useful for following the kinetics of specific IgE and IgG responses in mice. This was demonstrated with sera from mice immunized with ragweed antigen E.     

Glycoproteins of natural origin with an affinity for hepatitis B surface antigen.

Sera from certain animal species contain a substance(s) which binds hepatitis B surface antigen. The hepatitis B binding substance found in animals is not antibody, but appears to be a glycoprotein which reacted with antigen-coated beads and produced a "false positive" test for antibody. This glycoprotein could be selectively and quantitatively removed by reaction with purified hepatitis B surface antigen and centrifugation. Pili fractions isolated from Neisseria gonorrhoeae and Escherichia coli bound to hepatitis B surface antigen and produced false positive anti-hepatitis B surface antigen reactions. Mouse anti-bovine hepatitis B binding substance and rabbit anti-E. coli pili were capable of neutralizing bovine hepatitis B binding substance.     

Modeling and Optimization of High-Sensitivity,Low-Volume Microfluidic-Based Surface Immunoassays

Microfluidics are emerging as a promising technology for miniaturizing biological assays for applications in diagnostics and research in life sciences because they enable the parallel analysis of multiple analytes with economy of samples and in short time. We have previously developed microfluidic networks for surface immunoassays where antibodies that are immobilized on one wall of a microchannel capture analytes flowing in the microchannel. This technology is capable of detecting analytes with picomolar sensitivity and from sub-microliter volume of sample within 45 min. This paper presents the theoretical modeling of these immunoassays where a finite difference algorithm is applied to delineate the role of the transport of analyte molecules in the microchannel (convection and diffusion), the kinetics of binding between the analyte and the capture antibodies, and the surface density of the capture antibody on the assay. The model shows that assays can be greatly optimized by varying the flow velocity of the solution of analyte in the microchannels. The model also shows how much the analyte-antibody binding constant and the surface density of the capture antibodies influence the performance of the assay. We then derive strategies to optimize assays toward maximal sensitivity, minimal sample volume requirement or fast performance, which we think will allow further development of microfluidic networks for immunoassay applications.     

Fluoro-immuno-cytoadherence (FICA): A new method for the identification and enumeration of antigen-binding cells.

Antigen-coated particles of cross-linked dextran may be used for affinity chromatography of antibodies and for the fractionation of lymphoid cells with appropriate surface receptors. Furthermore, such particles serve as convenient substrates for quantitative immunofluorescence tests. The fluoro-immuno-cyto-adherence (FICA) is a simple technique which combines affinity chromatography and immunofluorescence, provides durable antigen-coated substrates and allows the identification, enumeration and characterization of lymphoid cells capable of binding an antigen, covalently linked via a spacer onto the surface of dextran beads. In the present study chicken thyroglobulin (TG) or bovine serum albumin (BSA) were coupled onto fluorescein and rhodamin-labelled or unlabelled Sephadex G-25 beads by means of spacer molecules. The specificity and degree of antigen-coating were controlled by indirect immunoflourescence. For the study of antigen-binding cells the different antigen-coated beads were mixed with suspensions of peripheral blood lymphoid cells from Obese strain (OS) chickens with spontaneous hereditary autoimmune thyroiditis, or with cells from BSA-immunized or unimmunized normal White Leghron chickens. Specific adherence of OS lymphocytes to TG-coated beads and of lymphocytes from BSA-immunized chickens to BSA-beads was found. The test and control preparations are observed simultaneously under the fluorescence microscope where the distinction of beads coated with different antigens can be made on the basis of the color of their fluorescence. Results obtained with the FICA technique are in good agreement with those of conventional rosette tests.     

Quantitative measurement of anti-ErbB-2 antibody by flow cytometry and ELISA.

To establish standard methods for measuring anti-ErbB-2 antibody, a flow cytometric and an enzyme-linked immunosorbent assay (ELISA) were developed and compared. In the flow cytometric assay, the antibody was measured by binding to human breast cancer cell line SKBR3 and the result expressed as mean channel fluorescence (MCF). In ELISA, the antibody was measured by binding to a recombinant, secreted human ErbB-2 containing the N-terminal 505 amino acids of ErbB-2 fused to myc and His tags (secE2/myc/His or secE2) and the result expressed as O.D.(405). A mouse anti-human ErbB-2 mAb 9G6 was used as the standard. Using flow cytometry, MCF of 9G6 binding increased with linearity between 0.6 and 10 microg/ml. Using ELISA, O.D.(405) increased with linearity between 0.015 and 1 microg/ml, indicating greater sensitivity of ELISA. The titer of an immune mouse serum was determined to be 400 and 8000 with flow cytometric and ELISA assay, respectively, consistent with the assay sensitivity. Based on standard curves generated with 9G6, antibody concentration in the same serum sample was calculated to be approximately 230 microg/ml by ELISA and approximately 270 microg/ml by flow cytometry. Therefore, excellent concordance in antibody concentration was obtained with different assays using linear regression and properly diluted samples. This concordance was verified with multiple serum samples.     

Heart transplantation with donor-specific antibodies directed toward denatured HLA-A*02:01: a case report

The development of solid-phase assays for antibody detection has aided in the frequent detection of human leukocyte antigen (HLA) antibodies in nonalloimmunized males. Some scientists have reported that these HLA antibodies are produced to pathogens or allergens and the reactivity with HLA coated beads is the result of cross-reactive epitopes. These antibodies may also be directed toward cryptic epitopes exposed on the denatured beads. In this report, we describe the case of a heart transplanted patient who exhibited anti-HLA-A*02:01 donor-specific antibodies detected with a bead-based assay (Luminex) and undetected with the complement-dependent cytotoxicity (CDC) test. Posttransplant monitoring, carried out with CDC and with Luminex on sera from this patient collected at the 2nd, 4th, 8th, and 12th posttransplant weeks and at 1 year confirmed the presence of anti-HLA-A*02:01 in all serum samples. Additional tests carried out with denatured and intact HLA molecules using single antigen beads demonstrated that the antibody was directed toward a cryptic epitope. One year after transplantation the patient is doing well. No sign of antibody-mediated rejection was observed throughout the follow-up. A comprehensive evaluation of the anamnesis and of antibodies is critical to avoid needless exclusion of organ donors.     

Binding of von Willebrand factor to collagen by flow cytometry

We developed a new method for the detection of large von Willebrand factor (vWf) multimers binding to collagen and for the determination of vWf antigen (vWf:Ag) using flow cytometry. Collagen is coated on to polystyrene beads, allowing detection of found large vWf multimers. In addition, rabbit antibody against vWf is coated on to the beads allowing detection of all vWf:Ag. In plasma samples from healthy persons and patients (with type 1, 2A, 2N, or severe von Willebrand disease or hemophilia), 4 different assays were performed: vWf:Ag by immunoelectrophoresis; vWf ristocetin cofactor (vWf:RCof); CBA; and vWf:Ag based on an enzyme-linked immunosorbent assay using polystyrene beads. We assayed the flow cytometric method using 2 bead sizes. The optimal bead size was 3.136 microns. The results of CBA and vWf:Ag closely correlated with those of vWf:RCof and vWf:Ag (immunoelectrophoresis), respectively, and showed a low limit of detection. Interassay variance of cytometric methods was lower than interassay variance of traditional assays. In addition, we used the new assays to monitor desmopressin therapy.     

Development and evaluation of a recombinant-glycoprotein-based latex agglutination test for rabies virus antibody assessment

The measurement of neutralizing antibodies induced by the glycoprotein of rabies virus is indispensable for assessing the level of neutralizing antibodies in animals or humans. A rapid fluorescent focus inhibition test (RFFIT) has been approved by WHO and is the most widely used method to measure the virus-neutralizing antibody content in serum, but a rapid test system would be of great value to screen large numbers of serum samples. To develop and evaluate a latex agglutination test (LAT) for measuring rabies virus antibodies, a recombinant glycoprotein was expressed in an insect cell system and purified, and the protein was coated onto latex beads at concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg/ml to find out the optimal concentration for coating latex beads. It was found that 0.5 mg/ml of recombinant protein was optimal for coating latex beads, and this concentration was used to sensitize the latex beads for screening of dog serum samples. Grading of LAT results was done with standard reference serum with known antibody titers. A total of 228 serum samples were tested, out of which 145 samples were positive by both RFFIT and LAT, and the specificity was found to be 100 %. In RFFIT, 151 samples were positive, the sensitivity was found to be 96.03 %, and the accuracy/concordance was found to be 97.39 %. A rapid field test—a latex agglutination test (LAT)—was developed and evaluated for rabies virus antibody assessment using recombinant glycoprotein of rabies virus expressed in an insect cell system.     

Inter and intra laboratory concordance of HLA antibody results obtained by single antigen bead based assay

Single antigen bead based assays (SAB) to identify antibodies to HLA are marketed as a qualitative test, however often used as a quantitative test as the results provide mean fluorescence intensity (MFI) which is found to correlate with the strength/avidity of the antibody. We studied the between and within laboratory variability in performing the SAB from one manufacturer. Ten samples were tested at four laboratories according to the manufacturer’s suggested protocol. Additionally same samples were tested on four consecutive days in one laboratory. All tests were performed using the same lot of beads and secondary antibody. Results were classified as positive at four different MFI cutoffs: 1000, 5000, 8000 and 10,000. MFI values across and within the laboratory were compared in a pair-wise fashion with Pearson’s correlations. Overall concordance for Class-I was 97% between laboratories and 98% within laboratory at all cutoffs. Pair-wise Pearson correlation between laboratories was 0.989–0.99, while within laboratory it was 0.998–0.999. For Class-II, overall concordance between and within laboratory was 98%. Pair-wise Pearson correlation between laboratories was 0.991–0.997, while within laboratory it was 0.997–0.999. There is good correlation between laboratories and within laboratory using the same manufacturer, same lot and same protocol while performing SAB.     

Increase of ovalbumin (OVA)-specific B cells in the peripheral blood of egg-allergic patients

Allergen-specific B cells were detected by antigen-coated magnetic beads. The frequency of ovalbumin (OVA)-specific B cells was significantly higher in patients with egg white-allergy than in age-matched nonallergic individuals. These B cells, isolated by the immunomagnetic-beads method, produced anti-OVA antibodies of mostly IgM class when they were transformed by Epstein-Barr virus and cultured for about 2 weeks. Based on a chronologic analysis, the increase of OVA-specific B cells was found to precede the increase of IgG and IgE anti-OVA antibodies in the serum. These observations indicated that OVA-binding B cells in the peripheral blood are already committed to producing IgM antibody and probably are the precursors of antibody-forming cells of the IgG or IgE class.     

A Standardized Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Antibodies to Toxoplasma Gondii

An enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Toxoplasma gondii infections on single serum dilutions was developed.

This test system is a standardized kit designed to detect circulating specific antibodies to Toxoplasma gondii in human sera. It consists of Toxoplasma gondii soluble antigen-coated microtitration multiwell plates, specific immunoglobulin-enzyme conjugate and other required reagents.

In a clinical trial performed on sera from 1,035 clinically suspected toxoplasmosis cases, the Sabin Feldman Dye Test (SFDT) and this ELISA system agreed closely. Relative to the SFDT, the sensitivity and specificity of the latter was 98.0% and 97.6% respectively with a correlation coefficient of 0.97. In a further study of 121 sera, the Indirect Fluorescent Antibody Test (IFAT), the Indirect Haemagglutination Test (IHAT) and this ELISA procedure showed over 90% agreement, with correlation coefficients of 0.98 and 0.95 respectively. Within the working concentration of specific antibody to T. gondii in human serum, there was a linear relationship between the ELISA values and the WHO international standard for human anti-Toxoplasma serum.     

Quantification and characterisation of IgG binding to mould spores by flow cytometry and scanning electron microscopy

The concentration of mould-specific IgG antibodies in serum may objectively indicate mould exposure and can help identifying exposed individuals. Although inhaled spores probably are the most important source of mould exposure, the commonly used methods for detecting mould-specific IgG antibodies are based on extracts from all mould components, with only low contribution from spores. We have developed a flow cytometric method using surface antigens on mould spores for quantifying mould-specific IgG antibodies in serum. Flow cytometric results were evaluated by comparison with ImmunoCap and ELISA measurements. The flow cytometric assay showed a broad linear dose-dependency and correlated moderately to strongly (r=0.41-0.97) with ImmunoCap and ELISA measurements. The IgG antibody binding was studied in detail by immunolabelling in scanning electron microscopy (SEM), revealing that morphology and IgG antibody binding differed among spores, both within and between mould strains. Germination studies by flow cytometry and SEM showed that IgG antibody binding to mould spores was altered during germination due to loss of coat. The present spore based antibody assay are simple and suitable for quantification of mould-specific IgG antibodies in serum, and includes specificity to other and possibly more relevant antigens than existing methods.     

The interaction of rheumatoid factor with hepatitis B surface antigen-antibody complexes.

A solid phase radioimmunoassay was developed in which the hepatitis B surface antigen was adsorbed to the surface of plastic beads. When the antigen-coated beads were incubated with human IgG antibody against hepatitis B surface antigen, immune complexes were formed on the solid phase surface. IgM rheumatoid factor was found to bind to the hepatitis B surface antigen-antibody complexes but not to the antigen or the IgG antibody alone. Since both hepatitis B surface antigen-antibody complexes and rheumatoid factor are commonly present in type B viral hepatitis, it is suggested that rheumatoid factor may play a role in the pathogenesis of this viral disease in man.     

Isoniazid in patient plasma may cause a false-positive result on the complement-dependent cytotoxicity test

Correct definition of clinically relevant anti-HLA antibodies is important for transplant organ allocation and outcome. We describe a candidate for kidney transplantation who was treated with isoniazid because of active tuberculosis. The patient's serum gave a positive antibody result on screening with the complement-dependent cytotoxicity (CDC) test but a negative result on screening with a bead-based assay (Luminex). The clinical history indicated no immunologic stimuli. Subsequent testing on fresh serum samples confirmed the discrepancy between CDC and Luminex results. An autologous cross-match test gave negative results, and the antibodies were sensitive to dithiothreitol treatment. We postulated that nonspecific binding of drug–antibody complexes to panel lymphocytes in the CDC test may have caused the observed lympholysis. This case, although isolated, emphasizes the importance of the combined use of CDC and solid phase assays. The CDC results alone would have led to the erroneous conclusion that the patient was highly sensitized.