Fluorescence Polarization Assay for Detection of Brucella abortus Antibodies in Bulk Tank Bovine Milk Samples

A simple, rapid, inexpensive fluorescence polarization assay for the detection of antibodies to Brucella abortus in bulk tank milk samples at the farm level or at dairies with a sensitivity and specificity of 100 and 95.9%, respectively, is described. The assay detects antibodies to B. abortus in 15 min by testing undiluted whey produced by chemical and physical manipulation of milk from bulk tanks. This sampling is noninvasive and therefore costs less and is less stressful than blood-based tests. The assay is specific and can detect antibodies at levels below that of the indirect enzyme immunoassay for milk and the fluorescence polarization assay for individual milk samples. Use of this test would make programs for surveillance of dairy animals and eradication of B. abortus more cost-effective.     

Clinical and Diagnostic Developments of a Gamma Interferon Release Assay for Use in Bovine Tuberculosis Control Programs

Currently, the Bovigam assay is used as an official supplemental test within bovine tuberculosis control programs. The objectives of the present study were to evaluate two Mycobacterium bovis-specific peptide cocktails and purified protein derivatives (PPDs) from two sources, liquid and lyophilized antigen preparations. PPDs and peptide cocktails were also used for comparison of a second-generation gamma interferon (IFN-γ) release assay kit with the currently licensed first-generation kit (Bovigam; Prionics AG). Three strains of M. bovis were used for experimental challenge: M. bovis 95-1315, M. bovis Ravenel, and M. bovis 10-7428. Additionally, samples from a tuberculosis-affected herd (i.e., naturally infected) were evaluated. Robust responses to both peptide cocktails, HP (PC-HP) and ESAT-6/CFP10 (PC-EC), and the PPDs were elicited as early as 3 weeks after challenge. Only minor differences in responses to Commonwealth Serum Laboratories (CSL) and Lelystad PPDs were detected with samples from experimentally infected animals. For instance, responses to Lelystad M. avium-derived PPD (PPDa) exceeded the respective responses to the CSL PPDa in M. bovis Ravenel-infected and control animals. However, a 1:4 dilution of stimulated plasma demonstrated greater separation of PPDb from PPDa responses (i.e., PPDb minus PPDa) with the use of Lelystad PPDs, suggesting that Lelystad PPDs provide greater diagnostic sensitivity than CSL PPDs. The responses to lyophilized and liquid antigen preparations did not differ. Responses detected with first- and second-generation IFN-γ release assay kits (Bovigam) did not differ throughout the study. In conclusion, antigens may be stored in a lyophilized state without loss in potency, PC-HP and PC-EC are dependable biomarkers for aiding in the detection of bovine tuberculosis, and second-generation Bovigam kits are comparable to currently used kits.     

Time-Resolved Fluorescent Resonance Energy Transfer Assay for Simple and Rapid Detection of Anti-Brucella Antibodies in Ruminant Serum Samples

Brucellosis is a globally significant zoonosis, the control of which is difficult and resource intensive. Serological tests form a vital part of a multifactorial approach to control and are often performed in large numbers. The aim of the present study was to develop a new assay to improve the efficiency, ease, and effectiveness of serological testing. An existing competitive enzyme-linked immunosorbent assay (cELISA) was adapted to a completely homogeneous time-resolved fluorescent resonance energy transfer (TR-FRET) assay. This was achieved by labeling an anti-Brucella monoclonal antibody with a long-lifetime donor fluorophore and Brucella smooth lipopolysaccharide with a compatible acceptor and optimizing the reading conditions. The assay was performed in a 96-well plate with a single 30-min incubation period and no separation (wash) steps and was concluded by a single plate-reading step. The performance of the assay was evaluated with a panel of serum samples from infected (n = 73) and uninfected (n = 480) sources and compared to the performance of the parent cELISA, an indirect ELISA (iELISA), and fluorescence polarization assay (FPA). The performance of the TR-FRET assay matched the performance of the iELISA, which had 100% diagnostic sensitivity and specificity, and surpassed the performance of the cELISA and the FPA. The results also demonstrated that the TR-FRET technique is effective with poor-quality serum samples from the field. To the knowledge of the authors, this is the first homogeneous TR-FRET assay to detect antibodies raised against an infectious disease. The technique appears to be sufficiently adaptable to meet the needs of many other similar testing requirements to identify infectious diseases.Brucellosis is a zoonosis of widespread distribution and significance caused by species of the genus Brucella. The disease is known to be especially prevalent in the Middle East and Mediterranean Basin (33), and there are disturbing signs of its reemergence across large areas of the globe, especially in central Asia. Few countries have successfully managed to eradicate the disease. In many of these countries, although the livestock sector is disease free, a significant wildlife reservoir remains and presents a risk of reintroduction (11).The principle etiologic agents of brucellosis are the classical smooth species Brucella abortus, B. melitensis, and B. suis. These species all have smooth lipopolysaccharide (sLPS), which is a major virulence factor (17, 35). Each of these species has a preferred host; however, many animals, including humans, are susceptible to each of the classical species, although the underlying mechanisms for host preference are not clearly understood (5). Brucellosis in ruminants is mainly manifested by reproductive failure due to abortion. There are few other clinical signs, and this causes difficulties with diagnosis. In humans, the disease is mainly presented as an undulating febrile condition, although there may be other, more serious complications. The symptoms of human brucellosis are particularly nonspecific, and this again presents serious diagnostic complications (2).There are many aspects to the effective control of the disease in both the human and the animal populations, including educational programs, effective animal tracing, vaccination of animals, and intersectorial cooperation. Within and, indeed, prior to the implementation of a holistic control program, establishing the prevalence of disease and identifying infected animals are crucial. Owing to a lack of specific symptoms, the most effective means of doing this is through serological testing, followed by, if possible, the isolation of Brucella from serologically positive animals. In areas where the disease has been eradicated, a surveillance system is vital in order to maintain freedom. Once again, serology also plays a vital role in this.The Organization International des Epizooties (OIE) prescribed and alternative serological tests for the diagnosis of brucellosis due to infection with smooth strains largely rely upon the detection of antibodies to the O antigen of sLPS (10, 32). The classical tests include the Rose Bengal test, the complement fixation test (CFT), and the serum agglutination test (SAT), all of which employ a whole-cell antigen as the key diagnostic reagent. More recently developed techniques, such as the indirect enzyme-linked immunosorbent assay (iELISA), competitive ELISA (cELISA), and the fluorescence polarization assay (FPA), use purified sLPS or O antigen. The immunodominance of the sLPS O antigen is the basis for the generally good sensitivity of these assays. The use of these antigens can lead to false-positive serological test results when animals are infected with bacteria possessing O antigens with a structure similar to that of the O antigen of Brucella species (7), such as Yersinia enterocolitica O:9. Owing to the widespread use of the S19 and Rev 1 vaccines, such tests also fail to reliably differentiate between vaccinated and infected animals.In all effective brucellosis control scenarios, the number of samples tested is high, and therefore, optimizing the efficiency of the testing regimen is critical to limit costs. ELISAs are readily amenable to high-throughput testing due to the standardized nature of the technology and reagents. This allows for many efficiency savings, including the introduction of automation (20). Although ELISAs have advantages over classical tests in this regard, they still require several steps to be completed, including separation (wash) steps. Although these steps can be automated, they are a vital part of the assay yet present a frequent source of imprecision, error, mechanical breakdown, and additional cost. Assays which have the advantages of the ELISA, such as assays that use a 96-well format, and that have an objective means of assessment of the results and good sensitivities and specificities but that reduce the burden of work and opportunity for error are clearly desirable.The aim of the project described here was to improve the efficiency of serological testing by developing a homogeneous homologue of the Brucella cELISA (from the Veterinary Laboratories Agency, Weybridge, United Kingdom) by using the principles of time-resolved fluorescent resonance energy transfer (TR-FRET). FRET occurs when two fluorophores (a donor and an acceptor) with the appropriate spectral properties transfer energy between them if they are within sufficient proximity to each other (9). The degree to which complementary antigens and antibodies have bound (and are therefore within close proximity) can be detected by labeling each with an appropriate fluorophore and measuring the amount of energy transfer produced after the initial excitation of the donor.Use of a donor fluorophore with a long fluorescent lifetime enables the specific transfer of energy to persist long after the nonspecific background fluorescence, due to the initial excitation, has ceased. Initiating fluorescence intensity (FI) measurements after the background fluorescence has reduced improves the sensitivity of the FRET technique (26). The fluorescence of both the donor and the acceptor fluorophores can be measured, and this endows the method with additional resistance to variable fluorescent effects due to sample matrices, such as serum.The introduction of competing agents, such as specific antigens or serum antibodies, that alter the degree of binding between the labeled reagents will be manifested as a change in the donor and the acceptor intensities. This process can be conducted with no solid-phase reagents or wash steps and with only a single incubation and a single read step.     

Immunofluorescence Assay for Detection of Human Metapneumovirus-Specific Antibodies by Use of Baculovirus-Expressed Fusion Protein

Human metapneumovirus (hMPV) has recently been identified as an etiological agent of acute respiratory infections. The hMPV fusion (F) protein has been indicated to be a major antigenic determinant that mediates effective neutralization and protection against hMPV infection. We developed a new immunofluorescence assay (IFA) using Trichoplusia ni (Tn5) insect cells infected with a recombinant baculovirus-expressing hMPV F protein (Bac-F IFA). A total of 200 serum samples from Japanese people 1 month to 41 years old were tested for immunoglobulin G antibodies to hMPV F protein by Bac-F IFA. The results were compared with those of the conventional IFA based on hMPV-infected LLC-MK2 cells (hMPV IFA). The titers obtained by the two IFAs correlated well (correlation coefficient of 0.88), and the concordance of seroreactivities between the two IFAs was 91% (κ = 0.76). For 192 of the 200 serum samples, the titers obtained by the Bac-F IFA were equal to or higher than those obtained by the hMPV IFA. These results indicated that the Bac-F IFA was more sensitive than the hMPV IFA and that the majority of the antibodies detected by the hMPV IFA reacted with the hMPV F protein. The Bac-F IFA is a more reliable, sensitive, and specific method for the detection of hMPV antibodies than is the hMPV IFA.     

Assessment of Imprecision in Gamma Interferon Release Assays for the Detection of Exposure to Mycobacterium tuberculosis

New gamma interferon (IFN-γ) release assays (IGRAs) to detect an exposure to Mycobacterium tuberculosis have recently been launched. The majority of the studies in temperate-climate countries agree that these methods have superior specificity and equal or even superior sensitivity over tuberculin skin tests (TSTs) in the diagnosis of latent tuberculosis (TB) infection (LTBI). However, reproducibility data of IGRAs are virtually missing. We assessed within-run, between-run, and total imprecision of two commercial IGRAs by testing samples from subjects with a stable state of TB infection or treated pulmonary TB, a sample from a healthy volunteer, and internal quality control samples. We calculated coefficients of variance (CV%s) to describe assays variability and compared the obtained results to the reported CV%s for other commercial immunodiagnostic methods. We illustrate an example of assay variability near the cutoff zone to demonstrate the necessity of a gray zone. Due to the strict adherence to the standard operation procedures (SOP) adopted in our laboratory, the total imprecision of enzyme-linked immunospot (ELISPOT)- and enzyme immunoassay (EIA)-based IGRAs was at a maximum CV% of 37.8% for the samples with moderate and high reactivities. Imprecision of testing samples with very low reactivity levels or nonreactive samples may, however, exceed 100%. In conclusion, despite multiple steps of the method performance, the analytical imprecision of IGRAs, which in our study design included also between-lot variability and had a component of normal biological variation, was well in accordance with the reported imprecisions of other manual immunodiagnostic tests. The recognition of the variability around the cutoff point advocates the use of a gray zone to avoid ambiguous result interpretations.Evaluation of immunometric tests for infectious diseases abides by the same rules as methods for clinical chemistry and is based on the same general principles (12). Evaluation of analytical performance includes, among other parameters, reproducibility data. As a prerequisite for CE mark registration to get a license to market the products for in vitro diagnostic use, manufacturers should provide reproducibility characteristics as a part of the overall performance data.Two new kits, namely, the T-SPOT.TB (Oxford Immunotec, Oxford, United Kingdom) (8) and QuantiFERON-TB Gold In-Tube (Cellestis Limited, Carnegie, Victoria, Australia) (www.cellestis.com/IRM/content/aust/qtfproducts_tbgoldintube_techinfo-perfparameters.html) kits, have been recently launched. The kits utilize the ability of sensitized CD8⁺ and CD4⁺ T lymphocytes to release gamma interferon (IFN-γ) when stimulated with synthetic peptides specific to Mycobacterium tuberculosis and detect exposure to Mycobacterium tuberculosis. While the first method measures the frequency of reactive lymphocytes in the peripheral blood mononuclear cell (PBMC) fraction, the latter measures the concentration of released IFN-γ into supernatants. These methods are collectively called IFN-γ release assays (IGRA). Although these were launched for diagnostics, clinically relevant information on assay reproducibility was available from only one test series (www.cellestis.com/IRM/content/aust/qtfproducts_tbgoldintube_techinfo-perfparameters.html) as of May 2009. From the literature search, we have found only a few publications that are related to this topic (4, 9, 14), whereas test sensitivity and specificity have been extensively tested and reviewed in recent meta-analyses (7, 10). The importance of the reproducibility parameter is emphasized by the demand to assess immunological conversions and reversions, in other words, a significant decrease in immunological responses that exceeds the total imprecision of the method. The clinical phenomenon of immunological reversion was reported to associate with, e.g., successful chemotherapy (2). A spontaneous reversion, which is a phenomenon that is not yet well understood, may be, indeed, a very important observation meaning pathogen clearance. However, the decrease in the response should be well documented and should clearly exceed the method''s total imprecision. Immunological conversion may mean a rise in the reactivity that is above the variation of technical and biological noise. Because the data on reproducibility are scarce, we assumed that ethical considerations may have constituted the major obstacle. Indeed, it may be difficult to obtain an ethical permission to collect blood samples consecutively from tuberculosis (TB) patients who may need urgent treatment.Both ethical considerations and unstable sample material make imprecision study of IGRAs difficult. In fact, IGRAs were the first diagnostic methods to exploit cell-mediated immunity and utilize the ex vivo activity of vital lymphocytes. For example, another recently introduced CE mark-registered test to evaluate the effect of immunosuppression on the function of lymphocytes (ImmuKnow; Cylex, Columbia, MD) provides only repeatability data (results obtained in different laboratories from the same venipuncture). The information on the between-run imprecision was not yet available in the kit instructions as of January 2009. The shortage of information reflects obvious practical problems in obtaining samples from critically ill subjects to study between-run imprecision as required.Imprecision data represent a very important parameter when the cutoff point and the width of the gray zone should be considered. Surprisingly, the interpretation guides for the results in both IGRA kit inserts do not discuss the topic of analytical uncertainty, i.e., the variation of a positive response around the cutoff point. Based on our pilot results, we have suggested earlier the use of a gray zone (11). This concept has been introduced recently in only one method (8), albeit without reference to the method imprecision.The aims of this study were (i) to provide an assessment of the total imprecisions of both IGRAs, (ii) to assess between-run imprecisions of both methods by using internal quality control (QC) samples, and (iii) to demonstrate an example from our daily routine for the need of a cautious interpretation of the result falling on a single cutoff point (per manufacturers'' instructions).     

Competitive Electrochemiluminescence Wash and No-Wash Immunoassays for Detection of Serum Antibodies to Smooth Brucella Strains

Brucellosis is a bacterial zoonotic disease of major global importance. Natural hosts for Brucella species include animals of economic significance, such as cattle and small ruminants. Controlling brucellosis in natural hosts by high-throughput serological testing followed by the slaughter of seropositive animals helps to prevent disease transmission. This study aimed to convert an existing competitive enzyme-linked immunosorbent assay (cELISA), used for the serodiagnosis of brucellosis in ruminants, to two electrochemiluminescence (ECL) immunoassays on the Meso Scale Discovery (MSD) platform. The first assay employed a conventional plate washing step as part of the protocol. The second was a no-wash assay, made possible by the proximity-based nature of ECL signal generation by the MSD platform. Both ECL wash and no-wash assays closely matched the parent cELISA for diagnostic sensitivity and specificity. The results also demonstrated that both ECL assays met World Organization for Animal Health (OIE) standards, as defined by results for the OIE standard serum (OIEELISA_SPSS). This report is the first to describe an ECL assay incorporating lipopolysaccharide, an ECL assay for serodiagnosis of a bacterial infectious disease, a separation-free (no-wash) ECL assay for the detection of serum antibodies, and the use of the MSD platform for serodiagnosis. The simple conversion of the cELISA to the MSD platform suggests that many other serodiagnostic tests could readily be converted. Furthermore, the alignment of these results with the multiplex capability of the MSD platform offers the potential of no-wash multiplex assays to screen for several diseases.Species of the genus Brucella cause serious chronic infections, collectively known as brucellosis. Brucellosis is a mammalian disease infecting many economically important animal species as well as humans. With a global distribution, brucellosis causes considerable animal and human health problems as well as huge economic costs. Brucella species are gram-negative, nonmotile, facultative intracellular coccobaccilli belonging to the α-2 subdivision of proteobacteria. The genus consists of six classical species, namely, Brucella abortus, B. melitensis, B. suis, B. ovis, B. canis, and B. neotomae, plus more recently discovered strains from marine mammals. Of the Brucella species, B. abortus, B. melitensis, and B. suis are of principal human health and economic importance. These species have smooth lipopolysaccharide (sLPS), which is considered a major virulence factor of disease (23), whereas B. ovis and B. canis have rough LPS (1).The World Organization for Animal Health (OIE) prescribed and alternative serological tests for diagnosis of brucellosis caused by smooth strains rely largely upon the measurement of the host antibody response to the O antigen of the sLPS (8, 22). Classical tests include the Rose Bengal test, the complement fixation test (CFT), and the serum agglutination test (SAT), all of which employ a whole-cell antigen as the key diagnostic reagent. More contemporary techniques, such as indirect enzyme-linked immunosorbent assay (iELISA), competitive ELISA (cELISA), and fluorescent polarization assay (FPA), employ purified LPS or O antigen as the diagnostic reagent. The immunodominance of the LPS O antigen is the basis for the generally excellent sensitivity of these assays (21). However, the use of this antigen can lead to false-positive serological results when animals are infected with bacteria possessing O antigens of similar structure (6), such as Yersinia enterocolitica O:9.ELISAs are readily amenable to high-throughput testing due to the standardized nature of the technology and reagents. This allows for many efficiency savings compared to the classical assays, including the use of effective automation (16). Despite the advantages of ELISA over the classical tests in this regard, ELISAs still require several steps to complete, including separation steps. Although these steps can be automated, they are a vital part of the assay and are a frequent source of imprecision, error, and mechanical breakdown. Assays which have the advantages of ELISA, such as a 96-well format, objective assessment, and good sensitivity and specificity, but which reduce the burden of work and opportunity for error are clearly desirable.The Meso Scale Discovery (MSD) electrochemiluminescence (ECL) platform uses electrochemical stimulation of reporter molecules conjugated to biological components to generate a light signal measured by photodetectors (2, 31), such as a charge-coupled device (CCD) camera. Carbon electrodes are integrated into the bottom of 96-well microtiter plates, to which biological components (for example, LPS) from traditional assays such as ELISA can be passively adsorbed. Biological conjugates, for example, monoclonal antibodies (MAbs), can be labeled with the reporter molecule ruthenium(II) tris-bipyridal [Ru(bpy)₃²⁺], which upon electrical stimulation emits light at 620 nm if it is within sufficient proximity to the carbon electrode excitation source. The reaction is enhanced by the addition of read buffer, which contains coreactants, including tripropylamine. Nonspecific signals are minimized as the stimulation mechanism (electricity) is decoupled from the signal (light).MSD ECL assays have the potential for separation-based (“wash”) and non-separation-based (“no-wash”) immunoassays due to the proximity-based nature of signal generation, where only labels near the surfaces of the electrodes are stimulated. The non-separation-based approach allows the addition of read buffer (MSD) directly to the components of the assay; this has an advantage over separation-based assays in that it removes a source of variation and reduces the time and labor required to perform the assay. In addition, MSD ECL assays can be multiplexed by spotting up to 10 different antigens onto discrete areas of the carbon electrode within each well of a 96-well plate; thus, the potential exists for multiplexed no-wash serological assays. The signal from each spot can be detected independently by a CCD camera, providing a quantitative measurement of the amount of light detected. Furthermore, the time taken to read a 96-well plate, approximately 70 s, is comparable to that for ELISAs and compares favorably to those for some other multiplex and ECL systems.We assessed the feasibility and potential of wash and no-wash MSD ECL assays for veterinary serodiagnosis of brucellosis. Biological components (B. melitensis 16 M sLPS antigen and BM40 [10], an anti-M O-chain epitope MAb) from the brucellosis cELISA produced by the Veterinary Laboratories Agency (26) were applied to the MSD platform. As with the parent cELISA, the wash and no-wash ECL assays allow competition between serum antibodies and the MAb BM40 [labeled with Ru(bpy)₃²⁺ for the MSD ECL assay] for sLPS bound to the carbon electrode. This affects the quantity of BM40 able to bind to this target. Thus, a positive result yields a low-intensity signal and a negative result yields a high-intensity signal. The new assays were validated against existing serological methods, using sera from Brucella-infected and noninfected ruminants.     

Antibodies to Spermatozoa: XI. The Use of Immunobeads for the Detection of Sperm Antibodies in Serum

ABSTRACT: Two procedures were developed and evaluated that used either larger or smaller volumes for the detection of sperm antibodies in serum by means of an indirect immunobead test (IBT). The immunobeads, coated with rabbit antibody to each of the major human immunoglobulins (IgG, IgA, or IgM), were mixed with preparations of donor sperm, onto which antibody had been coated by passive transfer from various serum samples. The results of the IBT could be evaluated in various ways: (1) positive or negative; (2) if positive, whether binding is to the tail, the head, or the head and tail of the sperm cells; (3) if positive, whether binding is by IgG, IgA, or IgM. The diverse IBT results were obtained from a group of 50 serum samples; these sera were also tested by two sperm agglutination methods; the gelatin agglutination test (GAT) and the tube-slide agglutination test (TSAT). There was an excellent agreement between the IBT and the GAT; it was not as good between the IBT and the TSAT. However, considering both agglutination methods together, 90% of the IBT-positive sera were agglutination-positive. In terms of morphological sites, tail binding occurred in 27 of 31 sera, head binding in 12 of 31 sera, and head-tail binding in 15 of 31 sera. The number with tail binding was very close to the number that were GAT-positive (26). As for the immunoglobulins, the most frequent class was IgG. IgA was 83% as frequent and IgM was only 25% as frequent as IgG. In a larger group with only IgG and IgA, of 31 IBT-positive sera, 26 showed IgG and 23 showed IgA; 18 showed both. Hence, only eight showed IgG exclusively, and only five showed IgA exclusively. One final point is that several sera with GAT titers of only 4 were IBT-positive, adding strength to the concept that such a low GAT titer does have antibody significance.     

Evaluation of Newly Developed Immunoperoxidase Monolayer Assays for Detection of Antibodies against Bovine Herpesvirus 4

This study describes the evaluation of immunoperoxidase monolayer assays (IPMAs) for detection of antibodies against bovine herpesvirus 4 (BHV4) DN-599 or BHV4 LVR 140 in sera of cattle. We compared the quality of these IPMAs with the quality of a BHV4 indirect enzyme-linked immunosorbent assay (ELISA). In addition, a preliminary serological survey of BHV4 antibodies was carried out to estimate the seroprevalence of BHV4 in Dutch cattle at different ages. The specificities of both BHV4 IPMAs were 1.00. The geometrical mean titers (detection limit) of the BHV4 IPMAs were twice as high as that of the BHV4 indirect ELISA. In experimentally infected cattle, BHV4 antibodies were detectable by IPMAs 16 to 18 days postinfection, which was almost 2 weeks earlier than in the indirect ELISA. The reproducibility of the BHV4 DN-599 IPMA (_κD value, 0.92) and of the BHV4 LVR 140 IPMA (_κD value, 0.87) were good. For field sera the overall agreement between the BHV4 indirect ELISA and the two BHV4 IPMAs, DN-599 and LVR 140, was 95 and 96%, respectively. The serological-survey study showed that the estimated seroprevalence of BHV4 in Dutch cattle was 16 to 18% and that the percentage of BHV4-positive animals varied by age category (between 6 and 43%). In summary, the two BHV4 IPMA formats have several advantages that make IPMA a useful alternative to the BHV4 indirect ELISA for detecting BHV4 antibodies in cattle.     

血清IL-33、IFN-γ与IgE在支气管哮喘患者中的表达及临床意义

目的观察血清白细胞介素-33(IL-33)、干扰素-γ(IFN-γ)及IgE在支气管哮喘患者中的表达及意义。方法选取94例急性发作期支气管哮喘患者,同时选取同期60例健康体检的人群为对照组,采用酶联免疫法测定血清中IgE、IL-33及IFN-γ水平。比较两组IgE、IL-33和IFN-γ的水平。分析IL-33、IFN-γ和IgE的相关性。结果支气管哮喘患者外周血中IFN-γ水平低于正常人群(t=4.533,P<0.001);IL-33、IgE水平高于正常人群(t=5.831、66.129,P<0.001,<0.001),差异有统计学意义。IgE水平与IL-33水平呈正相关(r=0.667,P=0.032),IFN-γ与IgE、IL-33呈负相关(r=-0.714,P=0.024;r=-0.623,P=0.038)。结论血IgE、IL-33和IFN-γ水平的变化在支气管哮喘患者发病过程中起到一定的作用。     

Development of a Human Gamma Interferon Enzyme Immunoassay and Comparison with Tuberculin Skin Testing for Detection of Mycobacterium tuberculosis Infection

A sensitive two-step simultaneous enzyme immunoassay (EIA) for human gamma interferon (IFN-γ) has been developed and used as an in vitro test for human tuberculosis (TB) in comparison with tuberculin skin testing. The EIA was shown to be highly sensitive, detecting less than 0.5 IU of recombinant human IFN-γ per ml within a linear detection range of 0.5 to 150 IU/ml. The assay was highly reproducible and specific for native IFN-γ. In addition, the assay detected chimpanzee, orangutan, gibbon, and squirrel monkey IFN-γs. Cross-reactions with other human cytokines or with IFN-γs derived from mice, cattle, or Old World monkeys were not evident. The assay was used to detect TB infection by incubating whole blood overnight with human, avian, and bovine tuberculin purified protein derivatives (PPDs), as well as positive (mitogen)- and negative-control preparations. The levels of IFN-γ in plasma supernatants were then determined. Blood from 10 tuberculin skin test-positive individuals responded predominantly to the human tuberculin PPD antigen and to a lesser extent to bovine and avian PPD antigens. By contrast, blood from 10 skin test-negative individuals showed minimal responses or no response to any of the tuberculin PPDs. Detectable levels of IFN-γ were present in all blood samples stimulated with mitogen. In vivo tuberculin reactivity was correlated with IFN-γ responsiveness in vitro. These results support the further study of the blood culture–IFN-γ EIA system as an alternative to skin testing for the detection of human TB infection.     

Use of Monoclonal Antibodies for the Detection of HLA and DR Antigens on Spermatozoa of Different Species

ABSTRACT: There are conflicting reports about the presence of HLA and DR antigens on human sperm. The difference may be due to the complexity of anti-sera used by earlier researchers. These limitations have been overcome by using monoclonal antibodies in the present studies. The microcytotoxicity test was used to determine the presence of HLA and DR antigens on sperm of different species. It was observed that about 60% of human spermatozoa express HLA antigens and a slightly lower percentage (48%) express DR antigens. These antigens are highly cross-reactive with that of monkey and, to a lesser extent, with that of buffalo. Histocompatibility antigens expressed on epididymal spermatozoa of rat and mouse are also weakly reactive with monoclonal antibodies directed to human HLA and DR.     

Expression and Identification of Immunological Activities of the HIV‐gp120N‐Human Interferon Gamma Fusion Protein

The human immunodeficiency virus type 1 (HIV‐1) envelope glycoprotein gp120 is a vaccine immunogen that has been studied extensively. To enhance the immune response of cells against HIV‐1 gp120, we tested the coexpression of gp120N with interferon‐γ (IFN‐γ) as an immune adjuvant. Two recombinant prokaryotic plasmids were constructed: the pET44b‐HIV‐1‐gp120N plasmid construct carried the HIV‐1 gp120N gene (pET44‐gp120N), whereas the pET44b‐HIV‐1‐gp120N‐IFN‐γ plasmid construct carried a fusion gp120N‐IFN‐γ gene (pET44b‐gp120N‐IFN‐γ). Target protein expression was achieved in E. coli BL21 (DE3) cells by chemical induction. To test the immunological activity of the proteins, mice were injected with a control, gp120N, or the fusion gp120N‐IFN‐γ protein. The serum and spleen cells of the mice were collected for immunological detection. Results showed that specific T lymphocyte proliferation and the expression of the Th1‐type cytokines (IL‐2 and IFN‐γ) were higher in the gp120N‐IFN‐γ group than the other two groups (P < 0.05). No difference was observed in the expression levels of the Th2‐type cytokines (IL‐4 and IL‐10; P > 0.05). These results suggest that IFN‐γ plays a prominent role as an immune adjuvant when coexpressed with HIV‐1 gp120N. IFN‐γ enhances the specific cell immune response of mice against HIV‐1 gp120. Anat Rec, 292:381–386, 2009. © 2009 Wiley‐Liss, Inc.     

Development of Human-Murine Chimeric Immunoglobulin G for Use in the Serological Detection of Human Flavivirus and Alphavirus Antibodies

Diagnosis of human arboviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the indirect IgG ELISA. Broad application of these assays is hindered by the lack of standardized positive human control sera that react with a wide variety of flaviviruses (e.g., dengue, West Nile, yellow fever, Japanese encephalitis, Saint Louis encephalitis, and Powassan viruses), or alphaviruses (e.g., Eastern equine encephalitis, Western equine encephalitis, Venezuelan equine encephalitis, and chikungunya viruses) that can cause human disease. We have created human-murine chimeric monoclonal antibodies (cMAbs) by combining the variable regions of flavivirus (6B6C-1) or alphavirus (1A4B-6) broadly cross-reactive murine MAbs (mMAbs) with the constant region of human IgG1. These cMAbs may be used as standardized reagents capable of replacing human infection-immune-positive control sera in indirect IgG ELISA for diagnosis of all human flaviviral or alphaviral infections. The IgG cMAbs secreted from plasmid-transformed Sp2/0-Ag14 cells had serological activity identical to that of the parent mMAbs, as measured by ELISA using multiple flaviviruses or alphaviruses.Arthropod-borne viruses (arboviruses) are responsible for a number of medically important human diseases. These viruses are maintained in nature through biological transmission between susceptible vertebrate hosts by blood-feeding arthropods, primarily mosquitoes and ticks. Although over 150 arboviruses are known to cause disease in humans, the majority of medically important arboviruses are found in three separate families, the Flaviviridae, the Togaviridae (genus Alphavirus), and the Bunyaviridae (24). Transmission of arboviruses can vary by season, a consequence of the feeding patterns of their respective arthropod vectors, as well as by specific geographic location, as is seen for dengue fever virus (DENV) and Japanese encephalitis virus (JEV) (20, 24). The primary clinical manifestation of arboviral disease in North America is encephalitis, although some arboviruses, such as yellow fever virus (YFV) are capable of causing severe hemorrhagic disease as well. Prior to the 1999 outbreak of West Nile virus (WNV) encephalitis in New York City, St. Louis encephalitis virus (SLEV) was the most important agent of epidemic viral encephalitis in North America, last causing a major epidemic in the mid-1970s (26, 28, 33). Since 1999, the distribution of WNV has rapidly expanded from New York to the rest of the United States and into Canada, Central America, and South America. As of April 2009, a total of 29,598 human WNV cases in the United States had been reported to the Centers for Disease Control and Prevention, of which 1,159 resulted in death (http://www.cdc.gov/ncidod/dvbid/westnile/surv&control.htm).Given the globalization of commerce and travel, virus-infected people, animals, and arthropod vectors are able to move more easily between locations with great speed (16). Thus, it is likely that other arboviruses will follow the example of WNV, resulting in new or novel disease outbreaks in regions of the world outside their normal geographic ranges. Therefore, a rapid and standardized approach to identification of arboviral infections is needed worldwide for the diagnosis and tracking of current and reemerging arboviral diseases.In the past, identification of antiviral antibody relied on four tests: the hemagglutination inhibition test, the complement fixation test, the plaque reduction neutralization test, and the indirect fluorescent antibody (IFA) test. Positive identification of a viral infection required a 4-fold increase in titer between acute- and convalescent-phase serum samples in these assays (20). Rapid serologic assays, such as the IgM capture enzyme-linked immunosorbent assay (MAC-ELISA) and IgG ELISA are now routinely used in diagnosis soon after infection. Early in infection, IgM antibody is more specific, while later in infection, IgG antibody is more cross-reactive. Inclusion of murine monoclonal antibodies (mMAbs) with defined virus specificities in these solid-phase assays has permitted a level of assay standardization that was not previously possible (30). In the diagnostic laboratory, the MAC-ELISA and the IgG ELISA are often used in tandem to identify positive specimens based on a 4-fold increase in titer between acute- and convalescent-phase serum samples and have replaced the more time-consuming and labor-intensive assays (11, 16, 21).Application of the ELISA in serodiagnosis of arboviral infection is most hampered by the limited availability of human infection-immune sera for use as virus-reactive, antibody-positive control specimens. For the most part, antibody-positive control sera are derived by pooling small volumes of antibody-positive diagnostic serum specimens. The specimens are typically obtained for only the most prevalent arboviral agents (20, 21). Lot-to-lot variability of these serum pools can be high, and constant recollection and recalibration of antibody-positive and -negative control sera are necessary to ensure that test parameters remain valid (10, 21). Of even greater concern is the lack of antibody-positive control sera that can be used in diagnostic ELISAs to identify arboviruses that currently cause rare or infrequent human infections (20).The replacement of variably reactive human control sera with group-specific human IgG antibodies would be a tremendous asset in the serological diagnosis of arboviral infections. Although a number of mMAbs demonstrating flaviviral, alphaviral, or bunyaviral group reactivity exist, they are unsuitable for use as positive serum controls in ELISAs designed to detect the presence of human antibodies. Moreover, the capture or detector antibodies used in these assays are often designed to react with other murine components of the ELISA, leading to an overwhelming false-positive response if mMAbs are employed as positive controls.Fortunately, advances in the humanization of mMAbs have made it possible to overcome these limitations (31). One such method involves the incorporation of the heavy (H)- and light (L)-chain variable (V) regions of a given mMAb into an expression plasmid containing the constant (Cμ) region of human IgM (10). Upon transfection of cells, the resulting plasmid construct expresses a human-murine hybrid IgM chimeric MAb (cMAb) molecule that retains the specificity of the “parent” mMAb but reacts like human IgM in the MAC-ELISA (10, 12, 32).We have previously reported on the construction and evaluation of an IgM cMAb with the specificity of the broadly flavivirus cross-reactive mMAb 6B6C-1. The 6ME2 IgM cMAb reacted with each flaviviral suckling mouse brain (SMB) or virus-like particle (VLP) antigen tested in the MAC-ELISA and displayed a strong preference for the WNV VLP antigen. The use of cell culture viral seed in place of the SMB or VLP antigens in the MAC-ELISA format resulted in enhanced reactivity, as measured by the maximum dilution of cMAb yielding a positive P/N value (positive/negative ratio; see below for details) against WNV, SLEV, DENV serotype 2 (DENV-2), and YFV. In this report we describe the development and characterization of two new IgG cMAbs for use in the indirect IgG ELISA. These cMAbs were created by incorporating the V regions of 6B6C-1 or the alphavirus group-specific mMAb 1A4B-6 into a plasmid construct containing the human IgG γ1 chain. The alpha- or flaviviral group reactivity of each cMAb was confirmed and subsequently evaluated in the standard indirect IgG ELISA. The cMAb demonstrating alphaviral (1GD5) or flaviviral (6GF4) group reactivities were selected for further use and were satisfactory replacements for antibody-positive human control sera against all alphaviruses or flaviviruses tested.     

An Enzyme Immunoassay for the Detection of Antisperm Antibodies

ABSTRACT: A simple and reliable enzyme-linked immunosorbent assay (ELISA) for the detection of antisperm antibodies has been developed in our laboratory. The antigen for the solid phase was produced by sperm sonication while antihuman globulin conjugated to alkaline phosphatase was used as the developing reagent. The conditions and reagents of the assay were chosen to give a mild treatment of the antigen, simple manipulation during washing steps, and nontoxic and readily available reagents. The results were compared to a conventional microscopical method routinely used in our laboratory that detects agglutinating antibodies to human spermatozoa. In 96% of all cases antibodies detected by the microscopical method were also detected by ELISA. Moreover there were some cases where no antisperm antibodies could be demonstrated by microscopy, but gave a positive reaction with ELISA. These were usually cases of unexplained oligospermia, agglutinates in the ejaculate, and bad motility or low viability of the sperms. These results, and also titration experiments of positive samples demonstrate the higher sensitivity of the ELISA by comparison with microscopical methods.     

Methodological Approach for the Detection of IgM Anticardiolipin Antibodies

The effects of 10% fetal calf serum (10% FCS), 10% adult bovine serum (10% ABS) and 1% bovine serum albumin (1% BSA) buffers on the reactivity of samples with or without IgM anticardiolipin antibodies (ACA IgM) were studied on cardiolipin-coated wells (target wells) and cardiolipin-free wells (control wells) in an ELISA test. With 1% BSA, target well reactivity was very low for samples containing ACA IgM. The 10% FCS induced a moderate but significant binding of polyclonal IgM on target wells particularly in the case of IgM hypergammaglobulinemia. With 10% ABS, whatever the IgM serum level, this non-specific binding did not occur on the solid phase either in the presence or in the absence of cardiolipin. Using 10% ABS, the study of 35 SLE sera showed that non-specific binding of IgM on control-wells occurred only with ACA IgM positive samples. Thus with the use of 10% ABS, we propose that subtraction of control well from target well values must not be performed for the detection of ACA IgM. The significance of cardiolipin-free well IgM reactivity of ACA IgM positive sera is discussed.     

两种检测方法测定抗甲状腺球蛋白抗体和抗甲状腺过氧化物酶抗体结果的比较

目的 应用化学发光免疫分析法（CLIA）和放射免疫分析法（RIA）分别测定血清抗甲状腺球蛋白抗体（抗TgAb）和抗甲状腺过氧化物酶抗体（抗TPOAb）.方法 采用CLIA法和RIA法测定304例不同类型甲状腺疾病患者和38名健康对照者血清抗TgAb和抗TPOAb水平.结果 CLIA法测定血清抗TgAb和抗TPOAb水平与RIA法测定的结果具有良好的一致性.结论 CLIA法测定血清抗TgAb和抗TPOAb可以用来替代RIA法.     

Multiplex Assay for Detection of Strain-Specific Antibodies against the Two Variable Regions of the G Protein of Respiratory Syncytial Virus

The role of strain differences in respiratory syncytial virus (RSV) disease has not been clearly defined. To investigate the possibility that strain differences contribute to susceptibility to repeat infections, we developed assays to detect antibodies to the two variable regions of the RSV G protein by cloning and expressing the internal variable region at amino acids (aa) 60 to 172 (g1) and the carboxy-terminal variable region at aa 193 to the carboxy terminus (g2) from different genotypes of RSV. The purified proteins were covalently linked to beads with different proportions of red and orange fluorescent dyes and reacted against serum specimens. Antibody reacting against the differently colored beads, and thus against different G polypeptides, was detected by use of flow cytometry and the Luminex system. This assay system detected group- and, to some extent, genotype-specific responses to RSV infection and can be used to investigate the role of strain differences in RSV disease.     

Development of a Human-Murine Chimeric Immunoglobulin M Antibody for Use in the Serological Detection of Human Flavivirus Antibodies

Current diagnosis of human flaviviral infections relies heavily on serological techniques such as the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA). Broad application of this assay is hindered by a lack of standardized human positive-control sera that react with the wide variety of flaviviruses that can cause human disease, e.g., dengue virus (DENV), West Nile virus (WNV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), and St. Louis encephalitis virus (SLEV). We have created a human-murine chimeric antibody combining the variable regions of the broadly flavivirus cross-reactive murine monoclonal antibody (MAb) 6B6C-1 and the constant region of human IgM to produce a standardized reagent capable of replacing human positive-control sera in a MAC-ELISA for the diagnosis of all human flaviviral infections. The human-murine chimeric IgM antibody secreted from plasmid-transformed Sp2/0-Ag14 cells had a level of serological activity identical to that of 6B6C-1 as measured by ELISA, immunoblotting, and MAC-ELISA for multiple members of the flavivirus genus, including WNV, SLEV, YFV, DENV, and JEV.Flaviviruses are positive-stranded RNA viruses, members of the family Flaviviridae, and are responsible for a number of medically important human diseases. Flaviviruses are arthropod-borne viruses (arboviruses) that are most commonly transmitted seasonally and in specific geographic locations. In the United States, flaviviruses are transmitted mainly by mosquitoes (17, 21). More than 70 different flaviviruses are known to exist; however, the majority are not currently associated with human disease (21). The tenuous nature of the status quo was proven by the emergence of West Nile virus (WNV) in the western hemisphere. Prior to the 1999 outbreak of WNV encephalitis in New York City, St. Louis encephalitis virus (SLEV) was the most important agent of epidemic viral encephalitis in North America, last causing a major epidemic in the mid-1970s (22, 24, 28). Since 1999, the distribution of WNV has rapidly expanded from New York to the rest of the United States and into Canada and Central and South America. As of August 2008, 27,841 human WNV cases in the United States have been reported to the Centers for Disease Control and Prevention (CDC) (http://www.cdc.gov/ncidod/dvbid/westnile/surv&controlCaseCount08_detailed.htm). Given the globalization of commerce and travel, virus-infected people, animals, and arthropod vectors are able to move easily between distant locations with great speed (13). Thus, it is likely that other arboviruses will follow the example of WNV, resulting in new or novel disease outbreaks in regions of the world outside their normal geographic ranges. Because of this, a rapid and standardized approach to the identification of arboviral infections is needed, worldwide, for the diagnosis and tracking of current and reemerging arboviral diseases.The most commonly employed serological technique for the diagnosis of human flaviviral infections is the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), which detects virus-reactive IgM, an effective marker of acute viral infection (17, 18, 21), in serum or cerebrospinal fluid from a person with a clinically compatible illness. In the diagnostic setting, the MAC-ELISA and a similar ELISA measuring virus-reactive human IgG are often used in tandem to provide clear diagnostic profiles (9, 13, 18).Application of the MAC-ELISA in the serodiagnosis of flaviviral infection is hampered by the limited availability of human infection-immune sera for use as virus-reactive, antibody-positive control specimens. For the most part, antibody-positive control sera are derived from small volumes of diagnostic serum specimens. The specimens are typically collected only from the most prevalent flaviviral agents (17, 18). The lot-to-lot variability of these specimens can be high, and constant recalibration of antibody-positive and negative-control sera is necessary to ensure that test parameters remain valid (8, 18). Of even greater concern is the lack of broadly cross-reactive antibody-positive control sera that can be used in the MAC-ELISA for the identification of atypical flaviviral infections (17).The availability of a flavivirus group-reactive human IgM antibody would be a tremendous asset in the serological diagnosis of flaviviral infections. Although a number of murine monoclonal antibodies (MAbs) demonstrating flavivirus group reactivity exist, they are unsuitable for use in the human MAC-ELISA. Fortunately, advances in the humanization of murine MAbs have made it possible to overcome these limitations (25). One such method, described by Hackett et al., involves the incorporation of the heavy (H)- and light (L)-chain variable (V) regions of a given murine MAb into an expression plasmid (pJH2-24-95B1; referred to below as pJH2) that contains the constant (Cμ) region of human IgM (8). Upon transfection of cells, the resulting plasmid construct expresses a human-murine hybrid (chimeric) IgM molecule that retains the specificity of the “parent” murine MAb but reacts like human IgM in the MAC-ELISA (8, 10).In this report we describe the development and characterization of such a human-murine chimeric IgM antibody prepared by using the IgM expression plasmid described by Hackett et al. (8). This chimeric IgM was created by incorporating the V regions of the broadly flavivirus cross-reactive murine MAb 6B6C-1 into a plasmid construct containing the human IgM μ chain. The murine MAb 6B6C-1 was originally raised against SLEV and is specific for the flaviviral envelope (E) protein (19, 23). The flavivirus group reactivity of chimeric 6B6C-1 IgM was confirmed, and the chimeric 6B6C-1 IgM was evaluated in the standard MAC-ELISA and shown to be a satisfactory replacement for antibody-positive human control sera against all flaviviruses tested.