Prospective Evaluation of a New Aspergillus IgG Enzyme Immunoassay Kit for Diagnosis of Chronic and Allergic Pulmonary Aspergillosis

Anti-Aspergillus IgG antibodies are important biomarkers for the diagnosis of chronic pulmonary aspergillosis (CPA) and allergic bronchopulmonary aspergillosis (ABPA). We compared the performance of a new commercial enzyme immunoassay (EIA) (Bordier Affinity Products) with that of the Bio-Rad and Virion\Serion EIAs. This assay is novel in its association of two recombinant antigens with somatic and metabolic antigens of Aspergillus fumigatus. In a prospective multicenter study, 436 serum samples from 147 patients diagnosed with CPA (136 samples/104 patients) or ABPA (94 samples/43 patients) and from 205 controls (206 samples) were tested. We obtained sensitivities of 97%, 91.7%, and 86.1%, and specificities of 90.3%, 91.3%, and 81.5% for the Bordier, Bio-Rad, and Virion\Serion tests, respectively. The Bordier kit was more sensitive than the Bio-Rad kit (P < 0.01), which was itself more sensitive than the Virion\Serion kit (P = 0.04). The Bordier and Bio-Rad kits had similar specificity (P = 0.8), both higher than that of the Virion\Serion kit (P = 0.02). The area under the receiver operating characteristic (ROC) curves confirmed the superiority of the Bordier kit over the Bio-Rad and the Virion\Serion kits (0.977, 0.951, and 0.897, respectively; P < 0.01 for each comparison). In a subset analysis of 279 serum samples tested with the Bordier and Bio-Rad kits and an in-house immunoprecipitin assay (IPD), the Bordier kit had the highest sensitivity (97.7%), but the IPD tended to be more specific (71.2 and 84.7%, respectively; P = 0.10). The use of recombinant, somatic, and metabolic antigens in a single EIA improved the balance of sensitivity and specificity, resulting in an assay highly suitable for use in the diagnosis of chronic and allergic aspergillosis.     

Enzyme Immunoassay for IgG Rheumatoid Factor Combining with Homologous IgG

An enzyme immunoassay (EIA) was developed to detect, in human sera, IgG rheumatoid factor (RF) combining with human IgG. Wells of the EIA plate were coated with the Fc fragment of human IgG. Binding of RF was detected by goat antibodies to F(ab')₂ of human IgG followed by rabbit anti-goat IgG conjugated with alkaline phosphatase and finally by the substrate. Using this procedure, RF was detected in 35%–85% of various pathological human sera, but only in 7% of normal human sera. An analogous procedure was also described for detection, in rabbit sera, of an RF combining with rabbit IgG.     

Enzyme Immunoassay for IgG Rheumatoid Factor Combining with Homologous IgG

An enzyme immunoassay (EIA) was developed to detect, in human sera, IgG rheumatoid factor (RF) combining with human IgG. Wells of the EIA plate were coated with the Fc fragment of human IgG. Binding of RF was detected by goat antibodies to F(ab')₂ of human IgG followed by rabbit anti-goat IgG conjugated with alkaline phosphatase and finally by the substrate. Using this procedure, RF was detected in 35%-85% of various pathological human sera, but only in 7% of normal human sera. An analogous procedure was also described for detection, in rabbit sera, of an RF combining with rabbit IgG.     

Rapid Enzyme Immunoassay of Chicken EGG Yolk IgG

A simple and rapid enzyme immunoassay for specific antibodies in chicken egg yolk is described. As a model system, the levels of anti-Salmonella IgG in the yolk of eggs obtained from various produce retailers were compared. Polyester cloth coated with Salmonella typhimurium lipopolysaccharide was used to capture specific egg yolk antibodies, which were then detected using an anti-chicken IgG-peroxidase conjugate. This assay, requiring less than 30 min to complete, revealed considerable differences in the relative levels of anti-Salmonella IgG in the egg yolks. Anti-Salmonella IgG levels were generally lower in eggs obtained from large produce retailers than in eggs obtained from a small local farm. This assay may provide a rapid and economical means of monitoring the levels of Salmonella contamination in chicken rearing facilities.     

Rapid Enzyme Immunoassay of Chicken EGG Yolk IgG

A simple and rapid enzyme immunoassay for specific antibodies in chicken egg yolk is described. As a model system, the levels of anti-Salmonella IgG in the yolk of eggs obtained from various produce retailers were compared. Polyester cloth coated with Salmonella typhimurium lipopolysaccharide was used to capture specific egg yolk antibodies, which were then detected using an anti-chicken IgG-peroxidase conjugate. This assay, requiring less than 30 min to complete, revealed considerable differences in the relative levels of anti-Salmonella IgG in the egg yolks. Anti-Salmonella IgG levels were generally lower in eggs obtained from large produce retailers than in eggs obtained from a small local farm. This assay may provide a rapid and economical means of monitoring the levels of Salmonella contamination in chicken rearing facilities.     

Semiquantitative Polymerase Chain Reaction Enzyme Immunoassay for the Diagnosis of Pertussis

 The two most commonly used targets for diagnosis of pertussis by the polymerase chain reaction have been the pertussis toxin promoter and the repeated insertion sequence IS481. A comparative assessment of these primers was performed on routinely collected nasopharyngeal swabs, stored at –20  °C, using novel semiquantitative enzyme immunoassays. Both sets of primers behaved similarly with bacterial suspensions, and the 17 culture-positive nasopharyngeal swabs were also positive with the pertussis toxin promoter primers, with one exception, which had been subject to prolonged storage. Significantly more of the 69 culture-negative swabs were positive with the pertussis toxin promoter primers (n=36) than with the IS481 primers (n=18). To determine the effect of inhibitors, a comparative assessment of three primer pairs against human DNA (β-globin and glyceraldehyde-3-phosphate dehydrogenase) was also performed.     

IgG4酶联免疫分析的建立及初步应用

用兔抗人IgG4的多克隆抗体(IgG4Ab)包被成固相板,以识别结合待测标本中的IgG4。用生物素标记IgG4Ab得BioIgG4Ab;用亲和素标记辣根酶得HRPA。在已包被IgG4Ab的固相板中加入IgG4标准品(或待测样品),反应、洗涤后,加入BioIgG4Ab,反应、洗涤后,加入HRPA,反应、洗涤后,板上形成IgG4Ab—IgG4—BioIgG4Ab—HRPA复合物,加酶底物显色,用酶标仪在490nm波长测定OD值,作标准曲线,根据标准曲线,查出标本中IgG4含量。该法测定范围:2.5～200ng/mL,最低检出量2.1ng/mL,批内和批间CV分别8.4%和11.2%。测得血清IgG4含量:青年人(30名)为37.7±2.3ng/mL;30名献血员为42.7±9.9ng/mL,49例重症监护患者明显升高为71.2±9.2ng/mL;49例肺部感染患者明显降低为28.4±9.2ng/mL。结果表明该法稳定,灵敏度适于检出人血清IgG4水平。     

Evaluation of an Immunoglobulin G Enzyme-Linked Immunosorbent Assay for Pertussis Toxin and Filamentous Hemagglutinin in Diagnosis of Pertussis in Senegal

The enzyme-linked immunosorbent assay is widely employed for the serological diagnosis of pertussis. It is generally concluded that a significant increase in specific immunoglobulin G (IgG) or IgA against the pertussis toxin (PT) or against filamentous hemagglutinin (FHA) in paired sera correlates with Bordetella pertussis infection. However, this type of diagnosis of pertussis has mainly been applied to unvaccinated children, with timely sampling of acute- and convalescent-phase sera. In current practice and in epidemiological studies, such criteria are not always fulfilled. The aim of this study was to analyze the significance of decreases in IgG antibody titers against PT and FHA between paired sera observed in suspected cases of pertussis infection. Serological results from paired sera were available for 460 children experiencing at least 8 days of cough. An anti-PT IgG decrease was observed in 25% of the children, more frequently than the anti-FHA IgG decrease. Fourteen percent of the serologic decreases were observed in children with culture-confirmed infection, and 59% of the decreases were observed in children with confirmation criteria according to World Health Organization recommendations. Most of the decreases were observed when serum samples were collected according to a standard recommended schedule. Serologic decreases were observed more frequently among vaccinated children than among unvaccinated children. This difference, which was highly significant (P < 0.00001), was explained by the different kinetics of the antibody responses between vaccinated and unvaccinated children. The importance of the antibody response for the evaluation of vaccine efficacy, namely a bias toward higher absolute vaccine efficacy when this response is not taken into account, is discussed. This study supports an earlier recommendation that a significant decrease in PT or FHA should be added to the diagnostic criteria for pertussis.     

Solid Phase Enzyme Immunoassays of Pertussis Toxin Using Peroxidase or Biotin Labelled Antibodies

Abstract

The quantitative determination of pertussis toxin (PT) is generally estimated by biological tests which are time-consuming, cumbersome and unsuitable for simultaneous testing of a large number of samples. The present work describes a rapid and sensitive ELISA procedure allowing PT assay based on a sandwich technique amplified via avidin-biotin Interaction. As low as 0.1 ng of PT in 0.1 ml sample could be detected by the procedure described.     

Performance of a Cytomegalovirus IgG Enzyme Immunoassay Kit Modified To Measure Avidity

The measurement of cytomegalovirus (CMV) IgG avidity accurately discriminates recent and past CMV infections. We sought to determine if the Wampole Laboratories CMV IgG enzyme immunoassay (EIA) could be modified to measure avidity. The evaluation panel consisted of 156 serum samples we used in 2002 to validate a laboratory-developed EIA, in which 78 serum samples exhibited low avidity, 7 exhibited intermediate avidity, and 71 exhibited high avidity. The qualitative agreement between the two avidity assays was 94% (147/156); all 9 sera with discordant results exhibited intermediate avidity in one of the assays. The avidity index (AI) values in the two assays showed excellent correlation (r = 0.96, P < 0.0001). The definition of high avidity was verified for the Wampole assay by demonstrating high avidity in 91/93 (98%) recently collected CMV IgG-positive/IgM-negative serum samples. The performance of the Wampole avidity assay in a reference laboratory setting was assessed using 470 consecutive serum samples submitted for CMV IgG avidity testing. Surprisingly, 101 serum samples were negative when screened for CMV IgG using the Wampole kit per the package insert; 98 of these 101 serum samples were tested using a CMV IgG chemiluminescent immunoassay, and only 5 were positive. Of the 369 CMV IgG-positive samples, 6% exhibited low IgG avidity, 6% exhibited intermediate avidity, and 88% exhibited high avidity; CMV IgM detection rates were inversely related to AI levels. These findings show that (i) the Wampole CMV IgG EIA can be modified to measure CMV IgG avidity, (ii) many samples are apparently submitted for avidity testing without knowledge of their CMV IgG status, and (iii) most CMV IgG-positive sera submitted for avidity testing exhibit high avidity.     

Evaluation of the Bartels Legionella Urinary Antigen Enzyme Immunoassay

The Bartels Legionella Urinary Antigen enzyme immunoassay (Intracel, USA) is intended for the presumptive diagnosis of past or current Legionnaires' disease by qualitative detection of Legionella pneumophila serogroup 1 antigen in human urine. This test was evaluated using single urine samples collected from 349 patients with lower respiratory tract infection of known aetiology. Specificity was estimated as 100% (181 samples, 95% CI: 98%–100%); sensitivity for Legionella pneumophila serogroup 1 was 98.8% (167 samples, 95% CI: 95.7%–99.9%). Assessing assay results using a Visual Interpretation Card provided by the manufacturer in place of a photometer gave rise to one false-positive result among the 78 control samples examined. Providing the endpoint of this assay is determined photometrically, the Bartels Legionella Urinary Antigen enzyme immunoassay appears to be a highly specific and sensitive kit for the diagnosis of infection caused by Legionella pneumophila serogroup 1. Electronic Publication     

Evaluation of an Enzyme Immunoassay for Detection of Histoplasma capsulatum Antigen from Urine Specimens

Detection of Histoplasma capsulatum urinary antigen (UAg) is important for the initial diagnosis of infection and for monitoring of patient responses to antifungal therapy. This study evaluated an analyte-specific reagent (ASR) enzyme immunoassay (EIA) for the detection of H. capsulatum UAg from Immuno Mycologics, Inc. (IMMY) (Norman, OK) in comparison with routine testing with the MiraVista (MVista) H. capsulatum quantitative EIA (MiraVista Diagnostics, Indianapolis, IN). Using prospectively collected urine specimens (n = 1,003), we observed an overall percent agreement between the two assays of 97.6% (979/1,003 samples). Compared with the MVista EIA, the sensitivity and specificity of the IMMY ASR EIA were 64.5% (40/62 samples) and 99.8% (939/941 samples), respectively, using a cutoff value of 0.5 ng/ml. Based on available clinical histories for 23/24 discordant samples, 5 IMMY assay-negative/MVista assay-positive samples were considered falsely positive. Furthermore, 10/23 discordant samples were positive by the MVista EIA but were below the limit of quantitation (<0.4 ng/ml). The clinical significance of these low positive results in the MVista EIA is unclear. In addition to the prospective study, we tested 11 urine specimens collected from patients with culture-confirmed Histoplasma infections, and 100% (11/11 samples) were positive by the IMMY ASR EIA. In conclusion, the IMMY ASR EIA may offer an alternative approach for the detection of Histoplasma UAg. Additional prospective studies are needed to better characterize the performance of the IMMY ASR EIA in conjunction with clinical and laboratory findings.     

Subclass Distribution of Human Anti-Staphylococcus aureus Alpha Toxin Antibodies: Suggestion of an IgG1, IgA1, IgG4 Switch Pattern

The subclass distribution of antibodies against alpha toxin from Staphylococcus aureus in man was investigated. Antibodies were restricted to IgG1, IgA1, and IgG4 and appeared to develop sequentially. These data suggest a distinct pattern of class and subclass switches in response to protein antigens. However, studies in immunodeficient (class- or subclass- deficient) donors show that the proposed patterns is not obligatory.     

Performance of Clostridium difficile Toxin Enzyme Immunoassay and Nucleic Acid Amplification Tests Stratified by Patient Disease Severity

Many clinical laboratories in the United States are transitioning from toxin enzyme immunoassays (EIA) to nucleic acid amplification tests (NAATs) as the primary diagnostic test for Clostridium difficile infection (CDI). While it is known that the analytical sensitivity of the toxin EIA is poor, there are limited clinical data on the performance of these assays for patients with mild or severe CDI. Two hundred ninety-six hospital inpatients with diarrhea and clinical suspicion for CDI were tested prospectively by toxin EIA, by C. difficile NAAT, and with a reference standard toxigenic culture. Following completion of laboratory testing, retrospective chart reviews were performed to stratify patients into mild and severe disease groups based on clinical criteria using a standard point-based system. One hundred forty-three patients with CDI confirmed by toxigenic culture were evaluated in this study. Among the patients with mild CDI, 49% tested positive by toxin EIA and 98% tested positive by NAAT. Among patients with severe CDI, 58% tested positive by toxin EIA and 98% tested positive by NAAT. Increased CDI disease severity was not associated with an increased sensitivity of EIA (P = 0.31). These data demonstrate that toxin EIA performs poorly both for patients with severe CDI and for those with mild CDI and support the routine use of NAAT for the diagnosis of CDI. The presence of stool toxin measured by EIA does not correlate with disease severity.     

Evaluation of Three Multiplex Flow Immunoassays Compared to an Enzyme Immunoassay for the Detection and Differentiation of IgG Class Antibodies to Herpes Simplex Virus Types 1 and 2

The diagnosis of herpes simplex virus (HSV) infections is routinely made based on clinical findings and supported by laboratory testing using PCR or viral culture. However, in instances of subclinical or unrecognized HSV infection, serologic testing for IgG class antibodies to type-specific HSV glycoprotein G (gG) may be useful. This study evaluated and compared the performances of three multiplex flow immunoassays (AtheNA Multi-Lyte [Zeus Scientific], BioPlex 2200 [Bio-Rad Laboratories], and Plexus HerpeSelect [Focus Diagnostics]) for the simultaneous detection of gG type-specific IgG antibodies to HSV types 1 and 2 (HSV-1 and HSV-2). Serum specimens (n = 505) submitted for routine gG type-specific HSV IgG testing by enzyme immunoassay (EIA) (HerpeSelect; Focus Diagnostics) were also tested by the three multiplex flow immunoassays. Specimens showing discordant results were tested by HSV type-specific Western blotting (WB). For HSV-1 IgG, the AtheNA, BioPlex, and Plexus assays demonstrated agreements of 94.9% (479/505 specimens), 97.8% (494/505 specimens), and 97.4% (492/505 specimens), respectively, with the results of EIA. For HSV-2 IgG, the AtheNA, BioPlex, and Plexus assays showed agreements of 87.9% (444/505 specimens), 97.2% (491/505 specimens), and 96.8% (489/505 specimens), respectively, with EIA results. Timing studies showed that the AtheNA, BioPlex, and Plexus assays could provide complete analysis of 90 serum specimens in 3.1, 1.5, and 2.9 h, respectively, versus 3.1 h by EIA. These findings suggest that the gG type-specific HSV IgG multiplex immunoassays may be beneficial to high-volume clinical laboratories experiencing significant increases in the number of specimens submitted for HSV serologic testing. The evaluated systems provide comparable results to those of EIA, while reducing hands-on time and eliminating the necessity to aliquot specimens prior to testing.Herpes simplex virus type 1 (HSV-1) and HSV-2 are common causes of disease worldwide, with transmission resulting from direct contact with virus-infected secretions. The prevalence of HSV-1 infection increases with age, and >70% of adults worldwide are seropositive for the virus (18). The incidence of antibodies to HSV-2 is dependent on age, sex, and risk factors (e.g., number of sexual partners) and may reach 60 to 95% in certain high-risk groups, such as patients infected with HIV (11, 13, 20). However, a relatively small percentage of patients (10 to 20%) know that they are infected with genital herpes (12, 13, 21), thereby contributing to increased transmission of disease.The diagnosis of HSV-associated disease is routinely made based on clinical findings and supported by laboratory testing using PCR or viral culture (13, 22). However, in instances of subclinical or unrecognized HSV infection, serologic testing for immunoglobulin G (IgG) antibodies to type-specific HSV glycoprotein G (gG) may be useful. Due to significant antigenic cross-reactivity among HSV structural proteins, only gG-based serologic assays have been shown to accurately differentiate between IgG class antibodies to HSV-1 and HSV-2 (2, 4, 9, 15), and FDA-approved, conventional, HSV type-specific enzyme immunoassays (EIA) and Western blot (WB) assays are commercially available. Although EIA and WB have demonstrated excellent sensitivity and specificity (2, 3, 15, 24), they require separate assays to be performed for the detection and differentiation of antibodies to HSV-1 and HSV-2. This may increase the potential for aliquoting errors, as well as the associated technologist and instrument time required for testing.Recently, a number of multiplex flow immunoassays (MFI) have been described for the serologic evaluation of various infectious diseases (6, 8, 16). This approach is similar to traditional EIA but allows for the simultaneous detection and identification of multiple analytes in a single reaction tube. MFI technology uses a liquid suspension array of up to 100 unique microspheres (5- to 6-μm beads), each conjugated to a different capture molecule (e.g., antibody, antigen, or nucleic acid). Each capture analyte is detected and quantitated following the addition of a fluorescently labeled reporter molecule (e.g., phycoerythrin) whose emission is measured by a flow-based detector. Since 2008, three multiplex flow immunoassays (AtheNA Multi-Lyte [Zeus Scientific], BioPlex 2200 [Bio-Rad Laboratories], and Plexus HerpeSelect [Focus Diagnostics]) have received FDA clearance for the detection and differentiation of IgG class antibodies to HSV-1 and HSV-2. These assays are fully automated and designed for high-throughput analysis of the HSV type-specific antibody response.Due to increasing test volumes (∼105% in the past 3 years) and the limitations of conventional methods for HSV antibody testing (e.g., limited throughput and labor-intensive testing), we undertook a study to evaluate and compare the AtheNA, BioPlex, and Plexus multiplex assays for the detection and differentiation of IgG class antibodies to HSV-1 and HSV-2. The objective of this study was to compare the results of MFI and EIA testing, using WB to further evaluate specimens showing discordant results.     

游离三碘甲状腺原氨酸酶免疫分析

将三碘甲状腺原氨酸（T3）与β－D半乳糖昔酶联结，制成T3-酶联结物（T3－E）。用T3－E作为示踪剂建立了游离T3（FT3）酶免疫分析（EIA）。本法灵敏度为0.29pmol／L，批内和批间变异系数分别为7.6％－8.7％和7.8％～14.9％，与二碘甲状腺原氨酸（T2）、反T3（rT3）和甲状腺素（T4）的交叉反应率分别为9.7×10-4、1.1×10-4和3.1×10-5。本法与FT3放射免疫分析（RIA）相关性好（r=0.91，P＜0.01）。用本法测定了36名正常人、16名健康孕妇、15例甲亢患者和15例甲低患者的血清中FT3值，其结果与临床相符。