Detection Of Antigen In Serum Research Articles

Immobilized chicken antibodies improve the detection of serum antigens with surface plasmon resonance (SPR)

Surface plasmon resonance (SPR) and other refractive index and mass sensitive methods are, due to complement activation by mouse monoclonal antibodies and with concomitant high background signal, only rarely used for the detection of antibody–antigen interactions in the blood serum milieu. In the present study chicken IgY and mouse IgG were immobilized to a sensor chip CM5 dextran matrix and compared for their background signal and detection of serum antigen. Ellipsometry with antibodies adsorbed to methylated silicon surfaces was used as a complementary detection method. As expected, fundamental differences in binding properties between the two kinds of antibodies were observed. Mouse antibodies bound large quantities of human serum. Human C1q was detected on mouse IgG and the complement system was activated, as seen from the rapid C3 and properdin depositions. Chicken antibodies bound low quantities of human serum and no human C1q. Moreover, C3 and properdin deposited only after prolonged serum incubations. Addition of EDTA to serum reduced the background signal modestly for both IgG and IgY. Serum samples with different concentrations of human C3 were injected over surfaces with immobilized chicken anti-C3, and the response was measured by SPR. Small concentration differences (<1.25 μg/ml) in a physiologically relevant range (1–40 μg/ml after 100 times dilution) could then be detected reproducibly. The SPR signal was totally obscured when a mouse monoclonal anti-C3 antibody was used for the detection.

Histoplasmosis in AIDS: advances in management.

Histoplasmosis is one of the most common opportunistic infections in HIV-infected patients who reside in endemic areas, and "imported infections" also occur elsewhere. A recent decline in the incidence of histoplasmosis appears to correlate with advances in antiretroviral therapy. Histoplasmosis occurs due to either dissemination of newly acquired infection or reactivation of latent foci of infection. Major risk factors include a CD4 count < or = 150/microL, positive complement fixation serology for the Histoplasma capsulatum mycelial antigen, and a history of exposure to chicken coops; in addition, suboptimal antiretroviral therapy seems likely to be a risk factor. Although there are a variety of clinical manifestations, most patients present with a several-week history of fever, chills, weakness, and weight loss. Diagnosis is based on positive cultures of blood, bone marrow, or other sites; detection of antigen in serum or urine; or characteristic histopathologic findings in biopsy specimens. Induction therapy consists of amphotericin B for acutely ill patients or itraconazole for patients with mild to moderately severe disease. Subsequent lifelong maintenance therapy with itraconazole is recommended. In patients with CD4 counts of < or = 150/microL, itraconazole is effective primary prophylaxis.

Partial Characterization of the Epitope on Excretory-Secretory Products of Fasciola hepatica Recognized by Monoclonal Antibody ES78

This report contains a partial characterization of the epitope recognized by monoclonal antibody (MAb) ES78 produced against excretory-secretory (ES) antigens of Fasciola hepatica. ES78 is currently used for the detection of ES antigens in serum and stool samples of cattle and humans with fasciolosis, using a highly sensitive and specific sandwich enzyme-linked immunosorbent assay (ELISA). The epitope was characterized by periodate oxidation, alkaline borohydride reduction, trichloroacetic acid precipitation, beta-mercaptoethanol treatment, and enzymatic proteolysis. These results, together with those of the 2-site ELISA, lectin immunoassays, and beta-galactosidase digestion, showed that MAb ES78 reacts with a partly protein/partly carbohydrate antigenic determinant that is found on several ES molecules of adult specimens of F. hepatica and contains at least 1 disulfide bond and beta-galactose probably as galactose-beta(1-3)-N-acetylgalactosamine disaccharide.

Detection of meningitis antigens in buffer and body fluids by ultrasound-enhanced particle agglutination

The standard test card agglutination of antibody-coated latex by Neisseria meningitidis, Streptococcus group B, Haemophilus influenzae type b and Streptococcus pneumoniae antigens has been compared with a technique involving local concentration of the coated latex in an ultrasonic standing wave. The detection of positive control antigen was enhanced, compared with the test-card procedure, over a 16 to 64 fold range on exposure to ultrasound. Sample filtration eliminated non-specific agglutination on ultrasonic exposure of latex in control serum, urine or concentrated urine. Tests of meningitis patient body fluids showed increased detection of antigen with ultrasound for CSF (11/14>7/14) serum (8/13>3/13) and concentrated urine (8/17>2/17) compared to test card assays. The ultrasound detection of antigen in serum or concentrated urine was comparable to that achieved with CSF on test cards. Serum dilution experiments showed that ultrasound could detect antigen in serum over a 1000 fold concentration range.

Microbial etiology of acute community-acquired pneumonia in adult hospitalized patients in Yaounde-Cameroon.

OBJECTIVE: To determine the microbial etiology of acute community-acquired pneumonia in Yaoundé. METHODS: Ninety-one consecutive adult patients admitted to hospital for radiologically confirmed acute community-acquired pneumonia were studied prospectively. Sputum microscopy and culture, blood cultures, pneumococcal antigen detection in serum and serologic analysis for agents of atypical pneumonia and for human immunodeficiency virus (HIV) were performed for most patients. RESULTS: There were 65 men and 26 women, mean age 36.5 years. Of 91 patients, 70.3% had at least one predisposing factor and 21.7% (20 of 81) were seropositive for HIV. A microbial etiology was identified in 48 (52.7%) cases. A single pathogen was identified in 42 (85.4%) and double pathogens in six (14.6%). Bacteremia occurred in 12 of 81 patients and was significantly more common in HIV-seropositive than in HIV-seronegative patients. Streptococcus pneumoniae was the commonest causative agent, identified in 22 of 91 (24.2%) patients, 10 of whom were bacteremic. Atypical pathogens were diagnosed in 14 of 65 patients with serologic tests. Mycoplasma pneumoniae and Coxiella burnetii were diagnosed in six of 65 (9.2%) cases each, and Chlamydia pneumoniae in three (4.6%) patients. Mycoplasma pneumoniae and Chlamydia pneumoniae occurred as a dual infection in one case. Seven of 91 patients died, and death was not associated with any particular etiology. CONCLUSION: Streptococcus pneumoniae remains the predominant etiologic pathogen of community-acquired pneumonia. For this reason, and also because ampicillin used empirically to treat patients with this disease in the same setting has been shown to be efficacious, we propose the use of an aminopenicillin in the initial treatment of acute community-acquired pneumonia in adults in Yaoundé. However, patients who fail to respond clinically to such treatment should benefit from either a macrolide or a tetracycline in order to cover for atypical pathogens.

Detection of cruzipain, the major cysteine proteinase from Trypanosoma cruzi and its C-terminal extension in biological fluids during experimental infection in mice.

Monoclonal antibodies (MoAbs) specific for unique epitopes of the catalytic domain of cruzipain (Crz) were used to develop a two-site sandwich ELISA specific for native Crz. In addition, the authors developed a sandwich ELISA that allowed the detection of the protease C-terminal domain (CT) using a combination of a MoAb specific for the CT and rabbit anti-Crz IgGs. Both assays were sensitive with detection limits of 2 ng/ml and 0.7 ng/ml, respectively. The assays were assessed for applicability in detection of antigens in serum and urine from experimentally infected BALB/c mice. The antigens were already detectable in serum by the third week after infection, reached their peak by week four, and decreased during the chronic phase of the infection. Throughout the infection the relative amount of CT detected was several-fold higher than that of native Crz, and the data demonstrate that the cT exposes highly immunogenic epitopes that are absent in native Crz. Since these observations have a potential application in diagnosis, the authors analysed the degree of cross-reactivity with antigens from T. rangeli, T. brucei, Leishmania mexicana and L. panamensis, and determined that the assays were highly specific. Measurable amounts of the CT were also recorded in urine samples.

Humoral and Mucosal IgA Antibody Response to a Recombinant 52-kDa Cysteine-Rich Portion of the Entamoeba histolytica Galactose-Inhibitable Lectin Correlates with Detection of Native 170-kDa Lectin Antigen in Serum of Patients with Amebic Colitis

Humoral and mucosal IgA responses to a recombinant cysteine-rich portion (designated LC3) of the Entamoeba histolytica galactose-inhibitable lectin's 170-kDa subunit were determined in patients with amebic colitis. All patients had 170-kDa amebic antigen in serum, compared with 1 of 50 cyst passers and 1 of 31 controls (P < .01). Seven days after treatment, serum and fecal 170-kDa antigen became undetectable in 12 of the 13 patients (P < .001). Serum anti-LC3 IgA was found in 83.8% of colitis patients, compared with 2% of controls and 12% of asymptomatic cyst passers (P < .001). Salivary and fecal anti-LC3 IgA levels were higher in patients than in cyst passers (P < .001). In conclusion, in amebic colitis, development of humoral and mucosal IgA responses to the recombinant LC3-encoded protein correlates with detection of amebic 170-kDa antigen in serum and feces.

Efficacy and Side Effects of Praziquantel in an Epidemic Focus of Schistosoma mansoni

Schistosoma mansoni was first reported in the area of Richard Toll (Senegal) in 1988 and spread rapidly in the community, after a series of human-engineered ecologic changes. A random population sample (n = 422) from Ndombo, a village near Richard-Toll, was studied in 1991 by stool examination (four Kato slides from two stool samples) and antigen detection in urine and blood. Stool-positive individuals were treated with 40 mg/kg of praziquantel. A house-to-house interview regarding side effects was conducted 24 hr after treatment. Two hundred ninety-eight subjects were re-examined 10 days (antigen detection) and 12 weeks (egg counts, antigen detection) after treatment. Before treatment, positive egg counts were found in 91% of the subjects, with 41% excreting more than 1,000 eggs per gram (epg) of feces. Treatment of 352 individuals caused serious but transient side effects (colic, vomiting, urticaria, and edema), the frequency of which increased with increasing egg counts. The parasitologic cure rate 12 weeks after treatment was only 18%, the frequency of egg counts with more than 1,000 epg decreased to 5%, and the mean egg count of those remaining positive was reduced by 86%. Antigen detection in serum 10 days and 12 weeks after treatment remained positive in 90% of the subjects, although titers decreased sharply. The low cure rates may be due to intense transmission and/or undeveloped immune responses in this recently exposed population. However, reduced drug susceptibility of the parasite strain has now been confirmed in one local isolate.

Immunodiagnosis of schistosomiasis mansoni in a low endemic area in Surinam by determination of the circulating antigens CAA and CCA

We evaluated the applicability of circulating antigen detection in serum and urine for the diagnosis of Schistosoma infections in a low endemic area. In total 389 individuals from Saramacca (Surinam) participated in the survey. Stool samples were examined using the Kato method, while circulating anodic antigen (CAA) and circulating cathodic antigen (CCA) were determined by highly specific monoclonal antibody-based ELISA's. Also schistosome specific IgM antibodies were measured by the indirect immunofluorescence assay, but the diagnostic performance of this test was found to be poor in this population. S. mansoni eggs were found in 29% of the examined cases, while CAA and CCA could be demonstrated in 23% and 17% of the serum samples and in 3% and 28% of the urine samples, respectively. Forty three percent of the study population was positive in at least one of these diagnostic assays, indicating that each individual test misses a substantial part of the subjects with an active infection. In most positive cases, intensities of infection were very low. As 204 individuals participated in all screening assays, diagnostic performance of each test was evaluated in this sub-population. The highest sensitivities were achieved with the urine-CCA assay and the parasitological examination, detecting 59 and 58 out of the 107 cases with an active infection, respectively. The serum-CAA assay detected 47 positive cases. Our results demonstrate that determination of circulating antigens, especially CCA in urine and CAA in serum, provides information additional to the parasitological examination, for the assessment of prevalence and intensity of Schistosoma infection in low endemic areas.

Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid

We evaluated the performance of CRYPTO-LEX (Trinity Laboratories, Inc., Raleigh, N. C.), a new mouse immunoglobulin M monoclonal antibody latex agglutination reagent which reacts with the capsular polysaccharide of the four serogroups of Cryptococcus neoformans. This test was compared with CALAS (Meridian Diagnostics, Cincinnati, Ohio) for the ability to detect cryptococcal antigen in serum and cerebrospinal fluid (CSF). A total of 580 clinical specimens (327 serum and 253 CSF samples), primarily from human immunodeficiency virus-infected patients, were tested in this study. Sixty-seven specimens (44 serum and 23 CSF samples) were positive for cryptococcal antigen with both tests, and 511 (282 serum and 229 CSF samples) were negative. The two latex reagents agreed for 326 of 327 serum specimens (44 positives and 282 negatives). One serum specimen with a titer of 1:2 was CALAS positive but CRYPTO-LEX negative. The titer correlation coefficient for the two tests was 0.884 when two highly discordant serum specimens were eliminated from analysis of the data. The two latex tests agreed for 252 of 253 CSF specimens (23 positives and 229 negatives). One specimen with a titer of 1:2 was positive with CALAS and negative by CRYPTO-LEX. The correlation coefficient of the two tests for CSF titers was 0.886. The sensitivity and specificity of CRYPTO-LEX were 97 and 100%, respectively, with a 99.6% correlation with CALAS. These data show that the performance of CRYPTO-LEX is comparable to that of CALAS for detection of cryptococcal antigen in serum and CSF.

Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid.

Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid.

Use of a monoclonal antibody in a dot immunobinding assay for detection of a circulating mannoprotein of Candida spp. in neutropenic patients with invasive candidiasis.

A dot immunobinding assay for the detection of a circulating mannoprotein (MP) antigen of Candida species in the sera of neutropenic patients in a hematological setting is described. The technique is based on the use of a monoclonal antibody which recognizes an oligomannoside epitope shared by distinct MP of pathogenic Candida species. The sensitivity of the assay for antigen detection in serum was between 2 and 5 ng/ml, and MPs from Candida albicans, Candida tropicalis, Torulopsis glabrata, and Candida parapsilosis, but not Candida krusei, could be detected. A retrospective analysis of sera from patients with proven invasive candidiasis versus sera from controls (Candida-colonized and noncolonized subjects) revealed that the novel assay has sufficient sensitivity, specificity, and predictive values to be of potential diagnostic significance.

Invasive aspergillosis in patients with acquired immunodeficiency syndrome: Report of 33 cases

purpose: Acquired immunodeficiency syndrome (AIDS)-associated invasive aspergillosis (IA) is a rare condition, which is mainly reported as isolated cases either antemortem or at autopsy. The role of AIDS itself is controversial, because many of the reported patients exhibited the classic risk factors such as neutropenia and steroid therapy. The aims of this study were to report 33 patients with IA during AIDS and their outcome, focusing on the risk factors and the value of diagnostic procedures. patients and methods: Thirty-three patients from 17 different medical centers in France were retrospectively included in the study. For pulmonary IA, we defined two types of patients: those with “confirmed IA,” describing all the patients with histologically proven disease, and those with “probable IA,” who had the development of a new pulmonary infiltrate on chest radiograph and a positive bronchoalveolar lavage (BAL) fluid culture for Aspergillus species without identification of other pathogens. For extra-pulmonary IA, the diagnostic criteria included both positive histology and culture. results: Of the 33 cases included in this series, 91% were recorded during the last 3 years (1989 to 1991), suggesting that aspergillosis is an emerging complication in AIDS. Approximately 50% of the patients did not exhibit any classic risk factor, i.e., neutropenia and steroid treatment; almost all patients had a CD4 cell count less than 50/mm 3. The mycologic culture from BAL was the method of choice for the diagnosis of invasive pulmonary disease because it was known to correlate well with histology findings obtained either antemortem or postmortem. Of 28 patients with a positive BAL culture for Aspergillus, 15 underwent a biopsy or autopsy and 14 were positive at histology. Serum antigen detection was positive in only 4 of 16 tested patients. Clinical and radiologic signs did not differ from those observed in neutropenic patients without human immunodeficiency virus, except for the higher incidence of neurologic complications in AIDS. Interestingly, we observed three cases of invasive necrotizing tracheobronchial aspergillosis with acute dyspnea and wheezing. The use of amphotericin B (0.5 mg/kg/d) and/or itraconazole (200 to 600 mg/d) was most often unsuccessful. Only four patients experienced clinical and radiologic improvement. The mean interval between the diagnosis of IA and death was 8 weeks (range: 3 days to 13 months). conclusions: This study suggests that aspergillosis is an important life-threatening condition in the advanced stage of AIDS. It requires an early diagnosis with BAL fluid culture and careful therapeutic evaluation.

Improved detection of HIV p24 antigen in serum after acid pretreatment.

HIV-1 p24 antigen was detected in 554 sera (509 from HIV-1 seropositive individuals and 45 sera from seronegative controls) using a conventional method with acid pretreatment of the sample in order to separate the p24 antigen/anti-p24 antibody immune complexes. In asymptomatic individuals there was a substantial increase in antigen detection (48.2% vs 8.4%). Similar results were also observed in ARC (59.1% vs 12.2%) and AIDS patients (85.7% vs 37.1%). It can be concluded that the acid treatment improves the sensitivity of conventional techniques to detect HIV-1 p24 antigen.

Detection of porin antigen in serum for early diagnosis of mouse infections with Salmonella typhimurium

Detection of porin antigen in serum for early diagnosis of mouse infections with Salmonella typhimurium

Detection of porin antigen in serum for early diagnosis of mouse infections with Salmonella typhimurium.

The monoclonal antibodies to porin, an outer membrane protein isolated from Salmonella typhimurium and sandwich enzyme linked immunosorbent assay (ELISA) has made possible the detection of porin from sera of S. typhimurium-infected mice. The specificity of the monoclonal antibodies was ascertained based on their cross-reactivity with porins isolated from S. typhi, Shigella flexneri and Escherichia coli and lipopolysaccharide (LPS) of S. typhimurium and E. coli. Serum samples were found to be positive for porin as early as 3 days after intravenous and 5 days after oral infection. In addition, a positive correlation was observed between the bacterial load and the concentration of porin detected in the sera. On the other hand, analysis of sera for anti-porin antibody showed diametrically opposite time kinetics with antigenaemia. These results indicate that porin accumulates in the serum of infected mice much earlier than the appearance of antibodies to porin. Thus detection of porin holds promise for early diagnosis of typhoid.

Pneumococcal finding in a sample from upper airways does not indicate pneumococcal infection of lower airways.

The presence of pneumococcus (Pnc) by antigen detection and culture was examined in nasopharyngeal aspirates (NPA) of 315 children hospitalized with middle or lower respiratory tract infection. Pnc was found in NPA from 115 (37%) patients, being demonstrated by antigen detection alone in 34 (30%), by culture alone in 26 (23%) and by both methods in 55 (48%) of Pnc-positive samples. Pnc findings in NPA were most common, 45-46%, in patients aged 1-4 years. Serological evidence of Pnc infection, based either on detection of Pnc antigen in serum or urine, or on demonstration of an antibody response to these antigens, was present in 31 (27%) of the 115 patients with and in 28 (14%) of the 200 patients without Pnc in NPA samples. In the 48 patients positive for Pnc in NPA samples both by antigen detection and culture the isolated Pnc strains were serotyped. In 45 (94%) of these the type/group of Pnc was the same by both methods indicating that the specificity of the antigen detection tests, latex particle agglutination and counterimmunoelectrophoresis, was high. To evaluate the diagnostic significance of Pnc antigen detection and culture in NPA, sensitivity, specificity and likelihood ratios were calculated; serological evidence of Pnc aetiology was used as a reference. For both methods, sensitivity was poor, less than 0.3, but specificity was good, greater than 0.8. It is concluded that the finding of Pnc by culture or antigen detection in NPA is no indication of Pnc respiratory infection. On the other hand, Pnc etiology is unprobable, if Pnc is not present in NPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Serodiagnosis of parasitic diseases

In this review on serodiagnosis of parasitic diseases, antibody detection, antigen detection, use of monoclonal antibodies in parasitic serodiagnosis, molecular biological technology, and skin tests are discussed. The focus at the Centers for Disease Control on developing improved antigens, a truly quantitative FAST-enzyme-linked immunosorbent assay, and the very specific immunoblot assays for antibody detection is highlighted. The last two assays are suitable for field studies. Identification of patient response in terms of immunoglobulin class or immunoglobulin G subclass isotypes or both is discussed. Immunoglobulin isotypes may asist in defining the stage of some diseases. In other instances, use of a particular anti-isotype conjugate may increase the specificity of the assay. Monoclonal antibodies have played important roles in antigen purification and identification, in competitive antibody assays with increased sensitivity and specificity, and in assays for antigen detection in serum, body fluids, or excreta. Molecular biological technology has allowed significant advances in the production of defined parasitic serodiagnostic antigens.

Frequency and Clinical Implications of Monoclonal Antibody Detection of Tumor-associated Antigens in Serum of Patients with Lung Cancer

We previously showed that a panel of monoclonal antibodies (MAb) (5E8, 5C7, and 1F10) that detect serum tumor-associated antigens (TAA) could distinguish patients with lung cancer from those without to a highly significant degree. However, among patients with lung cancer, the frequency and clinical importance of serum TAA expression were not established. Therefore, we analyzed the serum and initial clinical characteristics of 52 Philadelphia VA patients with newly diagnosed lung cancer seen over a 13-month period. A modified semiquantitative ELISA was employed to determine MAb reactivity. Our cohort was characterized by a mean age of 65 +/- 9 year (SD) and mean Karnovsky score of 74 +/- 10; marked weight loss was present in 28 subjects, and 39 presented with either Stage III or IV disease. The panel detected TAAs in 38 of 52 cases (sensitivity 73%; 95% Cl, 60-83%), including 13 of 22 squamous cell, 9 of 12 adenocarcinoma, 10 of 11 undifferentiated, and 6 of 7 small cell carcinomas. No significant differences were found between the reactive and nonreactive patients in terms of age, stage at presentation, histologic subtype, performance status, or weight loss. However, 1F10 and 5C7 were each associated with a greater risk of early death by Cox proportional hazard analysis (p = 0.017 and 0.006, respectively) even when other prognostic variables are accounted for. We conclude that specific serum TAA can be detected in the majority of lung cancer patients with all major histologic subtypes in a cohort with advanced tumors and poor prognostic indices.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity and specificity of serum antigen detection for diagnosis of meningococcal disease in children

A total of 72 samples of serum were examined in order to determine the sensitivity and specificity of meningococcal antigen detection in the diagnosis of meningococcal disease. Thirteen samples were from children with meningococcal meningitis (MM), 12 from children with meningococcal septicaemia (MS) and 47 from children with both MM and MS. A further 56 samples of serum from clinically well children were used as negative controls. Latex particle agglutination tests (LPA) were used to detect group B and groups A,C,Y,W135 meningococcal antigen and counterimmunoelectrophoresis (CIE) to detect group B, and groups A, C, D antigens. CIE proved slightly more sensitive than LPA in detecting antigenaemia but not significantly so. Detection of antigenaemia (by both LPA and CIE) had a sensitivity of 38% in diagnosing MM and of 69% in MS and MM + MS and a specificity of 100%. Increasing severity of disease was not statistically significantly associated with increased detection of antigenaemia.