Antigens In Body Fluids Research Articles

Antigen capture assay for detection of a 43-kilodalton Mycobacterium tuberculosis antigen

This study describes the development of an enzyme-linked immunosorbent assay (ELISA) to detect Mycobacterium tuberculosis antigens in body fluids. A double-antibody sandwich procedure that used human and rabbit anti-M. tuberculosis immunoglobulin G antibodies was followed. The ELISA was able to detect as little as 0.8 micrograms of protein of M. tuberculosis sonic extract. Of 253 cerebrospinal fluid specimens submitted for analysis, 11 (4.3%) false-positive results were recorded. Analysis of 317 pleural and ascitic fluid specimens resulted in 6 (1.9%) false-positive recordings. No false-negative results were recorded for any of the body fluids tested. This technique is rapid (5.5 h) and sensitive, may be developed and used in many laboratories with limited resources, and may prove useful in the diagnosis of extrapulmonary and pulmonary tuberculoses. Analysis of these body fluids by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western immunoblotting indicated that the antibody used in the ELISA detects a mycobacterial antigen of 43 kDa. Such antigens were not detected in body fluids of nontuberculous patients.

Diagnosis and management of Histoplasmosis

Histoplasmosis occurs throughout the world but is more common within the endemic areas of North America, particularly in fertile river valleys. Disease manifestations range from asymptomatic infection in the normal host with low-inoculum exposure to rapidly fatal, disseminated infection in the severely immunocompromised host, emphasizing the importance of cellular immunity in defense against Histoplasma capsulatum. Diagnosis depends on a high index of suspicion, knowledge of the clinical and epidemiologic features of the infection, and a thorough understanding of the uses and limitations of fungal cultural and serological laboratory procedures. Recently, a method has been developed for rapid diagnosis based on detection of a polysaccharide antigen in body fluids of patients with histoplasmosis. Amphotericin B remains the preferred treatment for more severe forms of histoplasmosis, particularly in the immunocompromised host, but oral treatment with ketoconazole or newer imidozoles appears to be effective in less severe infections in non-immunocompromised individuals.

Parasite excretory-secretory antigen and antibody to excretory-secretory antigen in body fluids and kidney tissue of Dirofilaria immitis infected dogs.

Excretory-secretory (ES) antigens of adult Dirofilaria immitis were detected by enzyme-linked immunosorbent assay (ELISA) in serum and urine from each of 12 experimentally infected dogs. Excretory-secretory antigens in serum were first detected 154 days postinfection. Serum antibodies directed against parasite ES antigens were detected by ELISA in all dogs. Kidney tissue elution studies were performed in 10 dogs, and antibody and parasite ES antigens were demonstrated in each case. Antibody or parasite antigen was not detected in serum, urine, or kidney eluates from uninfected dogs. At peak concentrations of the ES antigens in serum, there were correlations with the number of adult D. immitis present in the dogs (r2 = 23.8, P less than 0.05) and with the antigen concentration in kidney eluates (r2 = 73.5, P less than 0.001). Peak serum antibody concentrations were not correlated with either the number of adult worms or the antibody concentrations in kidney eluates. This study suggests that detection of parasite antigens in urine may be an important diagnostic aid. In addition, the correlation between the concentration of D. immitis ES antigens in kidney tissue and in serum without a similar correlation between serum and kidney antibody concentrations suggests that D. immitis ES antigens adhere to kidney tissue.

Recognition of a genus-wide antigen ofLegionella by a monoclonal antibody

A monoclonal antibody was produced against a cytoplasmic membrane protein that appears to be common to all species of the genusLegionella. The antibody was positive in polyacrylamide gel electrophoresis and Western blotting with extracts of all of 22 species type strains ofLegionella that were tested. The apparent molecular mass of the protein varied from 57.2 to 62.1 kilodaltons for the 23 species type strains ofLegionella. An enzyme-linked immunosorbent assay (ELISA) was developed with the monoclonal antibody to enable rapid screening of clinical and environmental isolates forLegionella. All of 23 species type strains ofLegionella that were tested were strongly positive with the monoclonal antibody in the ELISA. Among 27 other bacterial species and 84 strains that were tested, onlyBordetella ssp. andAcinetobacter lwoffii were cross-reactive in the ELISA. These two cross-reactive species are readily distinguishable fromLegionella by culture characteristics. The monoclonal antibody may also be useful in tests to detect the genus-wide antigen in body fluids of patients with legionellosis.

The ABH reactions of seminal stains

The ABO grouping results from approx. 1000 seminal stains have been collected and analysed. Most of the stains came from rape cases where the ABO and secretor status of both complainant and suspect were known. The results of the survey provided information concerning the usefulness of elution and inhibition as methods of body fluid grouping, the relative strengths of reaction of the A, B and H antigens in body fluids and the interpretation of the ABH reactions of body fluid stains.

Serotyping of streptococcus pneumoniae by latex agglutination

A simple method is presented for serotyping Streptococcus pneumoniae which uses easily prepared sensitized polystyrene latex particles. The technique is simple, fast and reliable and can detect pneumococcal antigens in body fluids.

Identification of a monoclonal antibody-defined breast carcinoma antigen in body fluids

The monoclonal antibody NCRC-11 defines antigens associated with secretory glandular epithelia as well as most epithelial malignancies. These components have been identified in, and isolated from, normal body fluids including urine and skim milk. The immunoadsorbent purified antigens from urine and milk were very similar to those purified from breast and ovarian carcinomas; by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and immunoblotting, NCRC-11 antibody-binding antigens from all sources were of high apparent molecular weight (> 400 kD) with the major component(s) present as a single band or a doublet. Also, by analysing epitope profiles, all purified antigen preparations were shown to react in a characteristic manner with a panel of monoclonal antibodies which were originally produced against human milk products or materials from tumours. Since it was shown that NCRC-11 antigens were released from tissues in a soluble form, the possibility that these antigens might represent a diagnostic marker for breast cancer was evaluated. The findings obtained indicated that NCRC-11 antigens were elevated in the serum of advanced breast cancer patients in comparison to healthy control females, so that access to the circulation was available to these products released from the tumour but not to those released from normal epithelia.

Production and characterization of monoclonal antibodies specific for Cryptococcus neoformans capsular polysaccharide

Cryptococcus neoformans is surrounded by a capsular polysaccharide. There are at least four known serotypes of the polysaccharide. The objective of this study was to produce monoclonal antibodies (MAbs) that could be used to study the distribution of epitopes among the serotypes of C. neoformans. BALB/c mice were immunized with cryptococcal polysaccharides of serotype A or D that were coupled to sheep erythrocytes. Splenocytes were isolated, and hybridomas secreting MAbs specific for cryptococcal polysaccharides were isolated. Two hybridomas, designated MAbs 439 and 1255, were produced from mice immunized with serotype A polysaccharide. One hybridoma, designated MAb 302, was produced from mice immunized with serotype D polysaccharide. All three antibodies were of the immunoglobulin G1 isotype. MAb 302 showed a specificity for serotypes A and D in Ouchterlony diffusion, agglutination, and opsonophagocytosis assays. MAb 1255 was reactive with polysaccharides and cells of serotypes A, B, and D. MAb 439 was reactive with polysaccharides and cells of serotypes A, B, C, and D. The reactivity of these MAbs closely matched the distribution of epitopes among cryptococcal polysaccharides predicted in previous studies of polyclonal antibodies reactive with cryptococcal polysaccharides. The ability to produce a MAb against an epitope shared by all four serotypes may have value for the detection of cryptococcal antigens in body fluids.

Detection of fungal antigen in body fluids for diagnosis of invasive aspergillosis

In clinical trials, the diagnostic utility of a radioimmunoassay (RIA) to detect Aspergillus antigen was demonstrated in sera obtained from patients with invasive pulmonary, endothelial, and disseminated aspergillosis, in cerebrospinal fluids from patients with Aspergillus meningoencephalitis, and in bronchoalveolar lavage fluids from patients with invasive pulmonary aspergillosis. The RIA was further evaluated in three blinded, controlled clinical trials. In one, sera were collected prospectively from patients with acute leukemia. Antigenemia was detected in four patients with invasive pulmonary aspergillosis (IPA) due to A. flavus, appeared early in the course of infection of three patients concurrent with the onset and evolution of lung infiltrates, and remitted with antifungal chemotherapy. Antigenemia was not detected in three patients before the onset of IPA, in eight leukemic controls, or in the 24 normals. In contrast, seven bronchoscopies were performed in five patients with IPA; fungi were not isolated in three. In a second study, antigenemia was found in coded sera from two patients with invasive aspergillosis but not in eight controls. In the third controlled study, 616 sera from 79 hematology patients admitted on 152 occasions were analyzed for circulating fungal antigen. The diagnostic utility of the RIA was confirmed and levels of antigenemia correlated with the patients' clinical course. These studies demonstrate the utility of the Aspergillus antigen RIA for diagnosis of invasive aspergillosis in hospitalized high risk patients.

Detection of Bacterial Antigens in Body Fluids by the Phadebact System

150 infected patients from 2 study centers had body fluids examined for presence of bacterial antigens (group B streptococcus, Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae type b) using the Pharmacia Phadebact system. The rates of detection of the pneumococcal antigens in the urine or serum of the patients with pneumococcal pneumonia, pneumococcal bacteremia and pneumococcal meningitis were 0.38, 0.47 and 0.5, respectively. The overall detection rate for H. influenzae infections was 0.92 and for group B streptococcal infections 0.87. Serum and urine from 100 non-infected patients were also tested for the presence of group B streptococcus, S. pneumoniae, N. meningitidis and H. influenzae type b antigens with only 1 false positive (H. influenzae type b).

The mode of action of interferons in viral infections and their possible role in the control of hepatitis B

Interferons can alter the course of virus infections by inhibiting virus replication at the intracellular level and by modifying the aspecific and specific immune response to viral antigens in body fluids and on cellular surfaces. Treatment of isolated cells with interferon renders them resistant to infections by viruses belonging to virtually any family. Knowledge of the mechanism of this effect is derived from studies employing both DNA (especially vaccinia virus and SV40) and RNA-viruses (especially picorna-, toga-, rhabdo-, reo- and retroviruses). Interferon induces multiple alterations in the level and state of intracellular regulatory molecules, leading to inhibition of virus replication at several possible steps. In the case of certain DNA viruses, transcription of viral DNA seems to be inhibited. In the case of RNA viruses the target for interferon action is mainly translation. The retroviridae constitute a special case and, in view of their analogy with the hepadnaviridae, are of particular relevance to the possible effects of interferon on the replication of HBV. Interferon inhibits one or more initial stages of primary infection of cells by transforming or nontransforming retroviruses, thereby preventing or delaying the synthesis and/or integration of viral DNA. In cells that already contain an integrated and fully expressed retrovirus genome, interferon treatment results in a reduced release of viral particles as well as a downward shift of the ratios between the numbers of infectious vs noninfectious particles. Immuno-modulatory properties of interferon which might alter the course of HBV-infection include: potentiation of cytotoxic activity of lymphocytes and macrophages; direct anti-inflammatory effects; enhancement or depression in antibody formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of immunoassay methods in the serodiagnosis of human filariasis.

To be more definitive as a serodiagnostic method, an ideal immunoassay for detection of filaria-specific antibody and filarial antigens in body fluids should identify active filarial infection and differentiate between past and present infection. It should perform well in the areas of precision, reproducibility, parallelism, and sensitivity and should use reference and quality control reagents prepared with human body fluids that contain defined amounts of filarial antigen. Moreover, a sufficient quantity of reference serum in stable form should be made available to permit interlaboratory cross-standardization. Difficulty with uniform radiolabeling of filarial antigen extracts and the interference of human antibody have combined to eliminate the competitive-binding immunoassay as a useful method. Of the noncompetitive methods evaluated, the immunoradiometric assay (IRMA) and its nonisotopic counterpart, the immunoenzymetric assay (IEMA), perform best. As a factor masking diagnostically important antigenic determinants, the variable amount of human antibody in blood interfered with all assay designs tested. Better characterization of the antigen(s) circulating in the blood or excreted into the urine of infected individuals will improve assay specificity and sensitivity and will facilitate the preparation of antibody probes more specifically targeted to filarial antigens for use in both the IRMA and IEMA.

Immunodiagnosis of human malignancy.

Definition of normal cell membrane antigens using immunologic techniques has permitted investigators to distinguish normal cells from malignant cells. Malignant cells express fetal antigens in concentrations different from normal mature cells. Mutant cells express differentiation as well as those antigens reflecting the altered cell genome. Normal cells can be transformed into malignant cells using either chemical agents or viruses. As these agents are incorporated into the genome, surface antigens are expressed that differ from normal cell isoantigens. These antigenic determinants can serve as tumor-associated or perhaps tumor-specific markers. Immunochemical methods now exist that permit isolation and purification of these antigens. Monoclonal antibodies have been generated that react with single epitopes associated with these antigens. Very sensitive and highly specific radioimmunoassays and enzyme immunosorbent assays can detect microgram quantities of these antigens in body fluids and as such permit serodiagnostic evaluation. Monoclonal antibodies can also be used for radioautography, immunofluorescent, and immunoperoxidase staining for diagnostic purposes of both cytologic and fixed tissue sections. The binding affinity and specificity of these monoclonal antibody reagents has successfully been used for in vivo tumor localization. Isotopically labeled monoclonal antibodies bind preferentially to tumor cells in the host and are useful for specific radionuclide scintigraphy. These recently developed immunodiagnostic techniques not only add specificity in terms of diagnostic accuracy but are useful for early detection of recurrent disease and in vivo localization of tumor deposits. Therapeutic implications are discussed.

Enzyme immunoassays for the detection of microbial antigens in body fluids: an overview

Abstract

Detection of group B streptococcal antigen in body fluids by a latex-coupled monoclonal antibody assay

A commercially available latex agglutination reagent, Directigen (Hynson, Westcott & Dunning, Div. Becton Dickinson & Co., Baltimore, Md.), in which a murine monoclonal antibody to group B streptococcal (GBS) antigen is the active component, was evaluated by using body fluid specimens from 94 sick infants. Antigen was detected in one or more admission specimens from 18 of 19 (94.7%) infants with symptomatic GBS infection. In 15 patients with GBS meningitis, cerebrospinal fluid, serum, and 10-fold-concentrated urine specimens were positive in 87, 50, and 100%, respectively. GBS antigen was detected in 50% of unconcentrated urines, but in no sera from infants with nonmeningitic GBS sepsis. Among specimens from 27 infants with invasive infection due to organisms other than GBS and from 48 culture-negative sick infants, false-positive latex agglutination reactions occurred in only 4 (9.5%) urine specimens and in no cerebrospinal fluid or serum specimens. The 94.7% sensitivity and specificity of the Directigen GBS test indicate that it is a useful reagent for the rapid diagnosis of invasive GBS infection in young infants.

Amélioration des conditions de dosage de l'activité peroxydasique lors de la réaction elisa

Summary We report here a highly efficient method for the ELISA last step (enzyme-substrate reaction) when horse-radish peroxidase is used. This method involves large concentrations of chromogen (ortho-phenylene-diamine) and H2O2 as a substrate in citric buffer. Under our conditions, results were better than those obtained with such chromogens as orthotolidine or 5-amino-salicyclic acid, and led to an increase in sensitivity of at least 70%. This marked improvement is useful for the direct detection of microbial antigens in body fluids when a high degree of sensitivity is needed, especially in virus diagnosis.

Commercial latex agglutination for detection of group B streptococcal antigen in body fluids

SEVERAL TECHNIQUES for the detection of bacterial antigens in body fluids have been shown to be helpful in providing a rapid, specific etiologic diagnosis prior to culture results in infants and children with meningitis and bacteremia. For the detection of group B streptococcal (GBS) antigen, these include countercurrent immunoelectrophoresis (CIE), ~4 latex agglutination 5-7 and slide coagglutination tests, 8 and enzyme immunoassay) Among these, latex agglutination is simple, rapid, and has the advantage of somewhat greater sensitivity than CIE and greater specificity than coagglutination. When a commercially available latex reagent (Streptex) was used for the detection of GBS antigen in cerebrospinal fluid (CSF), serum, and concentrated urine of infants with GBS infection, respectively 78.6%, 26.7%, and 93.3% of specimens were positive. 6 False positive results were rare. Recently a similar but more sensitive reagent (Wellcogen Strep B) has been prepared specifically for testing of body fluids. We describe the sensitivity and specificity of this latex agglutination assay in detecting GBS antigen in CSF and concentrated urine from infants with and without systemic GBS infection.

Rapid etiological diagnosis of childhood bacterial meningitis using a protein A-containing staphylococcal coagglutination test

Staphylococcal coagglutination test (COA) was applied for the rapid detection of soluble capsular antigens in body fluids of patients with H. influenzae type b (HIB) and pneumococcal (Pn) meningitis. Used COA kits were obtained from Pharmacia Diagnostics. And these results were compared with the results obtained by counter immunoelectrophoresis (CIE).HIB capsular antigen (polyribophosphate, PRP) was detected up to 16ng/ml by COA and up to 125ng/ml by CIE using Wellcome Reagents antiserum.HIB Meningitis: PRP was detected in 19 cases out of 23 cases (83%) in CSF specimens obtained within the first hospital day, equally by both COA and CIE. In urine specimens including concentrate ones, however, COA was more sensitive than CIE in identifying PRP (86% vs 43%). Overall, COA was able to detect PRP in 23/23 (100%), but CIE in 20/23 (87%).In serial specimens, PRP was detected up to the seventh hospital day in CSF, and up to the fifteenth day in five-fold concentrated urine by COA after starting appropriate antimicrobial treatment. By using COA, therefore, the causative organism may be identified in the cases with partially treated culture-negative meningitis.Pn Meningitis: Polysaccharide capsular antigens were detected in five out of eight cases (63%) by COA, and seven out of eight cases (88%) by CIE using Difco antisera in CSF obtained within the first hospital day. Therefore, COA showed less sensitivity than CIE in identifying Pn capsular antigens.In one case caused by Pn type 14, Pn antigen was not detected by CIE, but was detected by COA.From these results, I think that COA is a very useful tool for the rapid etiological diagnosis of bacterial meningitis in childhood.

Enzyme immunoassays for the detection of microbial antigens in body fluids

Enzyme immunoassays for the detection of microbial antigens in body fluids

Enzyme immunoassays for the detection of infectious antigens in body fluids: current limitations and future prospects.

Enzyme immunoassays are attaining increased usage for the direct detection of microbial antigens in body fluids. Advantages of enzyme immunoassays include a high degree of sensitivity resulting from the inherent magnification of the enzyme-substrate reaction and the use of objective end points without the need for radioactivity. Enzyme immunoassays have been developed for the reliable detection of several important microbial antigens in body fluids, including antigens of rotavirus, hepatitis B virus, and Haemophilus influenzae type b. However, standard enzyme immunoassay techniques are not sufficiently sensitive for the measurement of some antigens from other viruses, bacteria, and parasites in concentrations that commonly occur in body fluids during the course of infectious diseases. This review examines some of the limitations of currently available enzyme immunoassay technology and discusses approaches to increasing the sensitivity and specificity of enzyme immunoassay systems. Methods for improving these assay systems include the use of monoclonal antibodies, improved methods of enzyme-immunoreactant conjugation, more sensitive substrate systems, improved methods of antigen-antibody access, and the direct measurement of microbial enzymes. The use of such techniques should lead to the development of efficient enzyme immunoassay systems for the direct detection of a wide range of bacterial, viral, and parasitic infections.