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.     

Rapid detection of group B streptococcal antigen in human amniotic fluid.

Infants exposed in utero to group B streptococcus (GBS)-infected human amniotic fluid (HAF) are at high risk for serious infection. Latex particle agglutination (LPA) tests are not approved for detection of GBS in HAF. Two LPA systems, Patho-Dx Strep B and Wellcogen Strep B, were used to test unfiltered sterile HAF and filtered HAF containing concentrations of GBS carbohydrate from 0.2 to 100 micrograms/ml. Four different processing techniques were used to prevent nonspecific LPA: EDTA, nitrous acid, enzyme, and nitrous acid-heat. GBS (10(2) CFU/ml) was inoculated into filtered HAF, incubated, sampled serially, processed with enzyme, and tested by LPA. Unprocessed, unfiltered HAF showed 33% nonspecific agglutination when tested by LPA. Processing of HAF removed nonspecific agglutination and improved GBS antigen detection. Without processing, LPA could not detect less than 100 micrograms of GBS carbohydrate per ml. With nitrous acid or enzyme processing, as little as 0.2 microgram/ml could be detected. Results were easier to read after enzyme processing than after nitrous acid processing. Although both LPA systems were equally efficient, testing was easier with the Patho-Dx system. After enzyme processing, LPA could detect as few as 10(4) CFU/ml when agglutination was read with a 4 X hand lens. Substances in HAF induce false-positive reactions during LPA testing. Processing removes the interference and improves the detection of GBS. LPA testing of HAF may allow earlier identification and treatment of infants at risk for serious GBS infection.     

Detection of group B streptococcal antigen in necropsy specimens using monoclonal antibody and immunoperoxidase staining.

A murine monoclonal antibody, which recognises various serotypes of group B Streptococcus in formalin fixed, paraffin embedded tissue, was used to show the organism in necropsy specimens of newborn infant lung by an indirect immunoperoxidase technique. The method seemed to be complementary to that of Gram staining, and may be successfully used to identify group B Streptococcus antigen in histopathological material.     

Rapid detection of hepatitis B surface antigen by double antibody radioimmunoassay

HBsAg and anti-HBs can be detected by double-antibody radioimmunoassay in a reliable routine overnight assay of sensitivity approaching that of solidphase immunoradiometric assay. The test requires careful standardisation and regular preparation of I¹²⁵-HBsAg of appropriate quality, but otherwise uses only small quantities of commercially available reagents. In this paper, the reaction kinetics of the first incubation (test sample + limiting amount of rabbit anti-HBs), second incubation (reaction mixture + I¹²⁵-HBsAg) and precipitation reaction (reaction mixture + antirabbit globulin) were examined, to determine the minimum time required for the assay compatible with adequate sensitivity and user convenience; in addition, in order to reduce the time of radioactive counting, the maximum amount of I¹²⁵-HBsAg per test compatible with a satisfactory assay was examined. It was concluded that, by terminating each reaction before equilibrium was reached, results could be obtained in 2–3 hours with a detection limit similar to the standard overnight procedure. To allow routine examination of large numbers of samples, the possibilities of complete automation of this liquid-phase type of assay were discussed.     

Immunospecificity and quantitation of an enzyme-linked immunosorbent assay for group B streptococcal antibody.

Type-specific antigen was purified from the supernatant of type III group B streptococcal cultures, tyrosylated, and bound to microtiter wells for an enzyme-linked immunosorbent assay. The immunological specificity of the antigen and the assay was shown by (i) reaction only with homologous unabsorbed rabbit sera and (ii) inhibition after incubation of human serum with homologous but not heterologous purified antigen. The assay was quantitated by relating optical density readings to absolute amounts of human immunoglobulin G bound to the microtiter wells.     

Assay of antibody to group A streptococcal carbohydrate by enzyme-linked immunosorbent assay.

An indirect enzyme-linked immunosorbent assay system for determination of antibody levels to the group A streptococcal cell wall carbohydrate antigen is described. Optimal conditions for antigen preparation, purification, and conjugation to poly-L-lysine for adequate adsorption to the solid phase are presented. Antibody titers of unknown sera were determined by comparison to known reference standard pool sera. A highly significant correlation (p less than 0.0001) was found between enzyme-linked immunosorbent assay antibody titers and antigen-binding capacity in a previously described radioimmunoassay. Utilizing an isotype-specific anti-immunoglobulin reagent and immunoabsorbent-purified antibody to group A streptococcal cell wall carbohydrate antigen, we were able to detect nanogram quantities of antibody by the enzyme-linked immunosorbent assay technique. This system will provide for more generalized use of group A streptococcal cell wall carbohydrate antigen antibody determinations for the study of immune responses after streptococcal infections and their complications.     

Enzyme-linked immunosorbent assay for group B streptococcal antibodies.

We report here on the development of enzyme-linked immunosorbent assays (ELISAs) for antibodies to types II and III group B streptococci. Streptococcal antigens were prepared by trichloroacetic acid extraction and fractional alcohol precipitation. Microtiter wells were coated with antigen in 0.1 M carbonate buffer at pH 9.6. Lyophilization was found to be an essential step for efficient binding of the streptococcal antigens. After incubation with antibody-containing rabbit serum, bound antibody was detected with peroxidase-labeled goat anti-rabbit immunoglobulin G. Optimal antigen concentrations were 200 micrograms/ml for type II and 100 micrograms/ml for type III. An ELISA is also described that uses intact bacteria as antigen. Hyperimmune rabbit serum reacted at a titer in excess of 512 against trichloroacetic acid-soluble antigen and 4,096 against whole bacteria. Sera from human subjects were also tested. Most human sera contained antibody which bound to intact bacteria but not to trichloroacetic acid-solubilized streptococcal antigens. Antibody titers in human sera against intact bacteria correlated very well with opsonic antibody activity measured in a chemiluminescence assay.     

Rapid antibody capture assay for detection of group-A streptococci using monoclonal antibody and colloidal gold-monospecific polyvalent antibody conjugate.

A rapid one step, sensitive and specific antibody capture assay for detection of group-A streptococci from the throat swabs of children is described. Monoclonal antibody either MA-106 or MA-107 specific for group-A streptococci polysaccharide (APS) was used as the capture antibody on nitrocellulose paper and rabbit monospecific polyvalent antibody conjugated with colloidal gold to detect the presence of antigen. The lower detection limit of this assay is 15.6ng APS/ml. The assay is specific for APS and failed to recognize polysaccharides obtained from group-B,-C,-G streptococci as well as Staphylococcus aureus. Antigen extracted from throat swabs of children who were positive for beta-hemolytic plaques (other than group-A streptococci) as seen on blood agar culture gave negative readings, thereby confirming the specificity of the assay for APS.     

Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a monoclonal antibody immunoblot assay

Throat swabs from patients with pharyngitis and sputum specimens from patients with atypical pneumonia were tested for the presence of a Mycoplasma pneumoniae polypeptide with a molecular weight of 43,000 with the use of an M. pneumoniae species-specific monoclonal antibody in an immunoblot assay. This 43,000-dalton polypeptide was detectable in 33 of 33 throat swabs from patients with pharyngitis that were positive for M. pneumoniae by conventional culture as well as a culture-amplified enzyme immunoassay. The 43,000-dalton polypeptide was also detected in three of three M. pneumoniae culture-positive sputum specimens. It was not detected in 3 sputum specimens culture-confirmed for Legionella pneumophila, 10 sputum specimens from normal persons, or 25 throat swabs also from normal persons. This immunoblot assay could be completed within five hours and may be an alternative method for detecting M. pneumoniae antigen directly in sputum or throat swab specimens.     

Comparison of two antigen assays for rapid intrapartum detection of vaginal group B streptococcal colonization.

As part of a clinical investigation evaluating the efficacy of intrapartum antigen detection for screening for heavy vaginal colonization with group B streptococci (GBS), we compared the performance of modified Bactigen and Directigen GBS latex particle agglutination (LPA) kits. Paired vaginal swabs obtained from women in labor were rapidly transported to the laboratory and used for culturing (both swabs) and LPA testing (one swab by each method). GBS growth was estimated semiquantitatively and further designated as light or heavy growth. Performance specifications for each method were determined by comparing LPA and culture results from the same swab. A total of 4,251 paired swabs were evaluated during the study period. The performance specifications for detecting GBS growth of any degree for Bactigen and Directigen, respectively, were as follows: sensitivity, 20 and 24%; specificity, 99 and 99%. The performance specifications for heavy colonization for Bactigen and Directigen, respectively, were as follows: sensitivity, 57 and 62%; specificity, 99 and 99%. Neither LPA kit was a sensitive indicator of vaginal colonization with GBS or neonatal infection.     

Rapid detection of group B streptococcal colonization of the genital tract by a commercial optical immunoassay

The performance of a commercial optical immunoassay (OIA) was compared at two institutions with that of routine agar and broth culture methods for the detection of group B streptococcal (GBS) colonization of the genital tract. The Strep B OIA (Bio Star, USA) was used to test 962 vaginal swabs from pregnant women for the presence of GBS antigen. The prevalence of GBS vaginal colonization in this population was 22.4%. The OIA results were compared with those of culture on trypticase soy agar with 5% sheep blood (TSA) and broth enhanced culture (Lim broth). Sensitivity and specificity values of the OIA method compared to TSA culture alone were 82.5% and 91.8%, respectively. The sensitivity of the OIA method was equivalent to that of TSA culture (62.4% vs. 64.4%; p>0.5, ²=0.01) when the data were compared with broth culture. The extent of colonization affected the sensitivity of the OIA method: 100% of 4+, 94% of 3+, 96% of 2+, and 63% of 1+ TSA plates were detected by the OIA test. The commercial OIA method demonstrated sensitivity equivalent to that of TSA culture for the detection of GBS colonization. The OIA test offers two additional advantages over culture: reduced time required to obtain results (30 min vs. days) and the ability to detect GBS antigen in samples with compromised viability. The results of this study suggest that the Strep B OIA test can be a useful diagnostic tool in the management of early-onset GBS disease.     

Enzyme immunoassay for the detection of group A streptococcal antigen.

A competitive inhibition enzyme immunoassay for the detection of Streptococcus pyogenes directly from throat specimens or from solid bacteriological medium is described. Group A-specific polysaccharide adsorbed onto treated polystyrene beads, in conjunction with rabbit antibody to S. pyogenes, was used to determine the presence of the polysaccharide antigen. Inhibition values in excess of 65% were observed with 10(4) or more CFU of S. pyogenes per test. An inhibition of 25% was demonstrated with as few as 10(3) CFU per test. Heterologous microorganisms tested at 10(6) CFU per test reacted at levels of inhibition less than 25%. Two types of bacterial transport medium and swabs of different fiber compositions did not alter the assay performance. Accurate identification of S. pyogenes was achieved by testing single colonies picked directly from blood agar plates which had been incubated for 18 to 24 h. In addition, the assay was performed on throat specimens from children and adults having pharyngitis. A single-swab, blind study was conducted in which enzyme immunoassay reactivity was compared with results of blood agar culture and bacitracin sensitivity. When there were discordant results, serological identification was used as the confirmatory test. At an optimal cutoff value of 40% inhibition, sensitivity and specificity by enzyme immunoassay were 97.0% and 97.9%, respectively, as compared with confirmed culture results. The assay has an incubation time of 3 h and is a sensitive and specific method for the detection of S. pyogenes antigen.     

Identification of group B streptococcal antigen with lectin-bound polystyrene particles.

The lectin of the tomato, Lycopersicon esculentum, or of the potato, Solanum tuberosum, can be passively coupled to amide-modified polystyrene spheres to be used as a detection reagent for the specific identification of group B streptococcal cultures grown in selective or nonselective Todd-Hewitt broth for 5 and 4 h, respectively. Agglutination occurred when the lectin reagents were allowed to react with either the cell suspension, clarified broth, or antigen extracts from group B streptococci grown in Todd-Hewitt broth. No agglutination occurred when these lectins were allowed to react with strains of serogroup A, C, D, F, or G streptococci. False-negative agglutination responses may occur with certain serotype of group B streptococci grown on Columbia sheep blood agar. A 20-min staining time permitted the specific labeling of fixed smears of group B streptococci with fluorescein-conjugated Lycopersicon lectin. The lectin from the solanaceous plant Datura stramonium did not agglutinate group B streptococci or other clinically significant streptococcal serogroups.     

Specific detection of Haemophilus influenzae type b lipooligosaccharide by a polymyxin B monoclonal antibody assay

A monoclonal antibody (MAb)-based enzyme immunoassay was developed for detection of Haemophilus influenzae type b (Hib) lipooligosaccharides (LOS). The high affinity of polymyxin B for lipid A was used to bind the Hib LOS to microtiter wells. The immobilized LOS was detected with MAbs directed against the oligosaccharide component of Hib endotoxin. Hib LOS concentrations were measured in in vitro samples and in cerebrospinal fluid (CSF) sample obtained from rabbits with experimental Hib meningitis. The sensitivity of the assay was 1 ng LOS/ml sample and the results obtained with this assay correlated significantly with those obtained with the standard Limulus amebocyte lysate assay. This new assay provides a method for specific detection of Hib LOS in CSF samples and in aqueous laboratory fluids. This general methodology should also be useful for experimental research involving specific LPS/LOS molecules.     

Comparison of monoclonal antibody time-resolved fluoroimmunoassay with monoclonal antibody capture-biotinylated detector enzyme immunoassay for respiratory syncytial virus and parainfluenza virus antigen detection.

An all-monoclonal antibody, time-resolved fluoroimmunoassay was compared with several enzyme immunoassays for the detection of respiratory syncytial virus and parainfluenza virus type 1, 2, and 3 antigens in clinical specimens. The most sensitive enzyme immunoassay for parainfluenza virus type 1 was an all-monoclonal antibody assay with biotin-labeled detector antibody and streptavidin-peroxidase conjugate, but for respiratory syncytial virus and parainfluenza virus types 2 and 3 the most sensitive assay was a polyclonal antibody assay with horse capture antibodies and bovine or rabbit detector antibodies with anti-species peroxidase. All tests were evaluated with nasopharyngeal aspirate specimens from respiratory illnesses and with cell culture harvests of multiple strains of each virus isolated over many years. The time-resolved fluoroimmunoassay detected respiratory syncytial virus antigen in 92% of the specimens positive by culture, which was a decidedly higher sensitivity than either the monoclonal or polyclonal antibody enzyme immunoassay format (62 and 76%, respectively). For the parainfluenza viruses the time-resolved fluoroimmunoassay detected type-specific antigen in 94 to 100% of culture-positive specimens and again was more sensitive than the all-monoclonal antibody enzyme immunoassays (75 to 89%) or all-polyclonal antibody enzyme immunoassays (66 to 95%). Combined with results from a previously reported adenovirus time-resolved fluoroimmunoassay, these tests identified respiratory antigens in large numbers of clinical specimens.     

Detection of group B streptococcal antigen in early-onset and late-onset group B streptococcal disease with the Wellcogen Strep B latex agglutination test.

The Wellcogen Strep B latex agglutination test (Wellcome Diagnostics, Dartford, England) was evaluated as a method of detecting group B streptococcal antigen in urine, cerebrospinal fluid, and serum from neonates with early-onset (less than or equal to 7 days of age) and late-onset group B streptococcal disease. Urine was the best source of antigen, which was detected in 100% of six neonates with early-onset group B streptococcal disease who had urine available in the first 12 h of illness and in 88% of 17 group B streptococcus-infected neonates with urine available in the first 48 h of illness. Antigen was not detected in any samples from patients without group B streptococcal disease except in the urine of one patient with Proteus mirabilis meningitis. The Wellcogen Strep B latex test of the lot tested compares favorably with a noncommercially available latex agglutination test.     

Rapid detection of influenza virus by shell vial assay with monoclonal antibodies.

Of 45 influenza virus strains (43 type A and 2 type B) detected in conventional tube cell cultures (average time, 4 days), 25 (56%) were detected by immunofluorescence in the shell vial assay 24 h postinoculation. The specific fluorescence produced should allow this procedure to be readily adapted by laboratories with various degrees of experience with immunofluorescence methodology.     

Rapid detection of Bordetella pertussis by a monoclonal antibody-based colony blot assay.

Monoclonal antibodies to Bordetella pertussis filamentous hemagglutinin (FHA) and lipopolysaccharide (LPS) were used in a colony blot enzyme-linked immunosorbent assay designed for rapid detection of B. pertussis. Bacterial colonies from Bordet-Gengou agar plates were blotted onto nitrocellulose filter disks, lysed by immersion in chloroform, and reacted with monoclonal antibodies. Following reaction with peroxidase-conjugated rabbit anti-mouse immunoglobulin antisera and 4-chloro-1-naphthol, blue dots representing single colonies appeared on the filters. Blotting of single B. pertussis colonies could be performed after incubation for 40 h, i.e., before the colonies were visible by eye on the agar surface. Ten of ten B. pertussis strains showed positive blotting reactions with antibodies specific for B. pertussis FHA and LPS. Fourteen of fourteen B. parapertussis strains reacted with two of the FHA-specific antibodies but not with two of the LPS-specific antibodies. Strains of B. bronchiseptica showed a variable reaction pattern. No cross-reactions were observed with strains of Streptococcus mitis, S. pyogenes, S. pneumoniae, Staphylococcus aureus, Branhamella catarrhalis, or Klebsiella pneumoniae. This assay may be useful for identification of B. pertussis and B. parapertussis in suspected cases of whooping cough.     

Characterization of monoclonal antibodies to human group B rotavirus and their use in an antigen detection enzyme-linked immunosorbent assay

Three monoclonal antibodies (MAbs)--B5C9, B5E4, and B10G10--to human group B rotavirus, an agent implicated in epidemic outbreaks of diarrhea in the People's Republic of China, primarily in adults, were prepared. MAb reactivity was decreased when virus preparations were treated with EDTA, suggesting reactivity with the outer-capsid protein(s). Competition experiments suggested that these MAbs recognize overlapping epitopes within a single antigenic site. A simple antigen detection enzyme-linked immunosorbent assay (ELISA) specific for the human group B rotavirus was established by using these MAbs as capture antibodies. Fifteen clinical samples obtained from three epidemic areas in the People's Republic of China and previously shown by Chinese scientists to contain group B virus were all positive in the MAb capture antigen detection ELISA, whereas none of the 57 samples lacking the group B virus reacted in the test. The results suggest that this MAb capture antigen detection ELISA will be useful to identify outbreaks caused by the human group B rotavirus and to monitor possible spread of the virus.