Enzyme immunoassay for detection of Giardia lamblia cyst antigens in formalin-fixed and unfixed human stool.

An antigen-capture enzyme-linked immunosorbent assay employing rabbit and mouse antisera to Giardia lamblia cyst antigens was developed for the diagnosis of Giardia infection through detection of G. lamblia-specific stool antigens in cell-free aqueous eluates of human stool. This is the first report of the use of anti-cyst antibodies in an enzyme immunoassay for G. lamblia. The assay gave a positive result with 54 of 59 stools from patients with symptomatic, clinically diagnosed giardiasis, giving the test a sensitivity of 91.5%. A negative reading was obtained with all of 25 stools from G. lamblia-negative control patients. The assay could detect as few as 20 sonicated cysts added to control stool eluate. The assay was more sensitive to cyst-derived antigens than to trophozoite-derived antigens. With two exceptions, the assay gave a negative result with stools from patients infected with Entamoeba histolytica (seven), Cryptosporidium sp. (four), or Blastocystis hominis (seven) and thus appears to be specific for G. lamblia antigens. Storage of stool eluates for more than 6 months at 4 degrees C as unpreserved aqueous eluates or as formalinized eluates did not affect the ability of the assay to detect the giardial antigens. The enzyme-linked immunosorbent assay proved useful for monitoring the levels of G. lamblia-specific stool antigens in the stool of patients undergoing antigiardial chemotherapy.     

Enzyme immunoassay for detection of antibody-coated bacteria.

To quantitatively evaluate factors potentially affecting antibody coating of bacteria in urine, we developed an assay with enzyme-linked rather than fluorescein-conjugated immunoglobulin. Using the enzyme immunoassay (EIA) in an in vitro system in which concentrations of serotype O44 Escherichia coli and antibody titer to E. coli Orr O44 O antigen were known, we compared specimens run in parallel with a fluorescent antibody (FA) assay. At greater than or equal to 10(5) bacteria per ml, antibody titer to homologous O antigen correlated directly with absorbance in the EIA. Both tests had sensitivities exceeding 95% in specimens containing greater than or equal to 10(5) bacteria per ml, but the FA test detected 23 of 27 positive specimens with less than 10(5) bacteria per ml compared with 21 of 43 detected by EIA (P = 0.002). However, nonspecific fluorescence caused false positives in 8% of negative tests run by FA compared with 1% of simultaneous EIA tests (P = 0.05). pH alterations and pretreatment of bacteria with antibiotics did not affect either test. Heterologous E. coli strains showed no cross-reactivity with O44 antiserum, but all Staphylococcus aureus isolates tested caused false positives in both assays, and one Klebsiella strain repeatedly caused a false-positive FA assay. The EIA appears to be a simple, quantitative, and specific technique for detection of antibody-coated bacteria in this experimental system.     

Detection ofGiardia lamblia cysts in stool samples by immunofluorescence using monoclonal antibody

The diagnostic potential of indirect immunofluorescence to detectGiardia cysts in stool samples using a cyst-specific anti-Giardia lamblia monoclonal antibody was evaluated in comparison to conventional light microscopy. One hundred fifty specimens from clinically suspectedGiardia infections and 50 control samples from microscopically provedGiardia infections were tested.Giardia cysts were found in 15 of 150 (10 %) samples tested by light microscopy, whereas immunofluorescence microscopy detected 35 of 150 (23 %) positive samples. Forty-six of the 50 reference samples previously shown to containGiardia cysts were positive. Apparently, the four discrepant samples contained very low numbers of parasites, as none could be detected by conventional microscopy. The results show thatGiardia lamblia cysts are detected significantly more frequently using the antibody marker. The doubled number of positive stool specimens and detection of as little as four cysts per sample suggest that microscopical examination of samples can be improved by immunofluorescent staining ofGiardia lamblia cysts.     

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.     

Enzyme immunoassay for the detection of streptomycin and dihydrostreptomycin in milk

Polyclonal antisera against streptomycin were prepared by using a streptomycin‐oxime derivative coupled to bovine serum albumin for the immunization of rabbits. The specificity and sensitivity of these antibodies were tested in a competitive assay using a streptomycinenzyme conjugate (prepared by coupling a streptomycin‐hydrazone derivative to horseradish peroxidase) in a double antibody solid phase technique. The only detectable cross‐reactivity of the assay system with other aminoglycoside antibiotics and other substances similar in structure was shown to be with dihydrostreptomycin of about 148.7%. The detection limit in buffer solution was 0.6 ng ml^‐1 for streptomycin and 0–4 ng ml^‐1 for dihydrostreptomycin. Employing rapid sample preparation procedures, streptomycin and dihydrostreptomycin were detected in milk at levels as low as 6 and 0.8 ng ml^‐1respectively.     

Enzyme immunoassay for detection of antigen in acutePlasmodium falciparum malaria

A monoclonal antibody specific for an epitope of a 50 kDa Plasmodium falciparum antigen was used in an enzyme immunoassay for detection of the corresponding exo-antigen in culture supernatant and in the sera of 31 patients suffering from acute malaria. The assay was specific for Plasmodium falciparum and did not appear to be strain restricted. A parasitaemia level below 0.001 % could be detected.     

Enzyme immunoassay and enzyme-linked fluoroimmunoassay for detection ofChlamydia trachomatis antigen

An enzyme immunoassay (EIA) and an enzymelinked fluoroimmunoassay (ELFIA) utilizing monoclonal antibody to major outer membrane protein of Chlamydia trachomatis L2 were developed for rapid detection of chlamydial antigen in clinical samples. The EIA and ELFIA could detect levels of purified chlamydial outer membrane protein as low as 1.0 and 0.2 ng/ml respectively. However, when EIA and ELFIA were compared to chlamydial isolation using 160 patient samples, the sensitivity rate was 68 % and 85% respectively. The sensitivity of the antigen detection method might be increased by simply using less diluted samples than in the present study. Chlamydial antigen was also demonstrated by EIA and ELFIA in 15% and 23% of culture-negative samples. The reason for these false-positive findings remains undetermined.     

Enzyme immunoassay for detection of pneumococcal antigen in cerebrospinal fluid.

A solid-phase immunoassay utilizing horse antiserum against the C polysaccharide of Streptococcus pneumoniae and biotinylated rabbit antibodies to type-specific pneumococcal polysaccharides was developed to detect pneumococcal antigens in human body fluids and in broth cultures. Pneumococcal antigen could be detected in broth cultures of serotypes of S. pneumoniae containing as little as 10(2) to 10(3) organisms per ml. The assay system detected pneumococcal antigen in all 25 cerebrospinal fluid specimens obtained from patients with documented pneumococcal meningitis. There were no positive reactions noted in specimens from patients infected with Neisseria meningitidis group A or from patients without evidence of bacterial infection. The solid-phase enzyme immunoassay utilizing these reagents is a sensitive and specific assay for the immunodetection of a wide range of pneumococcal antigens.     

Enzyme immunoassay for detection of antibodies against isolated flagella ofLegionella pneumophila

Flagella ofLegionella pneumophila serogroup 1, strain Philadelphia (ATCC 33152), were isolated and used as antigen to detect antibodies by enzyme immunoassay (EIA) in sera of patients with significant titers againstLegionella as determined by the indirect immunofluorescence assay (IFA). Healthy blood donors served as a control group. Whereas the sera of blood donors were negative in both assays, sera of patients showed two patterns of reactivity. Sera with elevated titers againstLegionella pneumophilasero-group 1 orLegionella gormanii in IFA usually demonstrated high titers in EIA. However, high IFA titers againstLegionella bozemanii were associated with low titers in EIA. The data show that flagella protein ofLegionella pneumophila serogroup 1 strain Philadelphia is of limited value for the detection of antibodies againstLegionella other thanLegionella pneumophila serogroup 1, although in vitro studies revealed genus-specific epitopes. This might be due to unpredictable variabilities in the expression of flagella in vivo.     

Evaluation of rapid commercial enzyme immunoassay for detection of Giardia lamblia in formalin-preserved stool specimens.

Two hundred twenty-three formalin-preserved stool specimens were evaluated by using ProSpecT Giardia Rapid Assay (membrane bound) (Alexon, Inc., Sunnyvale, Calif.). Enzyme immunoassay (EIA) results were compared with those by conventional microscopic examination. Two hundred four specimens were negative by both methods, and 13 (6.3%) were positive. Five specimens were negative by initial microscopic exam and positive by EIA; three of these specimens were found to be positive upon extensive microscopic reexamination. The remaining two specimens were from patients who previously tested positive and who had recurrent symptoms of or responded to therapy for giardia. Therefore, we consider both cases to be true positives. One specimen exhibited a single cyst by microscopic exam and was negative by EIA Resolved results yielded a relative sensitivity of 95%, a relative specificity of 100%, a positive predictive value of 99.6%, and a negative predictive value of 100%, compared with a sensitivity of 74% for conventional microscopy.     

Enzyme immunoassay for detection of human immunodeficiency virus-specific immunoglobulin A antibodies.

Early diagnosis of human immunodeficiency virus (HIV) infection may be difficult in adults with acute or recent HIV infection and in infants with perinatally acquired HIV. Detection of HIV-specific immunoglobulin A (IgA) antibodies in infant serum by Western blot (immunoblot) has been suggested as a reliable method to identify HIV-infected infants, especially those over the age of 6 months, and as an adjunct to diagnosis of acute HIV infection in adults. We developed a simple enzyme immunoassay for detection of HIV-specific IgA, using standard commercially available reagents. Enzyme immunoassay was comparable to Western blot for detection of HIV-specific IgA in sera from adults (n = 216), older children (n = 49), and infants born to HIV-infected mothers (n = 65). Specificity was 100% and sensitivity ranged from 80 to 92%. IgA-enzyme immunoassay is a simple, highly sensitive method for detection of HIV-specific IgA antibodies and is easily adapted to the standard clinical laboratory.     

Enzyme immunoassay for detection of antibody to Epstein-Barr virus-specific early antigen.

Antibody to Epstein-Barr virus-induced early antigen could be measured in sera using enzyme-linked antibody. The sensitivity of this assay was comparable to that of the indirect immunofluorescent technique. Measurement of early antigen was accomplished on a producer cell line which was specifically treated to block late gene expression. It is now possible to measure Epstein-Barr virus-induced early antigen and viral capsid antigen antibodies by using the same cell line.     

Enzyme immunoassay for the measurement of histamine

This paper reports a competitive solid-phase enzyme immunoassay for measuring histamine in various biological samples. In this assay, the histamine to be quantified is chemically modified by 1,4-benzoquinone treatment and allowed to compete with a histamine-peroxidase conjugate for binding to a limited amount of an anti-histamine monoclonal antibody which was used to coat the wells of a microtitration plate. After incubation and washing, peroxidase activity associated with the solid phase is measured. With this method the histamine concentration in blood or various tissues may be determined easily, safely and reproducibly. Histamine concentrations from 0.3 to 20 ng/ml may be measured with the procedure reported here.     

Comparison of microscopy, rapid immunoassay, and molecular techniques for the detection of Giardia lamblia and Cryptosporidium parvum

Giardia lamblia and Cryptosporidium parvum are recognized as the most common protozoan infections in Saudi Arabia. Microscopic examination of stool samples, either direct or concentrated, for the recovery of G. lamblia cysts and trophozoites and C. parvum oocysts is still the most commonly used for the diagnosis of both parasites. We compared the conventional parasitological techniques of iodine-stained wet mount for G. lamblia and Kinyoun's acid-fast for C. parvum against ImmunoCard STAT® Cryptosporidium/Giardia and real-time polymerase chain reaction (PCR) detecting the 18S rRNA gene of G. lamblia and conventional PCR detecting the same gene of C. parvum at a tertiary hospital in Dhahran, Saudi Arabia. Out of 148 stool samples, 19 and 12 true positives were identified for G. lamblia and C. parvum, respectively, using a composite reference standard. In this case, true positives and negatives were considered as those with at least two positive or negative results out of the three tests. Both ImmunoCard STAT! and PCR methods were more sensitive than the microscopic tests of a single stool specimen of 85.7 % (CI?=?62.6–96.2 %) and 85.7 % (CI?=?56.2–97.5 %) for G. lamblia and C. parvum, respectively. However, specificity of microscopic tests was higher than other techniques for both parasites. Although PCR seems to be most sensitive for both G. lamblia and C. parvum, its low specificity may render its superiority over other techniques. When a single stool sample is used for detection of G. lamblia and C. parvum, better results can be obtained when coupled with serological testing. Although PCR is the most sensitive method for the detection of both G. lamblia and C. parvum, its use requires attention in relation to the increased possible false positives.     

Enzyme immunoassay for the detection of Chlamydia trachomatis antigen in urethral and endocervical swabs.

An enzyme immunoassay technique based on the direct detection of Chlamydia trachomatis antigen in urethral or cervical swabs was used for the rapid diagnosis of chlamydial genital infection. Urethral and cervical samples from 140 patients were tested in parallel by enzyme immunoassay and cell culture using iodine staining. The direct test had a sensitivity of 92.5% and specificity of 97.2% when compared with the cell culture system. The enzyme immunoassay technique provides a rapid and simple method for diagnosing chlamydial genital infection and may be performed on a large number of samples in laboratories which do not have tissue culture facilities or a trained microscopist.     

Comparison of microscopy, real-time PCR and a rapid immunoassay for the detection of Giardia lamblia in human stool specimens

Giardia lamblia is one of the most common intestinal parasites worldwide, with microscopy being the diagnostic reference standard for use with human stools. However, microscopy is time-consuming, labour-intensive and lacks sensitivity when single stools are examined. In the present study, microscopy, real-time PCR and a rapid immunoassay were compared for the detection of G. lamblia in human stools. All three methods were highly sensitive, with values of 99%, 100% and 98%, respectively. Specificity and positive and negative predictive values were >or=97%, except when using real-time PCR, for which the specificity and positive predictive value were 92% and 93%, respectively. The lower specificity of real-time PCR was associated mostly with failure to detect specimens regarded as true positives for G. lamblia DNA, although cross-contamination was suspected in a minority of cases because of the large amount of G. lamblia DNA present in most positive specimens. It was concluded that microscopy should remain the primary diagnostic tool for identifying G. lamblia in human stools, mainly because of its ability to simultaneously detect other gastrointestinal parasites. However, the simple and rapid immunoassay is a valuable tool to decrease turn-around time. Real-time PCR provides additional sensitivity, although there is a risk of cross-contamination. Based on this observation, and the need for other real-time assays to be developed to detect other intestinal parasites, real-time PCR is currently useful only as an additional test supplementary to microscopy.     

Monoclonal antibody-based enzyme immunoassay for Giardia lamblia antigen in human stool

A visually readable monoclonal antibody-based antigen-capture enzyme immunoassay for the detection of Giardia lamblia antigen in human stool specimens was developed and found to be 97% (30 of 31 stool specimens) sensitive for formalinized stools and 82% (49 of 60 stool specimens) sensitive for unfixed stool specimens by visual reading. The storage of specimens in 10% Formalin resulted in increased absorbance in 20 of 26 G. lamblia-positive specimens tested as both formalinized and unfixed specimens; the increase averaged 1,336%. The assay was specific for antigens of this organism and for antigens derived from the cyst, as opposed to the trophozoite, stage. The assay could detect the antigens of five cysts per well, but could not detect antigen in in vitro-cultured trophozoites. A mouse monoclonal antibody of the immunoglobulin G1 (IgG1) subclass, which was prepared against cysts of G. lamblia, was used as the solid-phase capture antibody. The antibody was reactive with the cyst wall, as determined by immunofluorescence. Polyclonal rabbit anti-cyst IgG was used as the secondary antibody, and peroxidase-labeled goat anti-rabbit IgG was used as the tertiary antibody in the assay format. Maximal capture of antigen from stool specimens occurred by 30 min. Optimal dilution of specimens was in the range of 1:60 to 1:600. Preliminary characterization of affinity-purified antigen recognized by the monoclonal antibody showed that it is heat stable (100 degrees C, 12 min) and resistant to sodium periodate treatment and that it may exist in multiple molecular weights from 45,000 to 110,000.