Diagnosis of amebic dysentery by detection of Entamoeba histolytica fecal antigen by an invasive strain-specific, monoclonal antibody-based enzyme-linked immunosorbent assay.

An invasive strain-specific monoclonal antibody against Entamoeba histolytica has been used in a capture enzyme-linked immunosorbent assay (ELISA) for the detection of invasive E. histolytica fecal antigen in clinical specimens and for the diagnosis of amebic dysentery in patients from Bangladesh. The fecal antigen capture ELISA (FAC-ELISA) did not cross-react with other parasite species in the clinical specimens or with noninvasive E. histolytica present in those specimens and in experimentally seeded stools. The limit of detection of the assay for invasive E. histolytica crude antigen diluted in phosphate-buffered saline or in stools was 0.58 and 3.9 micrograms/ml, respectively, which is the equivalent of approximately 72 and 487 E. histolytica trophozoites per well, respectively. The sensitivity, specificity, and efficiency of the FAC-ELISA were 87, 100, and 98%, respectively, for the detection of invasive E. histolytica antigens and 100, 100, and 100%, respectively, for the diagnosis of amebic dysentery. The FAC-ELISA is a potential alternative for the field diagnosis of amebic dysentery and for epidemiological studies to define the distribution of invasive E. histolytica.     

Diagnosis of typhoid fever by detection of Salmonella typhi antigen in urine.

A monoclonal antibody specific for group D Salmonella antigen 9 was used in an indirect enzyme-linked immunosorbent assay (ELISA) for detecting the antigen in urine specimens collected from patients with clinical typhoid fever in Jakarta, Indonesia. The ELISA had a sensitivity of 95% in identifying patients in whom Salmonella typhi was isolated from hemocultures, 73% in patients in whom S. typhi was isolated from stool specimens, and 40% in patients in whom the organism was isolated from bone marrow cultures. Among patients in whom S. typhi was isolated from blood cultures, the ELISA had a sensitivity of 65% when a single urine specimen was examined and 95% when serially collected urine specimens were examined. A dot blot immunoassay performed on a nitrocellulose filter in parallel had a sensitivity of 85%, versus 83% for the plate ELISA in which S. typhi was isolated from blood, bone marrow, and/or stool specimens. Since S. typhi antigen is intermittently excreted in the urine of patients with typhoid fever, serially collected urine from patients with typhoid should be tested for antigen 9.     

Evaluation of a monoclonal antibody-based test for detection of Helicobacter pylori-specific antigen in stool samples from mice

A test using monoclonal antibodies for detection of antigen in stool samples was compared with culture and histology for noninfected (n = 25), Helicobacter pylori-infected (n = 25), and Helicobacter felis-infected (n = 6) mice. Sensitivity and specificity were 96%. The monoclonal antibody-based test is therefore a noninvasive technique that is able to diagnose H. pylori infection in mice.     

A monoclonal antibody-based immunoassay for human lactoferrin

Monoclonal antibodies against human lactoferrin define at least 3 and possibly as many as 6 different epitopes. A sandwich enzyme-linked immunoassay, using monoclonals against different epitopes, has been optimised for the measurement of serum lactoferrin. In 35 samples from healthy adults the mean lactoferrin content of serum from blood clotted overnight was 0.54 +/- 0.26 micrograms/ml.     

Diagnosis of disseminated histoplasmosis by detection of Histoplasma capsulatum antigen in serum and urine specimens

The diagnosis of Histoplasma capsulatum infection by serologic testing for the presence of antibodies is limited by a high rate of false positive and false negative results and by the requirement that the patient have a normal immune response. We have developed a radioimmunoassay for the detection of H. capsulatum antigen in urine and serum specimens. Antigenuria was noted in 20 of 22 episodes of disseminated histoplasmosis that occurred in 16 patients, in 6 of 32 patients with self-limited infection, in 2 of 32 patients with cavitary histoplasmosis, and in 4 of 8 patients with a sarcoid-like illness caused by H. capsulatum. The detection of antigen in urine was reproducible in 38 of 41 (93 percent) retests of specimens. H. capsulatum antigen was also detected in the serum during 11 of the 22 episodes of disseminated histoplasmosis, in none of the 12 episodes of other types of histoplasmosis in patients with antigenuria, in 1 of the 33 patients with histoplasmosis who lacked the urinary antigen, and in none of the 50 controls. Antigenemia and antigenuria decreased after initiation of antifungal therapy and recurred in patients who had a relapse. We conclude that this radioimmunoassay for H. capsulatum antigen represents a useful new method for the rapid diagnosis of disseminated histoplasmosis.     

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.     

Interference in double-determinant monoclonal antibody-based assay for carcinoembryonic antigen (CEA)

We investigated the false positive phenomena in immunoassays of carcinoembryonic antigen (CEA) using mouse monoclonal antibodies, and studied the properties of interfering substances and the method to eliminate non-specific interference. Serum samples from 2,250 patients, which indicated more than 10 ng/ml of CEA with EIA test kit (Boehringer Mannheim Yamanouchi; BMY) in about 25,000 samples of CEA tests, were subjected to extraction by heating at 70 degrees C followed by CEA measurement of the supernatant. The overall correlation was good between CEA values of the original and extraction methods, but CEA values in 55 cases were found to be remarkably higher in the non-extracted method than the extraction method. CEA titers of 20 samples of the discrepant cases were measured by various commercial kits, and the result indicated that non-specific interference was unavoidable in any kit using a monoclonal antibody-based double-determinant immunoassay, in spite of difference in degree and frequency of interference. The interfering activity of two patients serum with remarkably discrepant values eluted at the void volume of a large molecular size of more than 1,000 kDa in gel chromatography of Sephacryl S-300. The non-specific interference was reduced by addition of mouse gamma-globulin to the buffer of BMY kit and abolished by pretreatment of the serum with heat-killed cells of Staphylococcus aureus, suggesting that interference might be caused by some species of immunoglobulin being able to bind mouse gamma-globulin.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Standardization of antigen E in ragweed pollen extracts using a monoclonal antibody-based enzyme immunoassay

A hybridoma-derived monoclonal IgG antibody specific for ragweed AgE was used to develop a competitive binding enzyme immunoassay suitable for quantitation of antigen E levels in ragweed pollen extracts. The assay was capable of detecting as little as 30 ng/ml AgE in crude pollen extracts. The monoclonal antibody was shown to react with AgE present in commercial pooled pollen extracts from a number of ragweed species as well as a laboratory extract from a single species. In contrast to previous conventional xenoantisera, it could distinguish true ragweed (Ambrosia sp) from false ragweed (Franseria sp). The use of monoclonal antibodies in assay systems such as this offers a reproducible and widely applicable method for allergen standardization.     

Application of a monoclonal antibody-based enzyme-linked immunosorbent assay for detection of an inflammatory response antigen in subclinical mastitic milk samples.

A monoclonal antibody to a 23.5-kDa bovine inflammatory antigen present in high levels in mastitic milk has been used in an antigen-capture enzyme-linked immunosorbent assay (ELISA) to screen milk samples from herds of cattle for subclinical mastitis. The results from 20 herds with a total of 2,612 quarter samples are presented. Good correlation was observed between the ELISA level and the milk cell count (MCC). The results demonstrated an average of 5% false negatives (1.8% associated with isolates of Staphylococcus aureus and/or Streptococcus spp.) and 7.7% false positives for each herd in relation to mastitic (greater than 400,000 cells per ml) or nonmastitic (less than 400,000 cells per ml) MCCs.     

Development of monoclonal antibody-based immunoassays for detection of Helicobacter pylori neutrophil-activating protein

One candidate preparation of human sequence recombinant transforming growth factor-β3 (TGF-β3) was formulated and lyophilized at NIBSC prior to evaluation in a collaborative study for its suitability to serve as an international standard. The preparation was tested by 8 laboratories using in vitro bioassays and immunoassays. The candidate preparation 09/234 was judged suitable to serve as an international standard based on the data obtained for biological activity and stability. On the basis of the results reported here, the preparation coded 09/234 was established by the WHO Expert Committee on Biological Standardisation (ECBS) as the WHO 1st IS for human TGF-β3 with an assigned value for TGF-β3 activity of 19,000 IU/ampoule.     

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.     

Diagnosis of pneumococcal pneumonia by detection of antigen in saliva

A sandwich enzyme-linked immunosorbent assay was used for the detection of pneumococcal C polysaccharide in saliva samples from patients with radiologically verified pneumonia. In 16 of 29 patients (55 %) with pneumococcal pneumonia, as verified by conventional culture methods, the antigen was present in saliva specimens. The test was negative in 35 of 36 patients (97 %) with non-pneumococcal pneumonia and in all saliva samples from a control group, consisting of 25 patients with no signs of respiratory disease. It is concluded that the detection of pneumococcal C polysaccharide in saliva offers a valuable complement to conventional diagnostic methods for pneumonoccal pneumonia.     

Diagnosis of pneumococcal pneumonia by antigen detection in sputum.

Pneumococcal polysaccharide was detected by counterimmunoelectrophoresis in the sputum of 20 of 26 (77%) adults with community-acquired pneumonia and a positive sputum culture for Streptococcus pneumoniae. The test was negative in 29 pneumonia patients with negative sputum culture for S. pneumoniae.Pneumococcal antigen was also detected in the sputum of six of nine adults with chronic bronchitis and a positive sputum culture, but was not detected in expectorated respiratory secretions of 22 pneumococcal carriers with colds. Pneumococcal antigen could also be detected in sputum by immunodiffusion; antigen titers varied from 1:2 to 1:256. These results strongly suggest that the detection of pneumococcal antigen in respiratory tract secretions indicates infection caused by S. pneumoniae.     

Sensitive and specific monoclonal antibody-based capture enzyme immunoassay for detection of nucleocapsid antigen in sera from patients with severe acute respiratory syndrome

A rapid antigen test for the diagnosis of severe acute respiratory syndrome (SARS) is essential for control of this disease at the point of management. The nucleocapsid (N) protein of SARS-associated coronavirus (SARS-CoV) is abundantly expressed in infected-cell culture filtrate as demonstrable by Western blotting using convalescent-phase sera from patients with SARS. We used monoclonal antibodies specifically directed against N protein to establish a sensitive antigen capture sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of SARS-CoV. The assay employed a mixture of three monoclonal antibodies for capture and rabbit polyclonal antibodies for detection of serum antigen in 32 cases of clinically probable SARS as defined by the World Health Organization during the epidemic in Guangzhou, China. Recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml. The linear range of detection in clinical specimens was from 100 pg/ml to 3.2 ng/ml. Using a panel of sera collected at different points in time, the amount of circulating N antigen was found to peak 6 to 10 days after the onset of symptoms. The sensitivity of the assay was 84.6% in 13 serologically confirmed SARS patients with blood taken during the first 10 days after the onset of symptoms (11 of 13). The specificity of the assay was 98.5% in 1,272 healthy individuals (1,253 of 1,272). There was no cross-reaction with other human and animal coronaviruses in this assay. In conclusion, a sensitive and quantitative antigen capture ELISA was established for the early diagnosis and disease monitoring of SARS-CoV infection.     

Development of a monoclonal antibody-based sandwich ELISA for the detection of ovalbumin in foods

Hen egg is one of the most frequent causes of food allergy in infants and adults. Ovalbumin (OVA) has been identified as a major egg allergen. In order to detect OVA in foods, a highly sensitive sandwich enzyme-linked immunosorbent assay (ELISA) based on two monoclonal antibodies (mAbs) was established. The 2 mAbs were selected out of 17 murine hybridomas secreting OVA-specific antibody. Using mAb17 as the capture antibody and mAb15 as the detection antibody, the detection limit of the ELISA method was 0.51 ng/mL, and the linear dynamic range was between 1.95 and 500 ng/mL. The recovery ranged from 85.6 to 115.2%, whereas the intra- and inter-assay coefficients of variation were less than 8.6 and 13.9%, respectively. Sample analysis verified that the produced anti-OVA mAb and the developed ELISA may provide a valuable tool for the sensitive determination of OVA in processed foods and for future studies on the mechanism of how OVA functions in anaphylaxis.     

Evaluation of a solid-phase monoclonal antibody-based enzyme immunoassay for insulin in human serum

A 'sandwich-type' enzyme immunoassay for the measurement of serum insulin is described in which a monoclonal antibody-alkaline phosphatase conjugate and an antibody-immobilized polystyrene solid phase are used. Serum samples of 50 microliters can be analyzed and the enzyme immunoassay is as sensitive as the conventional radioimmunoassay for insulin. The results obtained with ELISA correlate well with those of the radioimmunoassay (r = 0.9844) and the between-assay and within-assay coefficients of variation are less than 15% over the useful ranges of the assay (2-200 microIU/ml). The sensitivity is 2 microIU/ml and this can be increased by longer incubation times. The crossreaction with porcine insulin is 45%, with bovine insulin 30% and with human proinsulin 20%.     

A human leukocyte antigen identified by a monoclonal antibody

Leukocyte antigen of approximate molecular weight 200,000 daltons have beendescribed in mouse, rat, and man. We described here the reactivities of a monoclonal antibody. GAP 8.3, which identified such an antigen on human leukocytes. We found the leukocyte antigen H-T200 on T and B lymphocytes, granulocytes, monocytes, and platelets, but not on erythrocytes or nonhematopoetically derived cells. Resting and activated T cells had more antigen or their surfaces than did resting B lymphocytes and EBV-transformed B cells, respectively. The leukocyte antigen was detected on approximately 75% of bone marrow cells; cells of the erythroid series comprised the negative population. The GAP 8.3 antibody and its F(ab′)₂ fragments had no effect on in vitro stimulation of peripheral blood cells by mitogens or allogeneic cells. Antigen isolated from T cell lines had a higher electrophoretic mobility than did antigen from B cell lines; antigen from the mycloid line U937 comigrated with that from B cell lines. In addition, we detected very small but reproducible differences in the electrophoretic mobility of the antigen on two T cell lines.