Dot Enzyme Immunoassay Research Articles

Rapid diagnosis of Japanese encephalitis by using an immunoglobulin M dot enzyme immunoassay.

Japanese encephalitis (JE) occurs in rural settings in southern and eastern Asia, where diagnostic facilities are limited. For the diagnosis of JE virus (JEV) infection, we developed a nitrocellulose membrane-based immunoglobulin M (IgM) capture dot enzyme immunoassay (MAC DOT) that is rapid, simple to use, requires no specialized equipment, and can distinguish JEV from dengue infection. In a prospective field study in southern Vietnam, 155 cerebrospinal fluid (CSF) and 341 serum samples were collected from 111 children and 83 adults with suspected encephalitis. The JEV MAC DOT, performed on site, was scored visually from negative to strongly positive by two observers, and the results were compared subsequently with those of the standard IgM capture enzyme-linked immunosorbent assay. For the 179 patients with adequate specimens, the MAC DOT correctly identified 59 of 60 JEV-positive patients and 118 of 119 JEV-negative patients (sensitivity [95% confidence intervals], 98.3% [92.1 to 99.91%]; specificity, 99.2% [95.9 to 100.0%]; positive predictive value, 0.98; negative predictive value, 0.99). The MAC DOT also correctly identified three patients with dengue encephalopathy. Admission specimens were positive for 73% of JE patients. Interobserver agreement for MAC DOT diagnosis was excellent (kappa = 0.94). The JEV MAC DOT is a simple and reliable rapid diagnostic test for JE in rural hospitals.

Occurrence of S and F1C/S-related fimbrial determinants and their expression in Escherichia coli strains isolated from extraintestinal infections.

The presence of S and F1C/S-related fimbrial determinants was determined in 462 E. coli strains obtained from different extraintestinal infections and in 162 control isolates of E. coli by using two different DNA probes: an oligonucleotide probe consisting of three oligonucleotides that bind specifically to the S adhesin gene and a polynucleotide probe which is not able to distinguish between S, F1C, and S-related sequences. The expression of S and F1C phenotypes was tested by dot enzyme immunoassay with the corresponding monoclonal antibodies. S fimbriae genotypes were observed more frequently in septic (25%) and urinary (12%) isolates of E. coli than in faecal and water isolates (1%) and often occurred together with O2, O6, O18 and O83 antigens. F1C/S-related fimbrial DNA was detected with a higher frequency in UTI isolates (26%) than in septic (16%) and faecal (10%) isolates and was most frequently associated with O4, O6, and O75 serotypes. Since the production of S and F1C fimbriae was comparatively rare in all clinical and control isolates of E. coli, DNA hybridization assays which allow the sensitive and specific detection of fimbrial determinants even in the absence of their expression are preferable to phenotypic assays.

Occurrence of S and F1C/S-related fimbrial determinants and their expression in Escherichia coli strains isolated from extraintestinal infections

The presence of S and F1C/S-related fimbrial determinants was determined in 462 E. coli strains obtained from different extraintestinal infections and in 162 control isolates of E. coli by using two different DNA probes: an oligonucleotide probe consisting of three oligonucleotides that bind specifically to the S adhesin gene and a polynucleotide probe which is not able to distinguish between S, F1C, and S-related sequences. The expression of S and F1C phenotypes was tested by dot enzyme immunoassay with the corresponding monoclonal antibodies. S fimbriae genotypes were observed more frequently in septic (25%) and urinary (12%) isolates of E. coli than in faecal and water isolates (1%) and often occurred together with O2, O6, O18 and O83 antigens. F1C/S-related fimbrial DNA was detected with a higher frequency in UTI isolates (26%) than in septic (16%) and faecal (10%) isolates and was most frequently associated with O4, O6, and O75 serotypes. Since the production of S and F1C fimbriae was comparatively rare in all clinical and control isolates of E. coli, DNA hybridization assays which allow the sensitive and specific detection of fimbrial determinants even in the absence of their expression are preferable to phenotypic assays.

Multicentre evaluation of dengue IgM dot enzyme immunoassay

Background: The traditional methods used in the diagnosis of dengue infection do not lend themselves to field application. As such, clinical specimens have to be sent to a central laboratory for processing which invariably leads to delay. This affects patient management and disease control. The development of the dengue IgM dot enzyme immunoassay has opened up the possibility of carrying out the test in peripheral health settings. Objectives: This multicentre study was conducted to evaluate a new, commercial nitrocellulose membrane based IgM capture enzyme immunoassay. Study design: The sensitivity and specificity of the test were compared with in-house dengue IgM enzyme-linked immunoassays routinely performed by each of the selected centres. Known positive and negative dengue specimens, as well as specimens from non-dengue cases, were included in the evaluation. Results: Based on 402 specimens tested by the six centres, the sensitivity was 92.1% and specificity 88.1%, with an overall agreement of 92.8% when compared with IgM EIA assays performed on microplates. Conclusions: The results suggest that this commercial kit has a role to play in the diagnosis of dengue infection, especially in peripheral health settings.

Immunological cross-reactivity between salivary gland proteins of Hyalomma anatolicum anatolicum and Boophilus microplus ticks

Immunological cross-reactivity was established between salivary gland extract antigens of Hyalomma anatolicum anatolicum (H.a.a) and Boophilus microplus (B.m.), using native-polyacrylamide gel electrophoresis (native-PAGE), immunoblotting, DOT-enzyme immunoassay (DOT-EIA) and in vivo intra-dermal skin test revealing immediate type hypersensitivity swelling reaction. Native-PAGE revealed six proteins in common of molecular weights of 60, 66, 148, 264, 300 and > 300 kDa in the salivary gland extract (SGE) of both the ticks. Four additional bands of molecular weights of 56, 64, 120 and 220 kDa were present in H.a.a. but absent in B.m. Immunoblot assay of native-PAGE transblotted proteins of H.a.a., using mice anti- H.a.a. SGE and anti- B.m. SGE immune serum revealed a common protein of 66 kDa. In DOT-EIA, anti- H.a.a. or anti- B.m. SGE immune serum gave similar antibody titres against the SGE antigens of either tick species. Intradermal inoculation of the SGE antigens of either species on cross-bred calves ( Bos indicus × Bos taurus) immunized to H.a.a. or B.m. ticks produced a strong, immediate hypersensitivity skin swelling reaction.

Enhanced immunity to Hyalomma anatolicum anatolicum ticks in cross-bred ( Bos indicus × Bos taurus) calves using ascaris extract immunomodulator with the tick salivary gland extract antigens

Development of acquired immunity in cross-bred ( Bos indicus × Bos taurus) calves against Hyalomma anatolicum anatolicum, a tick vector of bovine tropical theileriosis was studied using ascaris extract (AE), an immunomodulator of IgE responses, along with the tick salivary gland extract (SGE) antigens in Freund's incomplete adjuvant (FIA) emulsion. Calves immunised with SGE + FIA showed significant rejection (47.4 ± 2.8%) of larvae, whereas only marginal rejection (12.47 ± 1.5%) of nymphs was observed. In contrast, calves immunised with the immunomodulator AE in addition to SGE + FIA showed significant enhanced rejection of nymphs (50.25 ± 5.2%), while the rejection of larvae was only slightly higher (55.8 ± 11.4%), and not statistically different. In addition, incorporation of the immunomodulator AE also resulted in significant enhancement in the percentage recovery of abnormally fed larvae and nymphs. This abnormal feeding was characterised by their white to pale-yellow colour insted of the dark-grey colour of normally fed ticks. It may possibly be attributed to their inability to gain access to the blood vessels owing to the host immunological reaction at the bite sites. Consequently, the ticks fed on extravascular fluid devoid of red blood cells resulting in their white to pale-yellow colour. Difference in the disease transmission potential of normally and abnormally fed ticks is of future interest. Sera from all the immunised calves after 2 weeks of immunisation were positive for anti-tick SGE antibodies in the dot enzyme immunoassay (DOT-EIA). The immunised calves were positive for immediate type hypersensitivity (ITH) skin reaction on intradermal inoculation of the tick SGE antigens. There was no apparent difference in the DOT-EIA antibody titres between the two immunised groups. However, ITH skin swelling was significantly higher in AE + SGE + FIA immunised calves. The study indicates that use of the AE as an immunomodulator along with the tick SGE antigens enhanced anti-tick immunity.

Serological diagnosis of visceral leishmaniasis by a dot-enzyme immunoassay for the detection of a Leishmania donovani-related circulating antigen

Field medicine in tropical areas needs laboratory assays which are inexpensive and easy to perform. To meet this need a semi-quantitative dot-enzyme immunoassay (EIA) was developed for the detection of an L. donovani-related circulating antigen and tested for clinical relevance in the diagnosis of visceral leishmaniasis (VL). The dot-EIA probes serum spotted on nitrocellulose for the presence of the antigen using a monoclonal antibody raised against L. donovani promastigotes, a peroxidase-conjugated rabbit anti-mouse immunoglobulin antiserum and chloronaphthol as peroxidase substrate. The intensity of dot staining by chloronaphthol is read by eye and scored. The dot-EIA was used to test the following groups: 69 patients with VL from Brazil, Kenya, China and France, nine patients with cutaneous leishmaniasis, 38 patients with tropical diseases other than VL, five patients with rheumatoid arthritis and 40 health blood donors. The specificity of the assay was 96.7% ( 3 92 false positive) and the sensitivity 98.5% ( 1 69 false negative). A quantitative EIA for the detection of serum antibodies which makes use of a crude, soluble L. infantum promastigote extract as capture antigen and which was used as the reference method, proved to be more specific (98.9%) but similarly sensitive (98.5%). It should be possible to produce a kit, suitable for large scale application at low cost in order to facilitate routine use of the dot-EIA in the diagnosis of VL.

Pathological studies in broiler chicks fed aflatoxin or ochratoxin and inoculated with inclusion body hepatitis virus singly and in concurrence.

Day-old broiler chicks found negative for maternal antibodies against inclusion body hepatitis (IBH) virus by agar gel precipitation test and viral antigen in cloacal swabs by dot enzyme immunoassay were divided into 6 groups of 20 chicks each. Group A was fed aflatoxin B1 at 1.25 ppm from 3 to 38 days of age; group O was fed ochratoxin A at 0.5 ppm from 3 to 38 days of age; group V was inoculated with 1 ml of IBH virus of titre log10 6.5 EID50 per 0.2 ml. Groups AV and OV were given aflatoxin B1 and ochratoxin A, respectively, and also infected with the virus. Group C served as control. There was mild enlargement and paleness of the liver up to 18 days post inoculation in group V; there were no lesions in group A; and there was gradual enlargement of the kidneys from 10 days post feeding of mycotoxin onwards in group O. In the combined groups AV and OV the gross lesions were slightly more severe. In group V, varying degrees of degenerative histopathological changes, congestion and haemorrhages were seen particularly in the liver, followed by the kidneys, bursa, spleen, myocardium and lungs, along with intranuclear inclusion bodies in the hepatocytes, mostly in the early stages of infection. Similar microscopic changes, but without inclusion bodies, were seen in groups A and O and the changes were pronounced in the later stages. In group O, the kidney lesions were more pronounced than the liver lesions. In the concurrently infected groups, AV and OV, the changes were similar but slightly more marked than in the corresponding individual groups. Inclusion bodies in hepatocytes were more frequent, more prominent and appeared earlier in the concurrent groups.

Development of a dot-enzyme immunoassay for the detection of circulating antigen in cattle infected with Taenia saginata cysticerci

A monoclonal antibody-based dot-ELISA was developed for the detection of antigen in the sera of cattle with Taenia saginata cysticercosis. After pretreatment of the sera with trichloroacetic acid, antigen could be detected in experimentally infected animals from five weeks after infection. The assay distinguished animals harbouring living cysts from those carrying only dead cysts. A reliable diagnosis of animals harbouring less than 100 cysticerci was not possible, but in cattle with more than 100 living cysticerci the sensitivity was 87·5 per cent; the specificity of the test was 93·5 per cent. These values were very similar to those obtained with a sandwich ELISA.

Comparison of two panels of monoclonal antibodies for determination of Chlamydia trachomatis serovars

A panel of monoclonal antibodies was developed for serovar typing of clinical isolates of Chlamydia trachomatis. The panel could distinguish all 15 established serovars from one another, although the hybridomas of the panel were developed by fusions of myeloma cells and spleen cells from mice immunized with antigen derived from the urogenital serovars D to L3. The typing assay was based on a dot enzyme immunoassay, and the monoclonal antibodies that were included in the panel reacted strongly in this assay. A collection of 289 clinical isolates from The Netherlands was typed. The observed serovar frequency distribution was 51 isolates of serovar D (17.6%), 103 isolates of serovar E (35.6%), 62 isolates of serovar F (21.5%), 28 isolates of serovar G (9.9%), 14 isolates of serovar H (4.8%), 2 isolates of serovar I' (0.7%), 20 isolates of serovar J (6.9%), and 9 isolates of serovar K (3.1%). These results were confirmed by typing these isolates with a panel of monoclonal antibodies purchased from the Washington Research Foundation, Seattle. No strain variation was observed within serovar D with both panels. However, restriction fragment length polymorphism analysis of the gene encoding the major outer membrane protein showed that 32 isolates were similar to the prototype D and 17 were similar to the variant D-. The two others showed a new restriction pattern. Our panel of monoclonal antibodies contained one monoclonal antibody that divided the serovar G isolates into two groups. This differentiation was confirmed by restriction fragment length polymorphism analysis, confining this difference to a known sequence variation in variable domain IV. These data support the subdivision of serovar G into serovars G (prototype strain UW-57) and Ga (prototype strain IOL-238).

Rapid Serodiagnosis of Typhoid Fever by Dot Enzyme Immunoassay in an Endemic Area

A dot enzyme immunoassay (EIA) using 50-kD outer-membrane proteins (OMPs) of Salmonella typhi was compared with the Widal test for the serodiagnosis of typhoid fever in 109 febrile children admitted to a hospital in an endemic area. In the culture-positive typhoid group, the initial dot EIA was positive in 40 of 42 cases and the initial Widal test was positive in 41. In the culture-negative clinical typhoid group, both the dot EIA and the Widal test were positive in 17 of 18 cases. In the nontyphoidal fever group, the dot EIA was negative in all of 49 cases and the Widal test was negative in 44. With culture used as the gold standard, the dot EIA is as sensitive as the Widal test (95% vs. 98%), has a similar high negative predictive value (96% vs. 98%), and is more specific (75% vs. 67%). In addition, the dot EIA offers the advantages of simplicity, speed, early diagnosis, economy, and flexibility (i.e., other diagnostic tests can be conducted simultaneously).

A dipstick Dot enzyme immunoassay for detection of rabies antigen

A dipstick Dot enzyme immuno diagnosis test was standardized to detect rabies viral antigens from brain, of rabies-suspected dogs, cattle, horses, cats and goats. This test was compared with the direct fluorescent antibody test (FAT). When compared to the direct FAT, the dipstick dot ELISA test did not produce non-specific false-positive results and was therefore specific and reliable. The advantages of the dipstick Dot-ELISA are discussed.

A direct dot-enzyme immunoassay to detect human ovulation

This paper describes an original dot-enzyme-linked immunosorbent assay (ELISA) for predicting ovulation in women, based on the detection of the pre-ovulatory estrogen peak in urine. A monoclonal anti-estrogen antibody is used which recognizes not only free estrogens but also some of their urinary metabolites (17-glucuro-and sulfo-conjugates) allowing a direct assay on early morning urines. Antigen is immobilized as a spot on a nitrocellulose membrane which is immersed in urine in the presence of this antibody. A peroxidase-labeled second antibody allows the detection of the first antibody bound to the membrane. Antigen and anti-estrogen antibody concentrations are chosen to obtain a maximal enzymatic coloration of spots corresponding to basal urinary estrogen levels and no coloration corresponding to the pre-ovulatory surge. Six menstrual cycleswere studied, comparing dot-ELISA results with patterns of: (1) urinary estrogens measured by RIA either directly or after hydrolysis and extraction, and (2) basal body temperatures. The validity of the pre-ovulatory signal obtained and the requirements for an adaptation of this methodology to a reliable home kit are discussed.

Determination of S Fimbriae Among Escherichia coli Strains from Extraintestinal Infections by Colony Hybridization and Dot Enzyme Immunoassay

449 E. coli strains obtained from septic infections (124 isolates), urinary tract infections (246) and water (79) were surveyed for S/F1C fimbrial DNA by DNA hybridization and for expression of S fimbriae by enzyme immunoassay. S/F1C fimbrial DNA was detected with greater frequency in septic (35%) and urinary (43%) isolates than in water isolates and was often associated with the O2, O4, O6, O18, O83, and O156 serogroups. Most O45 strains did not possess such sequences. Only 12% and 28%, respectively, of the septic and urinary strains possessing S/F1C fimbrial DNA expressed S fimbriae.

Immunoreactivity of a mammalian liver component with leprosy sera.

Sera from 77 leprosy patients in various stages of infection--tuberculoid (TT), lepromatous (LL), borderline tuberculoid and borderline lepromatous--15 contacts and 21 normal healthy individuals, were assayed in an indirect enzyme-linked immunosorbent assay and dot enzyme immunoassay using ethanol-soluble and thermostable extract of liver as the antigen. The highest incidences of reaction were found in untreated LL patients (100%) and in TT patients (91%), while the sera from borderline patients showed a comparatively lower incidence (43%). Some of the sera from contacts of leprosy patients (6/15) also showed high reactivity. Assays using lecithin as an antigen did not exhibit any reaction.

Biological activity of a human monoclonal antibody to Bordetella pertussis lipooligosaccharide

The heterohybridoma cell line HBp2 secreting human monoclonal antibody (hMAb) directed against Bordetella pertussis was generated by fusing SP2/HPT heteromyeloma cells with human spleen lymphocytes, after in vitro stimulation for 6 days. The hybridoma was maintained in culture for more than 1 year with continuous antibody secretion. The hMAb HBp2, an IgM, reacted with untreated and proteinase K-treated B. pertussis outer membrane antigens, whereas the reactivity was lost when the antigen was treated with sodium periodate. Human MAb HBp2 was shown to be specific to B. pertussis LOS by immunoblotting of whole cell extracts after SDS-PAGE. In a dot enzyme immunoassay, HBp2 reacted with all B. pertussis strains and clinical isolates tested except for four atypical variant strains of the LOS B phenotype. Human MAb HBp2 also reacted with a clinical isolate of B. bronchiseptica. No reaction was observed against B. parapertussis and other gram-negative species. Together these studies suggested that HBp2 is reactive with carbohydrate epitopes present on the LOS A. Binding assays with live bacteria demonstrated that hMAb HBp2 reacted with cell surface exposed epitopes on B. pertussis but the antibody did not bind significantly to the surface on intact B. bronchiseptica cells. When examined for bactericidal activity in the presence of complement, hMAb HBp2 showed high lytic capability against B. pertussis while no killing was obtained against B. bronchiseptica. These experiments established that LOS A is a target for human bactericidal antibodies. This antigen merits further investigation as a potentially important component in human immunity to B. pertussis infection.

Rapid Identification ofCandida Albicansby Dot-Enzyme Immunoassay

A highly sensitive and specific dot-enzyme immunoassay for the rapid identification of Candida albicans was developed using a murine monoclonal antibody (Mab), which adsorbed to cell surface-exposed determinants. This Mab reacted with 28 of the 28 C. albicans strains tested including the serotypes A and B and 2 C. stellatoidea. It did not react with 32 other isolates representing eight other Candida species commonly encountered in human materials. All the test could be performed in four steps in less than an hour. The yeasts were directly spotted on a strip of immunodyne membrane. Then the strip was incubated for 5 min. with the Mab, for 15 min. with a peroxidase-conjugate and for 30 min. with the enzyme substrate and 4-chloro 1 naphthol. This test proved useful for rapid and easy identification of C. albicans.

Rapid diagnosis of tuberculous meningitis with a dot enzyme immunoassay to detect antibody in cerebrospinal fluid

A simple dot enzyme immunoassay (Dot-EIA) was carried out to detect antibody to Mycobacterium tuberculosis antigen 5 in cerebrospinal fluid (CSF) specimens from 40 patients with a clinical diagnosis of tuberculous meningitis (TBM). The assay gave a positive reaction in all ten patients with culture proven TBM. In 30 culture negative patients with TBM, the assay was positive at a titre of 1:16 in 18 patients. In 40 patients with non-tuberculous neurological diseases (control group) the assay was negative at a titre of 1:16. The Dot-EIA had an overall sensitivity of 70% and a specificity of 100% in the diagnosis of TBM. This assay could be used as a rapid screening test to establish the diagnosis of TBM, particularly in patients in whom bacteriological investigations for Mycobacterium tuberculosis in CSF specimens are negative.

Four epitopes of Pseudomonas aeruginosa elastase defined by monoclonal antibodies

Monoclonal antibodies against the elastase of Pseudomonas aeruginosa were produced from spleen cells of BALB/c mice primed with purified elastase of P. aeruginosa and P3-X63-Ag8-U1 myeloma cells. The six clones established generated antibodies which reacted with a 33,000-Da peptide and recognized four different elastase epitopes by a competitive binding enzyme-linked immunosorbent assay. The monoclonal antibodies designated as ELA-17 and ELA-42 that recognize two different epitopes reacted by dot-enzyme immunoassay and by Western immunoblotting with all clinical and International Antigen Typing Scheme strains of P. aeruginosa positive for elastase.

Dot-immunobinding assay with monoclonal antibody for detection of Trichomonas vaginalis in clinical specimens.

A rapid and specific dot-enzyme immunoassay (DIBA) using monoclonal antibody was introduced for detection of Trichomonas vaginalis antigen in vaginal and urethral materials. The results of DIBA were compared with culture findings of the parasite in 245 female patients. Taking culture as the reference method, DIBA had a sensitivity of 92% and a specificity of 93%. The predictive value of a positive test was 98% and that of a negative test 77%. The efficacy of wet mount, culture and DIBA were compared in 134 women with trichomonal infection. The most sensitive method was cultivation with a positivity of 99.3%. DIBA was as sensitive as the wet mount (92%). In 51 sexual partners of women with trichomoniasis DIBA proved to be ineffective. The sensitivity of the assay with corpuscular antigen of T vaginalis was 1 x 10(4) cells/ml, with soluble antigen 47 micrograms/ml. Specificity of the assay was confirmed by lack of any cross-reactivity with Trichomonas tenax. Tritrichomonas mobilensis and Candida albicans.