Microenzyme-linked Immunosorbent Assay Research Articles

A case report of small bowel ileus possibly caused by Gnathostoma doloresi

Small bowel ileus due to the parasitic infection caused by omophagia of freshwater fish is relatively rare. We present a case of small bowel ileus possibly caused by inflammatory change associated with Gnathostoma doloresi infection. A 62-year-old man underwent partial resection of the small bowel under a diagnosis of ileus due to complete obstruction of the small bowel. He had eaten a few slices of raw freshwater fish four weeks before abdominal pain appeared, and he contracted creeping disease with several welts on the abdominal wall. Chronic inflammatory change suggestive of parasite infection was observed in the resected specimen. An immunoserodiagnostic study using microenzyme-linked immunosorbent assay led to a diagnosis of Gnathostoma doloresi infection. The postoperative course was favorable, and the patient was discharged 12 days after surgery. Only two cases of ileus due to Gnathostoma doloresi infection have ever been reported.

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.

Development and application of a dot-ELISA test for the detection of serum antibodies to Fasciola hepatica antigens in llamas

A microenzyme-linked immunosorbent assay (dot-ELISA) was developed to detect serum antibodies against Fasciola hepatica antigens in llamas. Sera from five F. hepatica-infected and 11 non-infected llamas were used in initial test development. Nitrocellulose filter disks containing F. hepatica excretory-secretory product were placed in 96-well microtiter plates, washed, blocked with Tween-20, then incubated with four-fold serial dilutions of llama sera. After incubation with rabbit anti-llama IgG followed by peroxidase-conjugated goat anti-rabbit IgG, addition of precipitable substrate resulted in purple dots on white background (positives) easily read by eye. The technique was further evaluated at titers of 1:512 using an additional six known positive and eight known negative llamas. Test results showed 6/6 known positive as positive and 8/8 known negative as negative. Sera were collected, at approximately weekly intervals, from three llamas experimentally infected with F. hepatica. The dot-ELISA detected antibodies to F. hepatica as early as the second week post-infection in all llamas. In a serologic survey of 256 llamas from an F. hepatica endemic area, the dot-ELISA detected antigen-specific serum antibodies to F. hepatica in 42 (16%) of the llamas. Although no difference was noted in antibody prevalence between sexes, prevalence increased in llamas over 6 months of age.

Cancer-associated quantitative differences in antigen levels of sialosyl Lewisx in stool from patients with colorectal carcinomas

A microenzyme-linked immunosorbent assay system employing monoclonal antibody SNH3 was developed for the detection of sialosyl Lewisx antigen in stool extracts from 80 patients with colorectal cancer, 13 patients with colorectal non-malignant disorders and 90 normal subjects. Sialosyl Lewisx antigen was detected in 35% of stool extracts from cancer patients but only in 7.7% and 2.2% of those from non-malignant patients and normal subjects respectively. Hemoglobin in the same stool samples was detected in 37.5% of cancer patients, 15.4% of non-malignant patients and 5.6% of normal subjects. The appearance of sialosyl Lewisx antigen in stool was not necessary correlated with that of hemoglobin, and overall 61.3% of cancer patients were detected by the combination of the two assays. The combination assay was also impressive in early detection of colorectal cancer (Dukes' A, 52%; Dukes' B, 57.1%). Therefore, the assay for sialosyl Lewisx antigen in stool would be useful for detecting colorectal cancer.

Detection of anti-helminth antibody by microenzyme-linked immunosorbent assay using recombinant antigen and anti-β-galactosidase monoclonal antibody

An enzyme-linked immunosorbent assay (ELISA) using antigenic β-galactosidase-helminth derived recombinant fusion protein (FP) obtained by the recombinant DNA technique provided a useful diagnostic tool for human helminthiasis. Helminth-infected human sera reacted strongly with FP that was immobilized with anti-β-galactosidase monoclonal antibody on microplates. However, FP did not react with sera from patients with other helminthiases. In detection of anti-helminth IgG antibody, the present ELISA system using FP and anti-β-galactosidase monoclonal antibody was highly specific compared to that using biochemically extracted soluble antigens.

The significance of free and immune-complexed hydatid-specific antigen(s) as an immunodiagnostic tool for human hydatidosis.

Micro-enzyme-linked immunosorbent assay (Micro-ELISA) systems were developed and evaluated for the detection of circulating (free or immune-complexed) hydatid antigens in the sera of patients with hydatidosis, by employing monospecific antibodies to hydatid-specific antigens of 8-kDa and 116-kDa. Fifteen (75%) of 20 sera from patients with hydatidosis had both 8-kDa and 116-kDa antigens freely circulating in their sera while three and two samples, respectively, had only 8-kDa or 116-kDa antigen. All the surgically confirmed cases of hydatidosis had detectable levels of both 8-kDa and 116-kDa circulating immune complexes in glycine HCl-treated sera. However, none of the sera from control subjects (patients with cysticercosis, ascariasis, ancylostomiasis, hymenolepiasis, amoebic liver abscess or viral hepatitis) had any detectable level of either type of circulating specific antigen. These results suggest that the demonstration of either 8- or 116-kDa antigen(s) in free or immune-complex form could confirm the diagnosis of hydatidosis.

Absence of hepatitis delta virus infection in chronic hemodialysis and kidney transplant patients in france.

Hemodialyzed patients and kidney recipients are frequently multiply-transfused and infected by hepatitis B virus (HBV). Hepatitis delta virus (HDV) has a symbiotic association with HBV. The prevalence of HDV infection has, surprisingly, not been assessed in patients in hemodialysis or renal transplantation setting. We retrospectively studied the prevalence of serum delta antigen and antidelta antibodies by a microenzyme-linked immunosorbent assay in 77 of the 80 HBsAg-positive patients who underwent a kidney transplantation over a 10-year period. Of these patients, 73% had active HBV replication as assessed by the presence of serum HBV DNA and 65% had a biopsy-proved chronic active hepatitis, including cirrhosis. None of our patients had any marker of HDV infection at the end of the hemodialysis period or at the end of follow-up in transplantation. These results establish that HDV superinfection has indeed been extremely rare during end-stage renal failure and kidney transplantation in France, in contrast to HBV reactivation or hepatitis C virus infection.

A microELISA assay for detection of anti-HLA activity of mouse monoclonal antibodies using an Astroscan 2100 automated plate reader

A microenzyme-linked immunosorbent assay (microELISA) method has been developed using an Astroscan 2100 system automated plate reader which was initially designed for tissue typing by a two colour fluorescent microcytotoxicity assay. A 96-well plate ELISA used for screening mouse monoclonal antibodies raised against surface HLA antigens has been modified for use with the Astroscan plate reader and 72-well typing trays. The existing substrate 4-methylumbelliferyl-beta-D-galactoside (4MUG) has been replaced with fluorescein-di-beta-D-galactopyranoside (FDG), to provide a wavelength (530 nm) detectable by the Astroscan or other automated plate readers designed for reading microcytotoxicity assay plates. The assay volumes have also been reduced tenfold for use with Terasaki microtest plates. The assay now has the major advantage of requiring only 5 microliters of test supernatant allowing hybridomas to be screened earlier during a fusion and on a wider cell panel. The use of the large panel which includes B lymphoblastoid cell lines (B-LCL) and mouse L cell transfectants expressing HLA genes, reduces the length of time the hybridomas need to be kept in tissue culture before selection. Other advantages include the reduction in the number of target cells required, smaller volumes of reagents throughout the assay and the ability to screen cytotoxic as well as non-cytotoxic monoclonal antibodies. The sensitivity of this microELISA proved to be comparable with the original assay and so provides an efficient screening method for monoclonal antibodies.

Donor selection for alloimmunized patients by platelet crossmatching of random‐donor platelet concentrates

Samples obtained from bag segments of stored platelet concentrates (PCs) were crossmatched with sera from alloimmunized patients to identify potentially compatible donors. Sera from 41 patients who were refractory to random-donor platelet transfusions were tested. All sera had lymphocytotoxic antibody (LCTAb), and 37 patients had LCTAb that reacted strongly against greater than 75 percent of the cells in the panel. At least 18 units of PCs were tested per patient. Sera from 33 patients were tested by microenzyme-linked immunosorbent assay (microELISA). Crossmatches with 703 (74 percent) of 944 PCs were positive, 23 percent were negative, 3 percent were indeterminant. Eight additional patients were also tested with a solid-phase system; 352 (85 percent) of 417 crossmatches were positive and 15 percent were negative. Patients with more reactive LCTAb had very few (often 0-2) in vitro compatible units. Thirty-four transfusions of pooled compatible PCs, all stored less than 3 days, were administered to clinically evaluable patients. Six of 20 transfusions selected by microELISA produced satisfactory increases (corrected count increments [CCIs] greater than 7500) 10 minutes after transfusion in 4 of 14 patients. Eight of 14 transfusions selected by solid-phase produced CCIs greater than 7500 in 5 of 8 patients. Although it is difficult to select large numbers of compatible units for heavily alloimmunized patients by these techniques, some transfusions were successful in patients who had very few HLA-compatible donors. This approach may be of particular value in emergency situations.

A Serodiagnostic Assay by Microenzyme-Linked Immunosorbent Assay for Human Anisakiasis Using a Monoclonal Antibody Specific for Anisakis Larvae Antigen

A serodiagnostic assay system to detect human anisakiasis is described. This assay uses a microenzyme-linked immunosorbent assay with monoclonal antibody An2 that recognizes the antigen specifically found in Anisakis simplex larvae. The data showed that sera from infected patients reacted strongly with the A. simplex larvae antigen that was immobilized with monoclonal antibody An2 on microplates. Patients' IgG, IgA, IgM, and IgE reacted strongly with An2-defined antigen at 4 or 5 weeks and at 1 or 7 days, respectively, after the onset of clinical symptoms of anisakiasis. However, sera from healthy persons did not react, even though they often ate raw fish. This indicates a high diagnostic specificity of this assay system for clinically manifested anisakiasis.

Thyroglobulin and Thyroid Peroxidase Share Common Epitopes Recognized by Autoantibodies in Patients with Chronic Autoimmune Thyroiditis

Monoclonal antibodies specific for human thyroid peroxidase (TPO) were prepared by the hybridoma technique using hyperimmune spleen cells from mice immunized with TPO purified from thyroid glands from patients with Graves' disease. Use of the microenzyme-linked immunosorbent assay method revealed that some of the monoclonal antibodies cross-reacted strongly with human thyroglobulin (Tg). Conversely, monoclonal anti-Tg antibodies cross-reacted with TPO, albeit to a lesser degree. Some anti-Tg autoantibodies in serum from patients with chronic autoimmune thyroiditis purified by Tg affinity chromatography bound TPO, and such binding was completely inhibited by Tg. Western blotting experiments revealed that thyroid microsomal 103K proteins recognized by mouse monoclonal and polyclonal anti-TPO antibodies were recognized by some monoclonal anti-Tg antibodies and anti-Tg autoantibodies, and conversely, that 19S Tg was recognized by some monoclonal anti-TPO antibodies. TPO was immunoprecipitated by anti-Tg autoantibodies isolated by Tg affinity chromatography. On the other hand, the specificity for TPO of the anti-Tg autoantibodies was not identical with that of anti-TPO autoantibodies. These cross-reactivities were not due to contamination of TPO with Tg or vice versa, or to contamination of the anti-Tg autoantibody preparations with anti-TPO autoantibodies. Taken together, these data indicate that Tg and TPO share common antigenic determinants and that some of those determinants are recognized by autoantibodies in the serum of patients with chronic autoimmune thyroiditis.

Detection of antibodies to Ureaplasma urealyticum in pregnant women by enzyme-linked immunosorbent assay using membrane antigen and investigation of the significance of the antibodies.

Optimal conditions of a microenzyme-linked immunosorbent assay using a group-specific membrane antigen of Ureaplasma urealyticum serotype 7 were established with rabbit antisera and applied for the evaluation of immunoglobulin M (IgM) and IgG antibodies in 139 serum specimens from pregnant women between 26 and 38 weeks of gestation, and the assay was compared with microorganism culture and investigated to determine the role of U. urealyticum in perinatal morbidity and mortality. U. urealyticum was isolated from 75 (54%) of 139 patients; 40 had a colonization greater than or equal to 10(6) cells per ml of swab (29%); 64 (85%) of 75 culture-positive patients had IgG antibodies (absorbance mean, 0.650), versus 4 (6%) of 64 culture-negative patients (absorbance mean, 0.103) (P less than 0.001). There was no cross-reactivity with Chlamydia trachomatis infection from patients from whom no mycoplasmas were isolated, but this cross-reactivity occurred in 24% of patients with other mycoplasma infections. There was a good correlation between quantitative evaluation of U. urealyticum colonization and antibody level (P less than 0.05). However, IgM antibody was found in 30% of culture-positive patients but also in 25% of the culture-negative group. Frequency of U. urealyticum colonization was greater in unmarried young women (less than 25 years old) with a history of genital infection, and a significantly greater frequency was detected in patients who smoked (P less than 0.01) and had a lower socioeconomic status (P less than 0.001). A lower infant birth weight was more associated with U. urealyticum colonization greater than or equal to 10(5) cells per ml. The enzyme-linked immunosorbent assay provided an additional means to diagnose and evaluate U. urealyticum infection in pregnant women.

Amniotic Fluid Thymosin α1 Levels Increase During Gestation

Thymosin alpha 1 is one of several cytokines produced by the thymus that modulates immune function. The presence of elevated serum levels of thymosin alpha 1 in pregnant women and their newborns has suggested that this peptide may play a role in perinatal immunology. In this investigation, we used a microenzyme-linked immunosorbent assay to assay amniotic fluid for immunoreactive thymosin alpha 1 and found levels that were remarkably higher than newborn serum levels (P less than 10(-4]. The increase of thymosin alpha 1 in amniotic fluid with fetal age was natural logarithmic (r = 0.838, P less than 10(-6]. Thymosin alpha 1 in amniotic fluid may account for some of the immunologic properties of this medium.

Microbiologic and serologic studies of Gardnerella vaginalis in intra-amniotic infection

Our objective was to investigate the role of Gardnerella vaginalis in intra-amniotic infection by use of comparative, quantitative cultures on selective media and by detection of maternal antibody response. Amniotic fluid was collected from patients with intra-amniotic infection and from matched control women. In addition to media for aerobes, anaerobes, and mycoplasmas, we used V agar-selective (Remel, Lenexa, KS) to isolate G vaginalis. Acute and convalescent maternal sera were collected and assayed for antibodies by a microenzyme-linked immunosorbent assay (ELISA) prepared against whole cells of G vaginalis. Gardnerella vaginalis was isolated in the amniotic fluid of 24 (28%) of the 86 patients with intra-amniotic infection, but this was not significantly different from the isolation rate in amniotic fluid of 86 matched controls (21%). No patient exhibited G vaginalis bacteremia. The ELISA performed on paired sera of selected patients showed that 25 had intra-amniotic infection (eight G vaginalis-positive, 17 negative), and 18 were asymptomatic (seven G vaginalis-positive, 11 negative). The amount of G vaginalis antibodies detected by ELISA in acute sera was similar in all four groups. Mean changes during convalescence were small (.053-.084 optical density units) and not significantly different. Although G vaginalis is found commonly in amniotic fluid of patients with intra-amniotic infection, the data do not support a pathogenic role for this organism; however, a facilitating role in polymicrobial infection cannot be excluded.

PREVALENCE OF COELIAC DISEASE IN TYPE 1 DIABETES MELLITUS: A STUDY PERFORMED VIA TESTS FOR ANTIGLIADIN ANTIBODIES

The association between diabetes mellitus and coeliac disease has been described a number of times. By using tests for antireticulin antibodies, Maki et al. found a greater prevalence of the disease in diabetic subjects. In our opinion, this method is not wholly reliable, which led us to study the problem using the test for antigliadin antibodies (AGA) by immunofluorescence (IFL-AGA) or microenzyme-linked immunosorbent assay (ELISA-AGA). This technique appeared to be more specific and more sensitive (Cacciari et al.) in identifying children with coeliac disease. We examined 132 subjects with type I diabetes (66 m, 66 f) with age ranging from 2 3/12 to 22 7/12 years. Antigliadin antibodies were assayed from 0 to 17 2/12 years after diagnosis of diabetes. 8 subjects were seen to be positive with either one technique or the other: all subjects for IgG, 7 out of 8 for IgA. These subjects underwent duodenal biopsy (4th portion). In 1 case crypt hyperplasia with total villous atrophy was observed, while 3 patients had a crypt hyperplasia with severe partial villous atrophy. These 4 patients were diagnosed as probably suffering from coeliac disease since other causes of alteration to the mucosa similar to those observed are practically unknown in children. All subjects were positive for IFL-AGA belonging to class IgG. No positive patients had shown signs of malabsorption or diarrhoea. Prevalence was 1 : 33. It is probable that genetics might shed light on the relationship between these two diseases, both diseases being associated with HLA B8 and DR3 antigens.

Cell-mediated immune responses to Chlamydia trachomatis in mothers and infants.

Cell-mediated immunity to Chlamydia trachomatis was studied in pregnant women with chlamydial infection of the cervix, in infants born vaginally to these women, and in infants presenting with chlamydial conjunctivitis. Uninfected pregnant women and their infants were studied as controls. McCoy cell cultures were used to isolate C. trachomatis from clinical specimens. Cell-mediated immunity was measured by lymphocyte proliferative responses in vitro to stimulation by chlamydial antigens. Chlamydial IgG antibody in serum specimens was detected by a microenzyme-linked immunosorbent assay technique. The mean lymphocyte proliferative responses to chlamydial antigens were greater in infected women than in uninfected women both during pregnancy and in the postpartum period. Lymphocyte responsiveness in infected pregnant women, however, was less than in postpartum women. Despite failure to detect chlamydial infection in exposed infants, lymphocyte proliferative responses were greater in umbilical cord blood and later in peripheral blood samples from neonates born to infected mothers than in infants born to uninfected mothers. These responses were also greater in infants with chlamydial conjunctivitis than in infants of uninfected mothers. These data suggest that cellular immune responses to chlamydial antigens are increased in infected mothers and infants and that infants may acquire chlamydial cell-mediated immunity transplacentally.

A microenzyme-linked immunosorbent assay for urinary albumin, and its comparison with radioimmunoassay

Interest in early albuminuria (‘microalbuminuria’) has intensified recently, particularly in the area of diabetes. We describe a sandwich type enzyme-linked immunoassay for urinary albumin and compare it with a conventional radioimmunoassay (RIA) using the same commercial antiserum. The assay ranges for ELISA and RIA were 7.5–500 μg/l and 20–5000 μg/l respectively and the corresponding sensitivities were 1.5 ng and 2.0 ng. The correlation coefficient between ELISA and RIA in the measurement of albumin in overnight urine samples from 74 children was r=0.89 ( P<0.001). ELISA has several practical advantages over radioimmunoassay, and we believe it has the potential to replace RIA in the screening and study of albuminuria.

A monoclonal antibody detecting neural and neuroendocrine differentiation.

A monoclonal antibody, named LICR-LON-E36, has been produced to the acidic fraction of soluble protein extracts of human brains. Using immunocytochemistry, it stained normal peripheral nerves, adrenal medulla, pancreatic islets, and enterochromaffin cells, in addition to recognizing an intracytoplasmic epitope in phaeochromocytomas, carcinoid tumors, some small-cell anaplastic lung, and a few breast carcinomas. Microenzyme-linked immunosorbent assay and electrophoretic nitrocellulose blotting experiments confirmed that the antibody is not directed at neuron-specific enolase. Its potential use as an immunological probe for neural and neuroendocrine differentiation is discussed.

Detection of antibody to staphylococcal lipoteichoic acid with a microenzyme-linked immunosorbent assay.

Sera from individuals with Staphylococcus aureus endocarditis and osteomyelitis and from some individuals with other forms of gram-positive endocarditis yielded higher readings in a microenzyme-linked immunosorbent assay against lipoteichoic acid from S. aureus than did sera from individuals with other types of serious staphylococcal infection or non-staphylococcal osteomyelitis, or from unselected inpatients.

Nonspecific induction of immunoglobulin M antibodies to periodontal disease-associated microorganisms after polyclonal human B-lymphocyte activation by Fusobacterium nucleatum.

The production of antibodies to oral bacteria was determined in lymphocyte cultures stimulated with sonicated Fusobacterium nucleatum, a potent inducer of polyclonal B-cell activation. After 9 days the cultures were examined by a microenzyme-linked immunosorbent assay for immunoglobulin M (IgM) antibodies to F. nucleatum, Bacteroides gingivalis, Actinomyces viscosus, and Streptococcus sanguis. Antibodies to these four bacteria were detected in cultures stimulated with polyclonal B-cell activation-inducing concentrations of F. nucleatum. However, significant concentrations of antibodies to F. nucleatum, but not to the other three microorganisms, were produced in cultures that received suboptimal polyclonal B-cell activation-inducing doses of F. nucleatum. Absorption studies indicated the specificity of the antibodies to each of the bacteria tested. IgM antibody production induced by F. nucleatum was enhanced by the addition of T cells. The production of IgM antibodies to the bacteria was reproducible in cultures from a single person tested on 3 consecutive days. The concentration of antibodies in replicate cultures, however, fluctuated greatly. To obtain consistent responses on successive days, multiple replicate cultures were required. These results suggest that F. nucleatum, which is frequently present in subgingival plaque, could induce the production of antibodies not only to F. nucleatum, but also to other microorganisms associated with periodontal diseases.