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A stool sample from a child with haemorrhagic colitis and haemolytic-uraemic syndrome gave a positive reaction with the RotaScreen latex agglutination test in the absence of other evidence of rotavirus infection. When this test is performed on bloody specimens, positive reactions should be interpreted with caution and confirmed by other means.
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Examination for CBH351 maize was conducted by the qualitative polymerase chain reaction (PCR) method in maize grain and maize processed foods obtained in the Tokyo area. The numbers of samples possibly positive in the screening test were 7 of 22 (31.8%) for maize grain samples, 4 of 14 (28.6%) for semi-processed foods, 11 of 30 (36.7%) for canned products, 3 of 30 (10.0%) for maize snacks, 3 of 4 (75%) for tacos and 1 of 3 (33.3%) for tortillas. However, CBH351 maize was not detected in the confirmation test. Therefore, the results of the screening test were false-positive. Since the reaction might have been caused by the base sequences of the 3'-end of primers CaM03-5' and CBH02-3' used in the screening test, a new primer pair was designed. The PCR products obtained with the new primer pair TMC2-5'--TMS2-3' were specific for CBH351 and were not obtained with barley, wheat, rice, RRS, Bt11, or Event176. Thus, the new primer pair shows high specificity. CBH351 maize was detected from samples containing at least 0.05% CBH 351 maize DNA by using this primer pair.
Irrelevant IgE binding to cellulose discs is known to give false positive results in Phadezym RAST (Pharmacia) for the estimation of allergen-specific IgE in serum. We investigated FAST-Plus Test (3M Diagnostic Systems), an enzyme-linked sandwich type Fluoro-Allergo-Sorbent Test in which a particular allergen was coated to polystyrene well. Phadezym RAST and CAP RAST (Pharmacia) using cellulose-derivative discs as adsorbent were used as reference methods. Patients' sera which gave negative blank reactions to uncoated filter paper disc in the Phadezym RAST system were assayed for specific IgE to 6 allergens using FAST-Plus Test, CAP RAST and Phadezym RAST, and the results of the former two were compared with those of Phadezym RAST using a comparable class system. FAST-Plus Test showed variable correlations with Phadezym RAST, the correlation coefficients ranged from 0.41 to 0.97 (r = 0.462 in house dust 1, r = 0.713 in house dust 2, r = 0.412 in Candida albicans, r = 0.952 in Dermatophagoides peteronyssinus, r = 0.969 in Dermatophagoides farinae and r = 0.682 in Japanese cedar), although most of the results were within one class difference. Similar correlations were obtained between CAP RAST and Phadezym RAST. Of 3004 patients' sera tested in the past two years using Phadezym RAST, 132 (96 cases) displayed positive blank reactions to the uncoated filter paper disc. Of the 96 cases, 80 sera were assayed for binding of IgE to the uncoated cellulose-derivative disc in the CAP RAST system. 18 showed positive results up to 7 IU/ml.(ABSTRACT TRUNCATED AT 250 WORDS)
Most samples of cerebrospinal fluid from 49 patients with or without coccidioidomycosis yielded positive agglutination with the Hyland ("CM") latex-particle agglutination test. Furthermore, simple dilution of sera from patients with or without coccidioidomycosis yielded a strong agglutination reaction with the latex particle agglutination test reagent. These reactions were found with three lots of the latex-particle agglutination test reagent. One lot latex particles was found to give agglutination reactions merely by addition of saline solution or water, though a second lot did not. It is advisable, therefore, that the latex-particle agglutination test not be applied to cerebrospinal fluid, and that it be applied cautiously, particularly with diluted sera.
BACKGROUND: Anti-CD4 autoantibodies are present in up to 10% of HIV infected patients and, until present, they were not recorded in other pathological circumstances. AIM: To test the presence of anti-CD4 autoantibodies in HIV-infected children, using an indirect ELISA test. DESIGN: Cross-sectional controlled study. PATIENTS AND METHODS: The study group: 9 HIV infected dystrophic children--3 AIDS (P2A), 6 P1C. Control groups: 9 HIV seronegative dystrophic children; 14 HIV seronegative eutrophic patients presenting chronic hepatopathies, or dialysed patients. METHODS: 1. Indirect ELISA using as antigen sCD4 molecules; 2. Western blot (sCD4); 3. "Antigenic displacement": sera preincubation with sCD4, followed by indirect ELISA. RESULTS: Indirect ELISA revealed anti-CD4 reactivities in all the dystrophic patients (9 HIV+ and 9 HIV-). WB sCD4 did not confirmed none of these reactivities. Sera preincubation with sCD4 did not significantly modified ELISA CD4 reactivities. CONCLUSIONS: False positive results (FPR) rate following testing of the presence of anti-CD4 in dystrophic children recommends prudence for the interpretation of such tests. "Antigenic displacement" could be an accessible and easy to perform method in order to eliminate the FPR.
We have developed a competitive enzyme immunoassay for the measurement of purified toxin A of Clostridium difficile. However, when we applied this assay to the detection of C. difficile toxin in stool specimens, we noted a high rate of nonspecific activity in fecal specimens which did not contain toxin. We found that the low specificity (26%) of the assay was due to the presence in stool specimens of interfering factors which desorbed the antigen coated on the solid-phase surface. These factors could be detected by measurement of the desorption of biotin-labeled proteins attached to the solid-phase surface. In addition, these interfering factors were partially inactivated by heating at 56 degrees C for 10 min and partially inhibited by phenylmethylsulfonyl fluoride (2 mM) or soybean trypsin inhibitor (10 mg/ml). These data suggested that the desorbing activity was due to proteolytic activity in the fecal specimens. Fetal calf serum (50%) was found to be the most effective measure in preventing the interfering effect. By using 50% fetal calf serum as a diluent, we increased the specificity of the antibody inhibition enzyme immunoassay to 93%. Interfering factors in stool specimens could be a cause of false-positive results in other competitive immunoassay systems. The use of diluents which neutralize protease activity can result in a marked improvement in the specificity of competitive immunoassay systems.
Sera of normal subjects and AIDS patients living in Minsk and Odessa were tested for antibodies to hazardous viral infections Lassa, Marburg, and Ebola. Four to 16% of examinees were seropositive to Ebola virus, 0.8 to 2.3% to Lassa, and up to 0.8% to Marburg virus. Common B-epitopes were found in viruses belonging to different families: Lassa, Ebola, and HIV. Antibodies specific to these viruses antigens were found in the reference sera to influenza A and B, respiratory syncytial virus, and adenovirus. Sera of convalescents after malaria and of AIDS patients contained antibodies to Lassa virus.
The accuracy of the latex particle agglutination test (LPAT) was assessed in blood stained cerebrospinal fluid (CSF) specimens from 166 paediatric patients, aged from three months to 13 years. A commercial LPAT kit was used to detect Haemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis A, B, and C soluble antigens. Culture of CSF specimens was used as the standard and all laboratory procedures were performed blind. The mean CSF erythrocyte count was 66,406 cells/mm3 in the cases and 11,560 cells/mm3 in the controls. The sensitivity and the specificity of LPAT were 83.8 and 94.0%, respectively, suggesting that LPAT is a useful diagnostic tool even in blood stained CSF specimens.
Four groups of cattle were experimentally immunised by four mycoplasma species of "mycoides-like" group, Mycoplasma (M) capricolum, M. mycoides subsp. mycoides (LC), M. mycoides subsp. capri and M. species group 7 of LEACH (PG50). They were then bled weekly during 2 months to establish antibodies kinetics against homologous and heterologous antigens. The standard method of complement fixation test (CFT) used in Europe and a new ELISA test for diagnosis of contagious bovine pleuropneumonia were performed in comparison with passive haemagglutination test (PHA) against antigens used for experimental immunisation. Cross reactions obtained are rather equal to the degree of similitude between these mycoplasma species. With CFT-cross reactions are transitory and occur only while homologous titers are very high, particularly with "PG50" and the two caprine mycoides strains. ELISA results using a threshold of positivity of optical density of 0.20, were similar to that obtained with CFT except ELISA specificity is not so different from CFT one. This experimental model could explain some natural situations.
BACKGROUND: Conflicting results were obtained in the assay of anti-transglutaminase (anti-tTG) autoantibodies in patients with chronic liver disease. In order to establish whether this was attributable to methodological differences, anti-tTG antibodies were assayed in a large number of patients suffering from liver cirrhosis (LC). METHODS: 54 patients with LC and 29 patients suffering from celiac disease (CD), used as controls, were tested for IgA and IgG anti-tTG with 11 different commercial methods. RESULTS: In the patients with LC, positivity ranged from 0% to 33.3% for IgA anti-tTG and from 0% to 11.1% for anti-tTG of the IgG class. The largest number of false positives was found with methods that used tTG in association with gliadin peptides as antigen substrate. A significant association was found between IgA anti-tTG antibodies and serum immunoglobulin concentration. CONCLUSIONS: The results of the various methods of assaying anti-tTG antibodies in patients with LC are highly variable, and the positives found are generally false positives, partly due to the high immunoglobulin concentration.
Mouse monoclonal antibodies (MoAbs) of different classes and subclasses directed against antigens not expected to be present on human cells have been screened by indirect immunofluorescence using flow cytometry for binding to human non-lymphocytic leukaemic cells and normal peripheral blood leucocytes. Antibodies of the IgG2a and IgG3 subclass, but not of the IgG1, IgG2b or IgM class bound to the blast cell and monocyte populations in a peripheral blood mononuclear cell preparation from a patient with acute myelomonocytic leukaemia. F(ab')2 fragments of an anti-salmonella antibody of IgG2a subclass failed to bind, indicating that the results were not due to cross-reactivity with antigens of the cell membrane, thus implicating the Fc region in binding. Furthermore, binding was partly blocked by inclusion of 10% heat-inactivated normal rabbit serum in the assays. IgG2a bound to varying degrees to the leukaemic cell populations in seven of the nine non-lymphocytic leukaemic specimens tested, but no binding to normal peripheral blood mononuclear cells or granulocytes was detected. The results emphasize the importance of including appropriate controls when screening MoAbs for binding to various types of human cells.
The autoinoculation test has been used as a diagnostic procedure in patients with chancroid-suspected genital ulcerations. In four of 13 patients with a positive autoinoculation test, herpes simplex virus type 2 was isolated from the primary lesions. In two of the cases this virus was also isolated from the inoculation site, and one patient developed a recurrent herpetic eruption here. Two patients were proven to have chancroid. Three of the remaining seven patients developed recurrent herpes genitalis later. It is concluded that the autoinoculation test should be avoided as a diagnostic procedure because of lack of specificity and risk of inducing recurrent herpes simplex at the site of inoculation.
An enzyme-linked immunosorbent assay (ELISA) was performed for diagnosis of paratuberculosis in goats, using as antigen a protoplasmic extract (PPA-3). The test was developed on the basis of the results obtained with two serum reference pools, positive and negative respectively. To avoid day-to-day variations, dilutions of the positive serum pool were included in each plate to obtain an arbitrary system, transforming absorbance into immunoglobulin (Ig) G anti-Mycobacterium paratuberculosis units. The ELISA was used on sera of two reference groups of animals. One group consisted of 35 goats suspected of being infected with paratuberculosis, which was confirmed by histological findings and isolation of M. paratuberculosis. The negative group consisted of 61 healthy goats from a farm free of paratuberculosis. The test showed a sensitivity of 100% and a specificity of 91.8%. Absorption of sera with a Mycobacterium phlei suspension did not modify either the sensitivity or the specificity of the test. Sera from the negative group were analysed by Western blotting, and four of them recognized two fractions with a molecular weight of 17.3 and 28.1 kDa.
The indirect fluorescent antibody (FA) technique for the diagnosis of Legionnaires' disease was used to investigate an outbreak of respiratory disease in a military population. The outbreak was later shown to be caused by an adenovirus. High titres were obtained using the ether-killed antigen supplied by the Center for Disease Control (CDC), Atlanta, but not with a formolised yolk-sac antigen prepared in out laboratory. The reactivity of these sera with the CDC antigen was removed by absorption with a partly identified gram-positive bacterium, whereas sera from persons with true Legionnaires' disease were unaffected by such treatment, suggesting that such reactivity is not specific for Legionnaires' disease. The lack of reactivity with negative control sera and strong reactivity from a small group of patients in whom seroconversion was demonstrated has led us to conclude that formolised yolk-sac antigen is a reliable antigen for the diagnosis of Legionnaires' disease by the FA method. Measurement of the sensitivity and specificity of the formolised yolk-sac antigen is still in progress.
The serum of a patient with prolymphocytic leukemia and a hyperviscosity syndrome contained high levels of IgM, with a monoclonal peak on protein electrophoresis, and had a high titer for fibrin(ogen) degradation products, using a rabbit anti-fibrinogen reagent, without other evidence for disseminated intravascular coagulation. A monoclonal IgM lambda cryoglobulin was identified. It retained reactivity with the fibrin(ogen) degradation products reagent, but failed to react with human IgG by latex fixation. Binding activity for rabbit IgG (and absence of binding activity for human, goat, and bovine IgG) was also demonstrable by double diffusion in agarose, and by enzyme-linked immunoassay. By enzyme-linked immunoassay, binding of both F(ab)2 and Fab and, to a lesser extent, Fc fragments of rabbit IgG was found. Increased binding of heat-denatured rabbit IgG was observed, without inhibition by similarly denatured IgG of other species. The false-positive fibrin(ogen) degradation products reaction in the serum of this patient was due to a unique monoclonal IgM lambda cryoglobulin with warm-reactive antibody activity for an epitope displayed on native and denatured rabbit IgG, but absent on native or denatured human, goat, or bovine IgG, and present in both F(ab)2 and Fc regions of the IgG molecule.
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