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PubMed1985

Clinical validation of an antibody-capture anti-rubella IgM-ELISA.

An antibody-capture IgM-ELISA using monoclonal antibodies for conjugate was subjected to clinical validation with respect to sensitivity and specificity. In 103 serum specimens, known to contain anti-rubella IgM by a sucrose density gradient method, IgM was found by the ELISA in 99 sera. In a second study, 16 out of 17 acute rubella infections were detected by the IgM-ELISA. In 17 out of 17 vaccinees, a specific IgM response could be demonstrated. Specificity of the antibody-capture ELISA was found to be high; no interference was seen in 60 rheumatoid-factor positive sera, in 100 highly positive IgG sera or 10 sera with anti-CMV IgM. Only one out of 100 sera with heterophile antibodies showed a positive response. In acute rubella infections, IgM was shown to be detectable from 1 to 4 days after onset of illness up to about 12 wk, with peak values at about 1 wk after onset.

Antibodies, Viral↗
PubMed1987

A one-step enzyme-linked immunosorbent assay to detect anti-CMV antibodies; development and clinical validation.

A simple one-step ELISA to detect anti-CMV antibodies was developed. The test was based on the inhibition principle, and used anti-CMV coated microtitre plates, CMV nuclear antigen and anti-CMV Fab'-HRP conjugate; total assay time was 1.5 h. The use of Fab'-HRP conjugates improved discrimination between positive and negative sera as compared with IgG-HRP conjugates. In an in-house clinical study, the one-step ELISA showed a 98.4% correlation with the latex agglutination test. The test could be demonstrated to be suitable for both serum and citrate- or EDTA-plasma specimens; heparin-plasma gave somewhat higher absorbance values. In an external clinical validation, the one-step ELISA showed a 99.5% correlation with the latex agglutination test, and was found to be highly suitable for screening of blood donor specimens in a blood bank setting.

Antibodies, Viral↗
PubMed2001

Titre and combination of ICA and autoantibodies to glutamic acid decarboxylase discriminate two clinically distinct types of latent autoimmune diabetes in adults (LADA).

UNLABELLED: ABSTRACT AIMS/HYPOTHESIS: This study aimed to define the immunological parameters which could be used to identify patients with the distinct metabolic features of adult latent autoimmune diabetes. METHODS: Sera of 312 patients with short-term diabetes (duration < 5 years) over 35 years of age at diagnosis were screened for ICA, GAD- and IA2-Ab by antibody assays validated in workshops. The antibody status was correlated with age, BMI, residual beta-cell function, measured by fasting C-peptide, onset of diabetes-related complications and markers of the metabolic syndrome (hypertension and hyperlipidaemia). RESULTS: A total of 51 antibody positive patients were identified. These patients had lower fasting C-peptide and less neuropathy and hypertension compared with matched antibody-negative patients. However, only patients with two or more antibodies had reduced residual beta-cell function compared with antibody-negative or single antibody-positive (ICA or GAD-Ab only) patients. Patients with two or more antibodies were also leaner and had diabetes-related complications or hypertension less frequently than single antibody-positive or antibody negative-patients. IA2 antibody status did not substantially contribute to the diagnosis or differentiation of LADA patients. CONCLUSION/INTERPRETATION: We concluded that the combination of ICA and GAD antibodies and high titre of GAD antibodies are characteristic of patients with insulin deficiency with the clinical features of Type I (insulin-dependent) diabetes mellitus (LADA-type 1). Single antibody positivity and low titre antibodies are markers for LADA-type 2 associated with the clinical and metabolic phenotype of Type II (non-insulin-dependent) diabetes patients.

Adult↗
PubMed1987

A sol-particle immunoassay for determination of anti-rubella antibodies; development and clinical validation.

A sol-particle immunoassay (SPIA) was developed for determination of anti-rubella antibodies. The test is based on antibody-coated gold sol particles, which may agglutinate in the presence of rubella haemagglutinin antigen. The agglutination which gives a decrease in optical density can be inhibited by specific antibodies present in sera incubated with the immunochemical reagents. The test is performed in microtitration plates and results can be read in a plate-reader. Clinical validation studies were performed comparing the anti-rubella SPIA with the haemagglutination inhibition test and with the latex agglutination test. The sensitivity of the SPIA was calculated to be 99%; the specificity of the test was at least 99.5%. The anti-rubella SPIA is highly suitable for large-scale screening to determine the rubella immune-status.

Antibodies, Monoclonal↗
PubMed2002

Antimitochondrial antibody profiles: are they valid prognostic indicators in primary biliary cirrhosis?

OBJECTIVE: Retrospective studies have reported that subtypes of antimitochondrial antibodies (AMAs) discriminate between a benign and a progressive course in patients with primary biliary cirrhosis (PBC). Four AMA profiles (A-D) were defined: profiles A and B associated with a benign course and C and D with a progressive course. We aimed to confirm whether AMA profiles predict prognosis in a large sample of North American patients with PBC. METHODS: Stored pretreatment sera from patients with PBC from two centers were tested for AMA profiles using standard techniques. Proportions of patients in each profile group reaching the endpoints of liver transplantation or death from liver disease were compared. Kaplan-Meier curves were constructed comparing AMA profiles. RESULTS: All 472 patients studied had AMA positive, biopsy-confirmed PBC. Mean age at diagnosis was 53 yr, 90% were female, mean follow-up was 7.6 yr (range = 0.5-23), and 51% received ursodeoxycholic acid for >6 months. Profile A was not detected; 16.7% had profile B; 51.1%, profile C; and 32.2%, profile D. Duration of follow-up was comparable among the different profile groups. The proportions of patients reaching endpoints of death from liver disease or transplantation did not differ among the AMA profiles. No difference in the Kaplan-Meier curves between the different profile groups was observed (p > 0.05). CONCLUSION: AMA profiles do not predict prognosis in patients with PBC.

Autoantibodies↗
PubMed1996

[The characterization of influenza viruses by the immunoperoxidase method using monoclonal antibodies. Its setup and validation].

The immunoperoxidase method for the rapid classification of influenza viruses in type and subtype was applied and validated for the first time in Cuba. The method is based on a rapid culture in MDCK-L cells and on the use of monoclonal antibodies for the classification in type and subtype. A pool of antibodies against influenza A and another against influenza B and HA1-71 and HA2-76 monoclonal antibodies are used for the subtyping in H1 and H3. The validation was carried out by applying this method to 21 international reference strains and to 23 human influenza virus strains that were isolated and previously classified by hemagglutination inhibition. All the strains reacted to the monoclonal antibodies according to their hemagglutinin type and subtype. 6 reference strains and 9 isolations were characterized within the H1N1 subtype: 9 reference strains and 10 isolations in the H3N2 subtype; and 6 reference strains and 4 isolations in type B. There were neither unspecific nor crossed reactions among the controls established. There was 100% of sensitivity, specificity and coincidence. The technique used proved to be fast and convenient for the characterization in type and subtype of the isolated influenza virus strains. It may substitute the classic hemagglutination inhibition method when it is required the rapid characterization of outbreaks or epidemics of acute respiratory infections, which is very important due to the high morbidity they cause mainly in risk groups and to their economic repercussion.

Antibodies, Monoclonal↗
PubMed2004

Identification and validation of cell surface antigens for antibody targeting in oncology.

Recent clinical successes with antibodies have reinvigorated interest in the identification and validation of new antigens for antibody therapy, including cell surface proteins for targeting in oncology, the focus of this review. Target identification commonly involves the search for differences between tumor and non-tumor cell lines and/or tissue at the DNA, mRNA, protein or antibody reactivity levels. The next stage, target validation, utilizes antibodies to profile the expression of antigen in normal and tumor tissue and to verify that the antigen is selectively expressed on the surface of tumor cells. Supportive evidence for protein expression is often sought by mRNA profiling and, sometimes, analysis for genomic defects. Unfortunately, concordance between mRNA and protein levels has been found in only about approximately 20% of cases and therefore must be evaluated for individual targets of interest. Antigens judged suitable for antibody targeting are then advanced to the next stage, namely, in vitro and then in vivo screening of antibodies for anti-tumor activities. Subsequent optimization of an antibody clinical lead for therapy is a desirable, if not obligatory, step to developing an antibody as an anti-cancer therapeutic. No single approach or even combination of methods has emerged as the preferred way to identify surface antigens suitable for targeting in oncology. Major options at each step in the process are reviewed here, including their strengths and limitations.

Animals↗
PubMed2002

Development and validation of a new monoclonal antibody to mammalian aromatase.

The biosynthesis of oestrogens from androgens is catalysed by the aromatase complex, an essential component of which is the aromatase cytochrome P450 (P450 arom) protein. Expression of a functional P450 arom is essential for normal fertility in males and females and the sequence of the protein is highly conserved. We have raised a new monoclonal antibody against a conserved peptide and validated it on fixed tissue sections of the rat, common marmoset (Callthrix jacchus) and human. The monoclonal antibody was used successfully for Western analysis and specifically reacted with a 55 kDa protein in microsomal extracts. On sections of ovaries in all three species, expression in follicles was specific to the mural granulosa cells of antral follicles and was present in corpora lutea. In the human and marmoset, staining of luteal cells was markedly heterogeneous and did not appear to vary consistently with the stage of the cycle. The intensity of immunostaining was elevated in corpora lutea from pregnant rats and following human chorionic gonadotropin rescue in the human. In the testis, the highest levels of expression were observed in the Leydig cells within the interstitium. In adult rat and marmoset, and possibly also in the human, some P450 arom was associated with the cytoplasm surrounding elongate spermatids but other germ cells were immunonegative. In conclusion, a new monoclonal antibody specific for P450 arom recognises the protein in rodent, primate and human. Its ability to work on fixed tissue sections will facilitate identification of individual cells expressing P450 arom within complex tissues.

Animals↗
PubMedDate not supplied

[Enzyme immunoassay system for the detection of low-avid lgG antibodies to human cytomegalovirus ("CMV-diagnost")].

The new Russian enzyme immunoassay system "CMV-Diagnost" based on the detection of low-avid IgG antibodies has been developed for the rapid diagnosis of cytomegalovirus infection. The system was found not only to determine the strained immunity in response to cytomegalovirus, but also to judge the current infection from the avidity index of detectable IgG antibodies with a high degree of validity. The antibody avidity index of less than 30% suggests an acute stage of primary cytomegalovirus infection. The minimum antibody threshold bodies (deltaOD has been established for the correct interpretation of data on low-avid antibodies. deltaOD of > or =0.6 optic units was for the developed test system "CMV-Diagnost. A correlation was found between the serum levels of low-avid antibodies and IgM antibodies to cytomegalovirus at the acute stage of the disease.

Adolescent↗
PubMed1990

Engineered human antibodies as immunologic quality control reagents.

This study demonstrates the utility of a panel of genetically-engineered human-mouse chimeric antibodies with variable region specificity for a hapten, NP, and constant regions analogous to the different human immunoglobulin isotypes as quality control reagents. They performed well as isotype-restricted human immunoglobulin proteins to test the specificity to total serum Ig immunoenzymetric assays and as antibodies to validate selected conjugated reagents used in human antibody immunoassays. The chimeric antibodies studied have several advantages over human paraproteins as quality control reagents for clinical assays. They possess single isotypes, are free of other human immunoglobulin isotypes, can be produced in large quantities in cell culture, and have a defined specificity for NP, an antigen that can be conjugated to BSA and isolubilized on most solid phase surfaces.

Animals↗
PubMed2003

The development and validation of an antibody-ELISA to detect Trypanosoma evansi infection in cattle in Australia and Papua New Guinea.

Trypanosoma evansi is exotic to Australia and Papua New Guinea (PNG). However, it might have been introduced to Papua (Indonesia); thus, there is a risk of it entering PNG and thence Australia. Because of logistical difficulties in PNG and northern Australia, surveillance for T. evansi must rely on serological tests. The accuracy of an Ab-ELISA using a detergent extract of T. evansi and three antigen fractions purified from the detergent extract using stepwise precipitation with saturated ammonium sulphate (AS) were compared. The ELISA using the AS 40-50% fraction had greater discriminatory power compared to the ELISA using the other antigen fractions. This ELISA then was compared with two commercial tests: the Card Agglutination Test for trypanosomiasis/T. evansi (CATT) and Suratex. CATT/T. evansi at 1/4 serum dilution has higher sensitivity and the ELISA has higher specificity. There is no likely benefit in combining antibody detection tests to improve the accuracy of diagnosis. Furthermore, the combination of Suratex (which was independent of the antibody tests) with the CATT or the ELISA did not improve the sensitivity. None of the tests was sufficiently sensitive to be used confidently to determine freedom from infection in animals imported into Australia from countries where T. evansi infection is endemic.

Agglutination Tests↗
PubMed1992

Quantitative analysis of human immunodeficiency virus type 1 antibody reactivity by western immunoblots: evaluation of relative antibody levels in seropositive individuals and mothers.

A quantitative analysis of antibody responses to human immunodeficiency virus type 1 (HIV-1) proteins using Western immunoblots and 125I-labeled protein A is reproducible and can be validated. The antibody levels obtained by Western immunoblots were compared with stoichiometric p24 radioimmunoassay over a wide range of antibody (correlation coefficient, .94; P less than .001). Antibody levels to gp160 and gp120 were validated using purified antigens. Analysis of antibody levels from 31 seropositive individuals revealed a statistically significant correlation between antibody levels to p24 and the other viral proteins except gp120. Anti-gag p24 antibody was strongly correlated with antibodies to other env products, specifically gp41 and gp160. Using the validated assay, HIV-1-infected mothers of infants were found to have highly variable levels of antibody to all viral proteins. Mothers of infected infants did not differ significantly from mothers of uninfected infants in antibody pattern or levels to any viral protein including gp120.

Adult↗
PubMed1995

Diagnostic validation of specific IgE antibody concentrations, skin prick testing, and challenge tests in chemical workers with symptoms of sensitivity to different anhydrides.

BACKGROUND: The diagnostic possibilities of sensitization to various acid anhydrides are limited because of the lack of standardized allergens for the different test systems. This makes the diagnosis of IgE-mediated sensitization caused by occupational exposure difficult. METHODS: We prepared conjugates of human serum albumin with phthalic, maleic, trimellitic, and pyromellitic anhydrides to be used for IgE estimation by enzyme-allergosorbent test, skin prick tests, and nasal and bronchial challenge tests. Nine anhydride workers, who complained of various respiratory symptoms, were studied. RESULTS: Of the nine workers, four had immediate-type skin test responses to one or more conjugates. All four subjects had elevated IgE concentrations in addition to two other workers. Three of six nasal challenges and four of nine bronchial challenges resulted in positive responses. All but one of the positive nasal or bronchial test responses were associated with elevated IgE levels. The seven positive challenge test results included five positive skin test responses. On the other hand, in all but two of the subjects with negative challenge test results, no specific IgE could be detected. In these two subjects the negative results were associated with low levels of IgE, and in one, with the absence of asthma. None of the results of tests with unconjugated anhydrides were positive. CONCLUSIONS: Anhydrides investigated in this study can induce IgE-mediated hypersensitivity, which can be diagnosed by using the respective human serum albumin in estimation of specific IgE and in skin, nasal, and bronchial challenge tests. Estimation of IgE was demonstrated to be more sensitive than skin prick testing.

Anhydrides↗
PubMed2000

Development and validation of a monoclonal antibody blocking ELISA for the detection of antibodies against both equine herpesvirus type 1 (EHV1) and equine herpesvirus type 4 (EHV4).

A monoclonal antibody blocking ELISA was developed for the detection of antibodies directed against either EHV1 or EHV4. For this purpose, we selected a monoclonal antibody directed against a cross-reactive, conservative and immunodominant epitope of both EHV1 and EHV4. High antibody titres were found in rabbit antisera and SPF-foal antisera infected with either EHV1 or EHV4. After experimental challenge of conventional horses with EHV1 or EHV4 significant increases in CF and ELISA titres were found, whereas VN antibodies did not always increase significantly. In 344 paired serum samples submitted for diagnostic purposes a good agreement (kappa = 0.75, confidence limits = 0.63-0.88) was found between VN test and ELISA regarding a significant increase in titres. Also, a good correlation was found between VN and ELISA titres (r = 0.76, p<<0.0005). The relative sensitivity and specificity of the Mab blocking ELISA as compared with the VN test were 99.9 and 71%, respectively. The rather low relative specificity of the ELISA may be explained by a relatively low sensitivity of the VN test. The ELISA also detected increases in titre after vaccination with an EHV1 subunit vaccine, and after primary field infections in weaned foals. We concluded that the Mab blocking ELISA is more sensitive, easier to perform, more rapid and more reproducible than the VN test. We consider this test as a valuable tool for serological diagnosis of both EHV1 and EHV4 infections.

Animals↗
PubMed2003

Validation of egg yolk antibody testing as a method to determine influenza status in white leghorn hens.

Determination of the avian influenza (AI) status of a flock has traditionally been done by detection of serum antibodies. However, for many diseases, detection of antibodies in egg yolk has been effective in monitoring the disease status of laying flocks. This study compared the utility of egg yolk vs. serum for determining AI status in laying hen flocks. Specific-pathogen-free white leghorn hens were inoculated via the respiratory tract with a low-pathogenic H7N2 AI virus or sterile allantoic fluid or subcutaneously with an inactivated oil emulsion vaccine produced from the same AI virus or normal allantoic fluid. Antibody levels were determined by the agar gel immunodiffusion (AGID) test, the hemagglutination-inhibition (HI) test, and the enzyme-linked immunosorbent assay (ELISA). Anti-influenza antibodies were detected in sera of all live virus-inoculated hens by day 7 postinoculation (PI) (AGID and ELISA tests), but detection of antibodies in egg yolk was delayed by a few days, with all being positive by day 14 PI. Sera from all vaccinated hens were positive by day 14 PI (AGID and ELISA tests), and egg yolk was positive by day 18 PI. The HI test was less sensitive than the ELISA and AGID tests in detecting anti-influenza antibodies in both sera and yolk. Serum and yolk from all control birds remained negative throughout the study. These studies show that currently used serologic tests can detect antibodies in serum and yolk samples from hens exposed to live AI virus or from those that have been vaccinated. Antibody is detected earlier in the serum than in the yolk and antibody is detected earlier from birds exposed to a live infection compared to birds vaccinated with an inactivated oil emulsion vaccine.

Animals↗
PubMed2002

Validation of 2 commercial Neospora caninum antibody enzyme linked immunosorbent assays.

This is a validation study of 2 commercially available enzyme linked immunosorbent assays (ELISA) for the detection of antibodies against Neospora caninum in bovine serum. The results of the reference sera (n = 30) and field sera from an infected beef herd (n = 150) were tested by both ELISAs and the results were compared statistically. When the immunoblotting results of the reference bovine sera were compared to the ELISA results, the same identity score (96.67%) and kappa values (K) (0.93) were obtained for both ELISAs. The sensitivity and specificity values for the IDEXX test were 100% and 93.33% respectively. For the Biovet test 93.33% and 100% were obtained. The corresponding positive (PV+) and negative predictive (PV-) values for the 2 assays were 93.75% and 100% (IDEXX), and 100% and 93.75% (Biovet). In the 2nd study, competitive inhibition ELISA (c-ELISA) results on bovine sera from an infected herd were compared to the 2 sets of ELISA results. The identity scores of the 2 ELISAs were 98% (IDEXX) and 97.33% (Biovet). The K values calculated were 0.96 (IDEXX) and 0.95 (Biovet). For the IDEXX test the sensitivity and specificity were 97.56% and 98.53%, whereas for the Biovet assay 95.12% and 100% were recorded, respectively. The corresponding PV+ and PV- values were 98.77% and 97.1% (IDEXX), and 100% and 94.44% (Biovet). Our validation results showed that the 2 ELISAs worked equally well and there was no statistically significant difference between the performance of the 2 tests. Both tests showed high reproducibility, repeatability and substantial agreement with results from 2 other laboratories. A quality assurance based on the requirement of the ISO/IEC 17025 standards has been adopted throughout this project for test validation procedures.

Animals↗