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Results for “antibody validation”

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At least 91 records · Page 5Linked to original sources
PubMed1988

The validity of radiolabeled anti fibrin antibody for deep vein thrombosis imaging.

Having previously established the specificity of a newly developed radiolabeled polyclonal anti fibrin antibody (AFA) for tagging fibrin depositions, clinical experiments for validation of this agent for imaging deep vein thromboses (DVT) were carried out in comparison with labeled human serum albumin (HSA) and labeled platelets. Patients with DVT were studied with dual tracers on a computerized scintillation camera, four with simultaneous application of 131I-AFA and 99mTc-HSA, and eight with 131I-AFA and 111In-platelets. The results showed that: a) Labeled AFA predominantly delineated thrombi while labeled HSA prevailingly delineated blood pool within a particular vein. b) Labeled AFA was found to be superior in both ways to labeled platelets for DVT imaging, i.e. by imaging effect and by semiquantitative "thrombus:blood pool" (obtained in counts/pixel) ratio index (RI). Average RI with SD obtained with labeled AFA (9 patients) was 2.44 +/- 0.48 and 1.55 +/- 0.27 with labeled platelets (7 patients). The results indicate that a monoclonal AFA, if labeled with 99mTc, 111In or 123I, might have potential for an optimum agent for DVT imaging.

Adult↗
PubMed2000

Schistosoma mansoni egg specific antibodies and circulating antigens: assessment of their validity in immunodiagnosis of schistosomiasis.

A total of 127 individuals of different age and sex; 92 from Kafr-Sendewa, Qualyobia Governorate, Egypt, in comparison to 23 cases with hydatidosis and fascioliasis as a parasitic control group, and 12 healthy control group from non-endemic area. All cases were screened by clinical examination, urine, stool, rectal snip, abdominal ultrasonographic examination and indirect haemagglutination test (IHAT). Accordingly, they were grouped into active intestinal schistosomiasis group, seropositive group by (IHAT), normal control group from the same endemic area, parasitic control group and normal control group. All cases were subjected to detection of IgG, IgM, IgG4, anti-soluble egg antigen (SEA) and anti-excretory-secretory egg antigen (ESEA) by ELISA tests; and circulating egg antigens by double-sandwich ELISA techniques. The results showed that IgG4 anti-SEA is the best diagnostic test, as it gave the best diagnostic efficacy (90%). Also, it is a good screening test which can be used in endemic area as it gave significant difference between the active intestinal schistosomiasis cases with each of the endemic control group (P < 0.001) and the seropositive cases (P < 0.05). Other valid diagnostic egg specific antibodies tests were IgM anti-SEA and IgG anti-SEA as their diagnostic efficacy were 80% and 76.6% respectively (P < 0.05). The diagnostic efficacy of circulating antigen detection (C.Ag) test was 70% (P < 0.05). In addition, it was the most specific test with 100% specificity. IG4-anti-ESEA gave the least cross-reaction with other parasites (17.3%). The mean optical density (OD) level of circulating antigen detection test was significantly higher in the organomegalic (hepatosplenomegaly) cases than the non-organomegalic cases (P < 0.05).

Adolescent↗
PubMed2005

The detection of anti-erythropoietin antibodies in human serum and plasma: part II. Validation of a semi-quantitative 3H-thymidine uptake assay for neutralizing antibodies.

Neutralizing antibodies to erythropoietin (EPO) can cause a loss of response to recombinant human EPO (rHuEPO) and lead to rare cases of sudden, unexplained, severe anemia in chronic renal failure patients treated with rHuEPO. An assay for neutralizing anti-EPO antibodies has been validated that is based on the inhibition of proliferation of human UT-7/EPO cells, an immortalized cell line, by neutralizing antibodies in serum test samples using 3H-thymidine as a marker for proliferation. The dependence of the human cell line on EPO for growth and proliferation in a concentration-dependent manner enabled the validation of a rHuEPO standard curve for cell proliferation that can be used to determine the presence of neutralizing anti-EPO antibodies in serum samples. Proliferation of the cells increases with increasing concentrations of EPO, forming an S-shaped standard curve, which is fit with a 4-parameter logistic model, between 2.5 and 50 mU/mL rHuEPO, with a percent coefficient of variation (% CV) from 8.7% to 22.1% and a % accuracy of 103.5% to 109.5%. Anti-EPO antibodies and serum with anti-EPO antibodies neutralize UT-7/EPO proliferation by 10 mU/mL rHuEPO in a concentration- or dilution-dependent manner with < or = 25% CV. Percent neutralization is calculated by determining the amount of EPO recovered from the original 10 mU/mL added using the formula [((10-concentration recovered)/10)x100%]. Stem cell factor (SCF) stimulated cell proliferation, but not as effectively as rHuEPO. Antibodies to SCF were not able to inhibit the proliferative response induced by EPO and vice versa, confirming the specificity of the assay for antibodies to EPO. High EPO levels can impact both the radioimmunoprecipitation and neutralization assays to produce a false negative result. However, the impact can be mitigated by the large dilutions used in the neutralization assay.

Antibodies↗
PubMed2005

The validation of new aromatase monoclonal antibodies for immunohistochemistry--a correlation with biochemical activities in 46 cases of breast cancer.

Intratumoral aromatase is a therapeutic target for the treatment of post-menopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. Immunohistochemistry (IHC) is considered one of the most suitable methods in this regard. A multi-centre collaborative group has been established to generate and validate new aromatase monoclonal antibodies. We have selected two monoclonal antibodies, #677 against native aromatase protein and F2 against formalin-fixed protein for this purpose. With these two monoclonal antibodies 43 cases of invasive ductal carcinoma, which had been previously assayed for aromatase activity by product isolation methodology, were immunostained in three laboratories in UK, USA and Japan and independently evaluated by three pathologists (H.S., T.A. and S.G.S.). Staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments of the tumors were assessed by estimating the proportion of positive staining cells and the relative intensity of staining in this fashion. Immunoreactivity could be detected in each component of the tissue specimens but a significant positive correlation with biochemical activity was detected only in malignant epithelium stained with 677 not in other components with #677 and not in any of the components. Staining using F2 as a primary antibody did not produce a positive correlation in any components with aromatase activity. These results suggest that we now have a monoclonal antibody against aromatase (#677) which may be used to stain archival materials. A methodology and scoring system is recommended whereby staining significantly correlates with aromatase activity of the resected tissue specimens of breast cancer.

Antibodies, Monoclonal↗
PubMed2003

Validation of new aromatase monoclonal antibodies for immunohistochemistry: progress report.

Intratumoral aromatase is a potential therapeutic target for the treatment of postmenopausal estrogen-dependent breast cancers. Therefore, reliable methods should be developed for routine application for the detection of intratumoral aromatase. A multi-center collaborative group has been established to generate and validate new aromatase monoclonal antibodies (MAbs). A recombinant GST-aromatase fusion protein was expressed in baculovirus and the purified protein was used for immunization of mice either as a native or formalin-fixed antigen. Hybridomas were generated using standard techniques and screened biochemically prior to immunohistochemistry (IHC) evaluation in human placenta, ovary and breast cancer tissues. Twenty-three MAbs selected by biochemical assays were further evaluated by IHC of paraffin-embedded tissue sections including normal ovary, and placenta, and a small series of 10 breast carcinomas. Of the 23 MAbs, 2 (clones 677 and F2) were determined to specifically stain cell types known to express aromatase in normal tissues. In breast carcinomas staining of malignant epithelium, adipose tissue, normal/benign and stromal compartments was detected. IHC was performed and independently evaluated by three pathologists (HS, TJA and SGS), each using the same evaluation criteria for staining intensity and proportion of immunopositive cells. With these two MAbs, interpathologist and intralaboratory variations were minimal in comparison with differences which could be detected between tissue specimens and antibodies.

Animals↗
PubMed1990

[The validation of an enzyme immunoassay for the detection of antibodies to bovine leukosis virus].

Validation of an enzyme immuno-assay for detection of antibodies against bovine leucosis virus is described in this paper. Internal standardisation of the test was done by means of a negative control serum. With absolute extinction of the negative control serum between 100 and 200 mE, a serum sample is rated positive, if its extinction is 1.5 times above the control. The methodological sensitivity of the enzyme immuno-assay described has proved to be four times as high as that of the immunodiffusion test. The results recorded at five diagnostic laboratories suggested a sensitivity of the test of 97.6 percent (92.1 to 100 percent) and a specificity of 98.1 percent (94.4 to 100 percent). The high efficiency of the test can be confirmed by immunoblotting.

Animals↗
PubMed1996

[Validation of a ultramicroELISA for detecting antibodies against hepatitis B surface antigen].

The results of a validation study of the ultramicroanalitical assay for the detection of antibodies against the hepatitis B surface antigen (UMELISA anti-HBsAg), which was carried out by comparing the results obtained with the Hepanostika anti-HBsAg, commercial diagnosis kit are presented. For this purpose, sera from the clinical assays of the Cuban recombinant vaccine against hepatitis B were used. With the first sera group (n = 30) it was obtained, 93.1% of sensitivity, 98.5% of specificity and a concordance of 94.3%. The correlation coefficient showed a similar trend of the results (p < 0.01) and no significant differences were found in the average geometrical titre (TPG) between both assays (p > 0.05). With the second group (n = 100), whose assays were carried out at the "Pedro Kouri" Institute of Tropical Medicine (PKI) and at the Immunoassay Center (IAC) simultaneously, it was observed a sensitivity of 96.25% in both centers, a specificity of 75% at the PKI and of 90% at the IAC, and a coincidence of 92% and 95%, respectively. The correlation coefficient presented similar values and there were no significant differences between the TPG obtained by the two methods (p > 0.05). The results attained show in general the validity of the new assay and the feasibility to put it into practice either for following up the infection, or for carrying out clinical assays of vaccine evaluations.

Enzyme-Linked Immunosorbent Assay↗
PubMedDate not supplied

Anti tissue transglutaminase antibodies as predictors of silent coeliac disease in patients with hypertransaminasaemia of unknown origin.

BACKGROUND: Unexplained hypertransaminasaemia can be regarded as an extraintestinal presentation of coeliac disease. AIM: To evaluate the reliability of immunoglobulin A anti tissue transglutaminase antibodies for identifying coeliac disease in those patients with raised transaminases of unknown origin. PATIENTS: Of 1,120 consecutive patients referred to the outpatient clinic for liver disease due to raised transaminases from September 1995 to December 1999, 110 were classified as having cryptogenic hypertransaminasaemia after the exclusion of every known cause of liver disease. METHODS: These 110 patients were tested for immunoglobulin A anti tissue transglutaminase and antiendomysial antibodies by enzyme-linked immunosorbent assay and indirect immunofluorescence, respectively. RESULTS: Ten patients resulted positive for both antibodies; in all of them duodenal biopsy showed a subtotal villous atrophy consistent with coeliac disease. They did not complain of any gastrointestinal symptom. Liver biopsy, performed in five, showed a histological picture of non-specific reactive hepatitis. CONCLUSIONS: Due to the high proportion (9.15%) of patients with cryptogenic hypertransaminasaemia affected by symptomless coeliac disease, serological screening for gluten-sensitive enteropathy must be included in the work-up of these patients. In this respect, anti tissue transglutaminase antibodies represent a valid alternative to antiendomysial antibodies with the advantage of being feasible everywhere thanks to the worldwide availability of enzyme-linked immunosorbent assay.

Adolescent↗
PubMed2001

Anticardiolipin antibodies are not associated with rheumatic heart disease.

The aim of this study was to examine the prevalence of anticardiolipin antibodies in rheumatic valve heart disease. Serum samples of 31 consecutive patients with rheumatic heart disease and documented valve involvement, as well as six patients with acute rheumatic fever were tested for IgG anticardiolipin antibodies by a validated ELISA. No anticardiolipin antibodies were found when a cut-off point set at mean +/- 5 s.d. was applied. We can conclude that anticardiolipin antibodies are not present in rheumatic heart disease patients and, as suggested by several observations, these antibodies do not appear to have a pathogenic role in this particular disease.

Adult↗
PubMed1998

Validation of a monoclonal antibody-based ELISA to detect antibodies directed against swine vesicular disease virus.

A simple, rapid and sensitive competitive monoclonal antibody-based ELISA for the detection of antibodies directed against swine vesicular disease virus (SVDV) was developed. The ELISA was validated using field sera originating from SVDV-infected and non-infected Dutch pig herds, reference sera obtained from the Community Reference Laboratory for Swine Vesicular Disease at the Institute for Animal Health, Pirbright Laboratory, UK, and sera from animals infected experimentally. When testing 4277 sera originating from non-infected Dutch pig herds and collected as part of the national screening program, this ELISA had only 0.6% false positive results, whereas approximately 2% of false positive results were obtained with a conventional blocking ELISA used until recently. A sensitivity relative to the virus neutralisation test of > 97% was achieved when testing sera collected from Dutch pig farms where an outbreak of SVDV had occurred. All international reference sera scored consistently correct. Sera collected sequentially from pigs experimentally infected with SVDV isolates representing all currently recognized antigenic groups, were scored positive slightly earlier by the ELISA compared to the virus neutralisation test. This monoclonal antibody-based competitive ELISA for SVDV antibodies designated the Ceditest ELISA for SVDV-Ab, is as sensitive but more specific than the ELISA used until recently. Because sera are tested at a single dilution (1:5), incubations are carried out at room temperature and test results are available within 3 h, this ELISA is simple, easy to automate and therefore very suitable for screening large numbers of serum samples.

Animals↗
PubMed1995

Detection of antigens in immune complexes by a dot blot assay.

A simple assay is described for detection of antigens as a part of immune complexes. The complexes together with free IgG molecules are absorbed on protein A (or G)-Sepharose. The Sepharose beads are transferred to nitrocellulose membrane where all IgG including their complexes with antigens are eluted by acidic buffer directly on the surface of the membrane. The antigens immobilized on the membrane after elution are detected by corresponding labeled antibodies. The validity of the procedure was assessed by experiments with complexes of avidin and rabbit antibodies. The assay was used also for detection of DNA in immune complexes in mice with experimental systemic lupus erythematosus (SLE).

Animals↗
PubMed1996

Clinical validation of renin monoclonal antibody-based sandwich assays of renin and prorenin, and use of renin inhibitor to enhance prorenin immunoreactivity.

Newly developed IRMAs to measure the plasma concentrations of renin and prorenin were validated for clinical use and compared with a classical enzyme kinetic assay. The IRMAs involve two monoclonal antibodies, one that reacts equally well with renin and prorenin and one that recognizes renin well but prorenin only minimally. Prorenin reactivity with the second antibody was enhanced by adding the renin inhibitor, Remikiren, to plasma. The complex of prorenin with this active-site ligand undergoes a conformational change, whereby prorenin is converted into a form that cannot be differentiated from renin by the IRMA. The linear working range of the assay was 4.0-3000 mU/L. The concentration of prorenin was calculated by subtracting the assay result obtained without Remikiren (i.e., renin) from the result obtained with Remikiren (i.e., renin plus prorenin). No more than 2% of prorenin present in plasma was detected as renin. The interassay CVs for renin quantification were 18%, 13%, and 8% at low, medium, and high concentrations, respectively. The interassay CV for calculated prorenin was 8% at both low and high concentrations. The IRMA results were highly correlated with those of an enzyme kinetic assay in healthy subjects; in patients with such conditions as primary hyperaldosteronism, renovascular hypertension, and low-, medium-, and high-renin essential hypertension; and in women undergoing gonadotropin stimulation.

Adult↗
PubMed2003

Rapid and quantitative detection of human septin family Bradeion as a practical diagnostic method of colorectal and urologic cancers.

BACKGROUND: Malignant tumor progression is a complex and multi-gene event which can not be easily detected or predicted. The detection of malignant cells using marker genes is hampered by the fact that these markers are only expressed by certain malignancies or lack sensitivity and/or specificity. We have reported a human septin family gene Bradeion, which shows strong cancer-specific expression in colorectal and urologic cancers as a result of carcinogenesis. MATERIAL/METHODS: Diagnostic efficacy and validity of Bradeion gene expression were tested by two independent systems, one is a protein detection method using monoclonal antibody based immuno-chromatographic membrane strip tests (a nitrocellulose test strip assay), and another is a gene expression detection method, quantitative RT-PCR. The technology has been established using Bradeion fusion proteins, in vitro cultivated human cancer cell lines, and also patients' test samples with controls. RESULTS: Bradeion test strip by combination with two monoclonal antibodies are valid for the detection of 1 ng/ml Bradeion, and successfully applied for patient urine samples with no false-positive results. Positive detection rates were over 70% of the patient urine samples so far tested (prostate cancer, renal cell carcinoma, and bladder cancer) in 15 to 30 minutes. Quantitative RT-PCR resulted in significantly high copy numbers of 0.4-3.0-3.0 x 10(5) per microg total RNA in patients' tissue samples, whereas those from normal tissue or other cancers found negative. CONCLUSIONS: The present study introduces the practical diagnostic methods using a disease-specific molecular marker, which provides safe, economical, and rapid clinical screening of cancer.

Animals↗
PubMed2004

Validation of a quantitative SPR assay for recombinant FVIII.

Surface plasmon resonance was employed to establish a quantitative assay for recombinant FVIII (rFVIII) products using rFVIII as standard. The anti-FVIII monoclonal antibody ESH4 was immobilized onto a carboxymethyldextran surface. A range of rFVIII concentrations were injected over the surface and the binding response enhanced by the addition of a further monoclonal antibody ESH8. Validation using National Institute of Biological Standards and Controls (NIBSC) sixth International rFVIII concentrate standard gave inter- and intra-assay coefficient of variations (CVs) of 7.5 and 3.68% respectively for ESH4-rFVIII binding alone. Enhancement of the binding signal by secondary addition of ESH8 produced inter- and intra-assay CVs of 2.75 and 1.5%. The ESH4 immobilized chip was found to retain binding capacity following regeneration for at least 75 cycles. The assay was found to be unsuitable for quantitation of plasma derived FVIII product but may prove useful for monitoring of rFVIII production.

Antibodies, Monoclonal↗
PubMedDate not supplied

Characterization of immunochemical reaction for human growth hormone with its monoclonal antibody by perfusion protein G affinity chromatography and capillary zone electrophoresis.

An extremely rapid assay technique for antibody-antigen interaction using human growth hormone and its monoclonal antibody as an example has been developed by utilizing Protein G bound to perfusion chromatography matrices. The antibody and antigen were mixed and incubated at different molar ratios by keeping the concentration of antibody constant. The mixture of antibody and antigen solution was then injected onto the Protein G column. The complex of antibody and antigen formed in the sample solution was retained on the Protein G column as was the free antibody. The peak-area and height of the retained complex and antibody linearly increased with the molar concentration of the antigen when it was not in excess of the corresponding stoichiometric amount of antibody, however, those of the retained peak were constant when the concentration of antigen was in excess of that of the antibody. The validity of this method was confirmed by the results of capillary zone electrophoresis. The method developed cannot only be used to determine the biological activity between antibody and its antigen quickly, but also to determine the stoichiometry of immunological reactions between the antibody and antigen when a stable complex of them can be formed.

Antibodies, Monoclonal↗
PubMed2003

Antibody-based therapeutics in oncology.

The recent clinical and commercial success of anticancer antibodies, such as rituximab (Rituxan) and trastuzumab (Herceptin) has created great interest in antibody-based therapeutics for hematopoietic malignancies and solid tumors. Given the likely lower toxicity for antibodies versus small molecules, the potential increase in efficacy by conjugation to radioisotopes and other cellular toxins and the ability to characterize the target with clinical laboratory diagnostics to improve the drug's clinical performance, it is anticipated that current and future antibody therapeutics will find substantial roles alone and in combination therapy strategies for the treatment of patients with cancer. It is also likely that conjugation strategies will add new radiolabeled and toxin-linked products to the market to complement the recent approvals of ibritumomab tiuxetan (Zevalin) and gemtuzumab ozogamycin (Mylotarg). However, although there are a large number of agents in both early and later stages of clinical development, only a handful will make it through regulatory approval and become successful products. This review considers the structure of anticancer therapeutic antibodies, the techniques used to reduce their antigenicity, factors that influence efficacy and toxicity, conjugation with isotopes and toxins and antibody target validation.

Antibodies, Monoclonal↗