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At least 91 records · Page 5Linked to original sources

PubMed1991

Biological assays for interleukin 1 detection. Comparison of human T lymphocyte, murine thymocyte and NOB-1 assays.

Various biological assays are used for qualitative and quantitative measurements of interleukin 1 (IL-1) in supernatants from cell cultures. The purpose of the present study was to compare the specificity and variability of three cellular IL-1 bioassays: the PHA co-stimulatory human T lymphocyte proliferation assay, the PHA co-stimulatory murine thymocyte (THY) proliferation assay, and the 2-step NOB-1 conversion assay. Three different ways of IL-1 unit calculation, based on a semi-logarithmic plot, a double-logarithmic plot, or a probit-analysis plot were also compared. The T lymphocyte assay can be used only to demonstrate qualitative differences in IL-1-like activity, whereas the THY assay is excellent as a semi-quantitative assay, with a low intra-assay variability, but also with a low specificity. The NOB-1 assay is probably more specific with respect to IL-1 measurement, although, with a high intra-assay variance. The THY and the NOB-1 assays both have a high inter-assay variability, and measurement of samples from longitudinal clinical studies must be done in one and the same analysis if quantitative differences are to be illustrated. Probit analysis for unit calculation is recommended. To generate a consensus view as to assay performance, collaborative laboratory studies are needed.

Animals↗

PubMed1997

Modified and improved anti-acetylcholine receptor (AchR) antibody assay: comparison of analytical and clinical performance with conventional anti-AChR antibody assay.

We developed a modified anti-acetylcholine receptor (AChR) antibody (Ab) assay based on a radioreceptor assay and a calibration curve. We compared the analytical and clinical performances of this modified assay with those of the conventional anti-AChR Ab radioreceptor assay. Serum specimens were from patients with myasthenia gravis (MG) (n = 156) and from control subjects (n = 106). The modified assay demonstrated lower within-assay (4.0-6.6%) and between-assay (5.3-7.8%) CVs, greater linearity, lower cost, and shorter assay time than the conventional method. ROC curve analysis indicated almost identical specificity and sensitivity (> 0.92) for these two anti-AChR Ab assays. The modified and conventional assays were also equivalent for blocking anti-AChR Ab assay. Moreover, the modified anti-AChR Ab assay, differently from the conventional assay, allowed us to reveal anti-AChR Ab concentration differences among different clinical grades of MG.

Adolescent↗

PubMed1997

The rodent uterotrophic assay: critical protocol features, studies with nonyl phenols, and comparison with a yeast estrogenicity assay.

The major protocol features of the immature rat uterotrophic assay have been evaluated using a range of reference chemicals. The protocol variables considered include the selection of the test species and route of chemical administration, the age of the test animals, the maintenance diet used, and the specificity of the assay for estrogens. It is concluded that three daily oral administrations of test chemicals to 21- to 22-day-old rats, followed by determination of absolute uterus weights on the fourth day, provide a sensitive and toxicologically relevant in vivo estrogenicity assay. Rats are favored over mice for reasons of toxicological practice, but the choice of test species is probably not a critical protocol variable, as evidenced by the similar sensitivity of rats and mice to the uterotrophic activity of methoxychlor. Vaginal opening is shown to be a useful, but nondefinitive, adjunct to the uterotrophic assay. The ability of test chemicals to reduce or abolish the uterotrophic response of estradiol is suggested to provide a useful extension of the uterotrophic assay for the purpose of detecting antiestrogens. The results of a series of studies on the environmental estrogen nonyl phenol (NP), and its linear isomer n-nonyl phenol, confirm that branching of the aliphatic side chain is important for activity. 17beta-Desoxyestradiol is shown to be of similar activity to estradiol in the uterotrophic assay and is suggested to represent the "parent" estrogen of NP. Benzoylation of NP and 17-desoxyestradiol did not affect their uterotrophic activity, in contrast to the enhancing effect of benzoylation on estradiol. Selected chemicals shown to be active in the immature rat uterotrophic assay were also evaluated in an in vitro yeast human estrogen receptor transactivation assay. Most of the chemicals gave similar qualitative responses to those seen in the uterotrophic assay, and the detection of the estrogen methoxychlor by the yeast assay evidenced a degree of intrinsic metabolic competence. However, the assay had a reduced ability (compared to rodents) to hydrolyze the benzoate ester of estradiol, and the estrogenic benzoate derivative of NP was not active in the yeast assay. These last results indicate that current metabolic deficiencies of in vitro estrogenicity assays will limit the value of negative data for the immediate future. The results described illustrate the intrinsic complexity of evaluating chemicals for estrogenic activities and confirm the need for rigorous attention to experimental design and criteria for assessing estrogenic activity.

Animals↗

PubMed1991

Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S. typhimurium mutagenicity and rodent carcinogenicity assays.

The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation. It is a multi-endpoint assay which utilizes E. coli WP2s(lambda). Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells'). Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested. Results with 133 compounds are presented. These include 111 compounds which have been tested in the S. typhimurium assay and 66 compounds for which both rodent bioassay and S. typhimurium assay data exists. The concordance for the Microscreen assay and the S. typhimurium assay was 71%. For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S. typhimurium assay. However, the S. typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%). The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S. typhimurium assay the association with carcinogenicity was non-significant (p = 0.086). The Microscreen assay was able to detect halogenated compounds better than the S. typhimurium assay. The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting.

Animals↗

PubMed2005

Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability.

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.

Animals↗

PubMed1997

Evaluation of a bedside whole-blood rapid troponin T assay in the emergency department. Rapid Evaluation by Assay of Cardiac Troponin T (REACTT) Investigators Study Group.

OBJECTIVE: To evaluate the performance of a new bedside whole-blood rapid assay for cardiac troponin T (cTnT) in patients presenting to the ED with symptoms consistent with acute coronary ischemia. METHODS: A prospective, observational trial was performed in 8 participating medical centers. Serial blood samples were obtained on presentation to the ED and 3 and 6 hours later. The cTnT whole-blood rapid assays were performed immediately at the site. Treating physicians and patients were blinded to the results of the rapid assays. Serum samples were analyzed at the core laboratory for serum cTnT, creatine kinase (CK)-MB, and myoglobin. The sensitivity of the rapid assay for detecting acute myocardial infarction (AMI) was compared with the sensitivities of serum cTnT, CK-MB, and myoglobin assays. RESULTS: Of 721 patients, 102 were diagnosed as having AMI. The median elapsed time from symptom onset to ED arrival was 3 hours. The sensitivities of the rapid assay for detecting AMI were 19.6%, 59.0%, and 69.7% at 0, 3, and 6 hours, respectively. The sensitivities of the serum cTnT assay (p-values for comparison with the rapid assay for cTnT) were 25.0% (p = 0.18), 69.6% (p = 0.04), and 79.8% (p = 0.02) at 0, 3, and 6 hours, respectively. The CK-MB and myoglobin sensitivities were 21.9%, 64.5%, and 81.0%; and 43.8%, 77.4%, and 71.4%, respectively. There were 7 patients with AMI who had negative rapid assay readings and positive serum cTnT levels; 4 of these patients were enrolled at the same site. Twenty patients not diagnosed as having AMI had at least one positive rapid assay. Fourteen of these 20 patients had a diagnosis of clinically relevant cardiac disease. CONCLUSIONS: The sensitivity of this whole-blood rapid cTnT assay for detecting AMI is comparable to that of current serum assays and offers the advantage of providing rapid bedside results. Discrepancies between serum and whole-blood assays for cTnT noted in this study may indicate the need for further education for the test reader prior to patient use.

Adult↗

PubMed1987

Comparison of reverse transcriptase assay with the Retro-Tek viral capture assay for detection of human immunodeficiency virus.

The reverse transcriptase (RT) assay for human immunodeficiency virus (HIV) is tedious, expensive, and nonspecific for HIV activity. Because the Retro-Tek viral capture assay (VCA) (Cellular Products, Inc., Buffalo, NY) is relatively quick, inexpensive, and specific for HIV antigens, we compared the sensitivity and specificity of the two assays in a blinded fashion to see if the VCA could replace the RT assay. Specifically, peripheral mononuclear cells were cocultured from 11 Western blot-positive acquired immunodeficiency syndrome (AIDS) patients. The culture supernatant fluids were tested every 4-7 days for at least 28 days by VCA for HIV antigens, and by RT assay for retrovirus activity. Ten seronegative healthy donors with no risk factors were also cocultured and tested. Seven (64%) of 11 AIDS patients were positive by VCA and confirmed by RT assay in each instance. Concordance between VCA and RT assay was 100% in each patient. Time-to-positive detection was 7-28 days for both assays. Once a culture became positive by either assay, subsequent culture supernatant fluids remained positive by both assays until the culture was discarded, usually at day 28. The ten healthy seronegative donors were negative by both assays. These results suggest that the VCA is preferable to the RT assay for detection of HIV.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Detection of anti-DNA antibodies: a comparison between two Farr assays, Crithidia luciliae and a human chromosomal substrate assay.

Four commercially available assays were compared with a 14C DNA Farr assay for their ability to detect anti-DNA antibodies in 119 sera from 109 patients with systemic lupus erythematosus (SLE) and 25 control sera. The 14C DNA Farr assay was the most sensitive and specific assay (SLE, 57% positive, controls, 0%). A commercial 125I DNA Farr assay was significantly less sensitive (SLE, 39% positive). The fluorescence human chromosomal preparation assay was as sensitive as the 14C DNA Farr assay (SLE, 58% positive) but less specific (controls, 8% positive). The immunofluorescence Crithidia luciliae assay was specific, but less sensitive (SLE, 37% positive) than the 14C DNA Farr assay. Adaptation of Crithidia to immunoperoxidase did not alter its sensitivity or performance. These results confirm that the 14C DNA Farr assay, locally refined and performed by experienced hands, is the most sensitive and specific assay for anti-DNA antibodies. The 125I DNA Farr was no more sensitive than the Crithidia assay but considerably more laborious. The human chromosomal preparation may be suitable as a rapid screening test for anti-DNA antibodies.

Animals↗

PubMed2000

Fluorescent coupled enzyme assays for D-alanine: application to penicillin-binding protein and vancomycin activity assays.

D-Alanine (D-Ala) is a ubiquitous constituent of bacterial cell walls. Assays for D-Ala can be used to investigate several aspects of cell wall biosynthesis and the effects of antibiotics on this process. High-sensitivity fluorescent assays for D-Ala were developed in a microtiter plate format based on d-aminoacid oxidase/horseradish peroxidase (DAO/HRP)-coupled reactions. For comparative purposes the classic chromogenic (UV-vis) assay using o-phenylenediamine (OPD) was also adapted to microtiter plates. OPD gave a lower limit of sensitivity of 2 nmol and was linear up to 60 nmol. Two commercially available fluorogenic HRP substrates were then tested in this assay. Amplex Red (AR) gave a lower limit of sensitivity of 2 pmol and was linear up to 400 pmol d-Ala. QuantaBlu (QB) based assays exhibited a lag in their response to D-Ala corresponding to 50 pmol D-Ala. This lag complicated calibration, but could be eliminated by addition of 150 pmol D-Ala to all assays. The QB assays were linear up to 3000 pmol D-Ala and gave a lower limit of sensitivity of 10 pmol. These assays are demonstrated for the characterization of the dd-carboxypeptidase activity of a soluble form of Escherichia coli penicillin-binding protein 5 (PBP 5) against the classic PBP substrate diacetyl-L-Lys-D-Ala-D-Ala. AR and QB based assays gave identical v/E(T) profiles, whereas OPD based assays gave slightly (10%) higher activity. This is consistent with the loss of a small amount of E. coli PBP 5 activity during the dilution necessary prior to its use in the highly sensitive fluorescent assays. These assays were then demonstrated for characterization of vancomycin binding to a D-Ala-D-Ala-based substrate.

Alanine↗

PubMed2002

Evaluation of the clinical performance of equimolar- and skewed-response total prostate-specific antigen assays versus complexed and free PSA assays and their ratios in discriminating between benign prostatic hyperplasia and prostate cancer.

BACKGROUND: Prostate-specific antigen (PSA) exists in human serum in two principal forms, free PSA (fPSA) and protein-complexed PSA, predominantly PSA-ACT (alpha(1)-antichymotrypsin). Equimolar response (EMR) total PSA (tPSA) immunoassays measure each of these forms equally while skewed-response (SKR) assays overestimate or underestimate the tPSA concentration. The advantages of EMR over SKR tPSA assays are controversial. METHODS: We used five nonhuman serum-based samples each containing a different proportion of fSPA:PSA-ACT (0:100 to 100:0, %:%) and patients' serum samples from men with histologically confirmed benign prostatic hyperplasia (BPH) (n=94) or PCA (n=30) and a wide range of fPSA concentrations to investigate the molar response status of six tPSA assays. Receiver-operator characteristic (ROC) curve analysis was used to compare the discriminatory power of these assays in distinguishing men with BPH from those with PCA. RESULTS: The Bayer Immuno-1 tPSA (BtPSA) assay demonstrated EMR characteristics and diagnostic accuracy similar to the Hybritech Tandem-E and Tandem-R tPSA assays. At 90% sensitivity, EMR tPSA assays had higher specificity than SKR tPSA assays. CONCLUSIONS: The BtPSA assay is an EMR tPSA assay and EMR assays provide improved diagnostic specificity over SKR tPSA assays.

Adult↗

PubMed2002

Quantifying the stress induced by distress in patients with lumbar disc herniation in terms of natural killer cell activity measurements: chromium release assay versus multiparameter flow cytometric assay.

STUDY DESIGN: A comparative study was conducted. OBJECTIVES: To attempt quantification of the stress in patients with lumbar disc herniation by measuring natural killer cell activity, and to evaluate the assay methodology. SUMMARY OF BACKGROUND DATA: Stressful events may have adverse effects on health. Distress in the patient with lumbar disc herniation also may be a stressful event. Previous findings have shown the activity of natural killer cells to be impaired in medical student volunteers and after bereavement or other stressful life events. However, the stress in patients with spinal disorders has not been evaluated. The standard assay for natural killer cell activity has been the chromium release assay. However, this assay requires the use of radioactive chromium. Flow cytometric assay is a nonradioactive method that analyzes the activated natural killer lymphocyte subsets: CD45+, CD56+, CD69+. METHODS: Peripheral blood lymphocytes were obtained from 20 patients with lumbar disc herniation (mean age, 30.8 years) and 20 healthy individuals (mean age, 38.9 years). Both chromium release assay and flow cytometric assay were performed for each patient. RESULTS: The mean natural killer cell activity in the patients (8.0% +/- 4.3%) was significantly lower than in the 20 healthy adult volunteers (12.6% +/- 4.5%). The mean natural killer cell activity in the healthy volunteers was 12.6% +/- 4.5% for the chromium release assay and 11.0% +/- 4.1% for the flow cytometric assay. The correlation (r = 0.77) of natural killer cell activity between the two methods was high. CONCLUSIONS: Natural killer cell activity in the patients with lumbar disc herniation was significantly lower than in healthy individuals, and the stress induced by pain and other distress may be present in patients with lumbar disc herniation. For measuring natural killer cell activity, flow cytometric assay is a reproducible assay, and it may be a viable alternative to the standard chromium release assay.

Adult↗

PubMed1997

Comparative study of cytomegalovirus (CMV) antigenemia assay, polymerase chain reaction, serology, and shell vial assay in the early diagnosis and monitoring of CMV infection after renal transplantation.

BACKGROUND: Early diagnosis of cytomegalovirus (CMV) infection, which is an important cause of morbidity and mortality in renal transplant recipients, remains of great importance. This prospective study was performed in kidney transplant recipients to determine the diagnostic value of the CMV antigenemia assay in comparison with polymerase chain reaction (PCR), serology, and shell vial assay. METHODS: Seventy-five consecutive renal transplant recipients were enrolled in this study and monitored by both antigenemia assay and serology. The initial 34 of the 75 patients were subjected to PCR and shell vial assay. RESULTS: Antigenemia, PCR, and shell vial assay became positive before the onset of CMV-related symptoms in 31/34 (89%), 13/16 (81%), and 2/16 (13%), respectively. None of the 34 patients who had symptomatic CMV disease showed a significant increase in IgG or IgM before the onset of symptoms. Antigenemia and PCR assays turned positive, 7 and 11 days (median), respectively, before the onset of clinical symptoms. Serology and shell vial assay became positive 21 and 25 days (median), respectively, after the onset of CMV-related clinical symptoms. To examine the clinical value of these assays, "good correlation" was defined based on the correlation between the clinical course and the results of the assays. Good correlation with the antigenemia assay was observed in 33 (96%) out of 34 renal transplant recipients who recovered from their CMV disease after ganciclovir therapy. Only one of 16 (7%) patients showed good correlation by shell vial assay, whereas PCR and serology did not show a good correlation. Consequently, antigenemia was considered the best way to monitor CMV infections after kidney transplantation. CONCLUSIONS: Only the CMV antigenemia assay can be successfully employed after renal transplantation for the early diagnosis and extensive monitoring of active CMV infection.

Adolescent↗

PubMed2004

Evaluation of real time polymerase chain reaction assays for confirmation of Neisseria gonorrhoeae in clinical samples tested positive in the Roche Cobas Amplicor assay.

OBJECTIVE: Development of a rapid, sensitive, and accurate assay for confirmation of Neisseria gonorrhoeae in clinical samples. METHOD: Two real time polymerase chain reaction (PCR) assays, developed on the LightCycler for amplification of the N gonorrhoeae cppB gene, were utilised for confirmation of this bacterial pathogen in samples positive by the Roche Cobas Amplicor assay. Performance characteristics of the two assays were compared with other commercial nucleic acid amplification assays, including the Abbott LCx and Roche 16S rRNA tests. RESULTS: All related Neisseria as well as other bacterial species tested negative by both cppB gene based assays, whereas 120 N gonorrhoeae clinical isolates from various geographical regions gave in positive results. Both assays had a sensitivity of one copy per reaction. 122 clinical samples positive and another 50 samples negative for N gonorrhoeae by Roche Cobas Amplicor were selected from a specimen pool of more than 3000 women tested previously. Overall, 73 of 122 (59.8%) samples were confirmed as positive. The two real time assays had sensitivities of 99% and 100% and specificities of 98% and 100%, respectively. The 16S and LCx assays produced similar results to the real time assays, indicating a similar sensitivity to and specificity of both real time assays. CONCLUSION: The data from this study highlight the need to confirm N gonorrhoeae positive Cobas Amplicor PCR results as an important part of the testing algorithm of all diagnostic laboratories utilising this assay.

DNA, Bacterial↗

PubMed2003

Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists.

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.

Animals↗

PubMed2001

The fibrin assay comparison trial (FACT): correlation of soluble fibrin assays with D-dimer.

Soluble fibrin (SF) is regarded as an indicator of acute fibrin formation and a precursor of fibrin thrombi. Using a set of clinical plasma samples, and fibrin derivatives, five assays for measurement of SF, including two chromogenic assays, two ELISA systems, and one latex-enhanced photometric immunoassay were compared. Correlation between SF assays was moderate (Spearman's rho values between 0.344 and 0.805). Re-calibration with serial dilutions of desAABB-fibrin monomer resulted in adjustment of the numerical scale of the assays without improving correlation. All SF assays reacted with purified crosslinked fibrin derivatives. Using clinical plasma samples, Spearman's rho of SF assays with D-dimer consensus values based upon results of 23 quantitative D-dimer assays were between 0.491 and 0.911. Although all SF assays react with desAABB-fibrin monomer complexes, SF assays are heterogeneous concerning reactivity with fibrin compounds observed in clinical plasma samples. The prospect of a common calibrator for SF assays therefore seems to be remote. Since SF assays react with crosslinked fibrin derivatives, it is not possible to clearly distinguish between acute fibrin formation, and fibrin dissolution on the basis of the results of current SF assays.

Calibration↗

PubMed1993

Analytical performance evaluation of EMIT II monoclonal amphetamine/methamphetamine assay: more specificity than EMIT d.a.u. monoclonal amphetamine/methamphetamine assay.

We evaluated a new EMIT II monoclonal amphetamine/methamphetamine assay for screening human urine by comparing it with the EMIT d.a.u. monoclonal amphetamine/methamphetamine assay and a fluorescence polarization assay. The EMIT II assay has a cutoff of 1 mg/L d-methamphetamine. The EMIT II and EMIT d.a.u. assays were run on a BM/Hitachi 704 analyzer; for the fluorescence polarization assay we used a TDx analyzer. All EMIT II positive samples were also analyzed by the fluorescence polarization assay. We used gas chromatography/mass spectrometry (GC/MS) for confirmation of the presence of amphetamine or methamphetamine. Within-run CVs for the Level 1 (1 mg/L) and Level 2 (3 mg/L) calibrators for the EMIT II assay were 0.47% and 0.53%, respectively. Corresponding between-run CVs were 1.48% and 1.60%, respectively. Of the 1007 samples screened for amphetamines, 50 were positive by the EMIT d.a.u. assay; 21 samples (not a subset of the 50 samples) were positive by the EMIT II assay. However, 19 samples that tested positive by EMIT II also tested positive by the EMIT d.a.u. assay. Subsequent testing of the EMIT II positive samples by the fluorescence polarization assay detected in six positive samples. By means of chiral derivatization wer identified two specimens containing primarily l-isomers of amphetamine and methamphetamine. Sympathomimetic amines were identified in several of the samples not containing amphetamine or methamphetamine.

Amphetamine↗

PubMed1995

Comparison of an indirect immunofluorescence assay and a modified sensitive immunoblot assay for the study of the autoantibody in pemphigus vulgaris.

Some patients with pemphigus vulgaris (PV) have positive direct immunofluorescence (DIF) but are negative by indirect immunofluorescence (IIF). The purpose of this study was (1) to compare the sensitivity of an IIF assay with an immunoblot (IB) assay, (2) to compare the IIF and the IB assay in PV patients in whom the clinical picture and DIF were consistent, but the IIF was negative and (3) to compare the IIF and the IB assay in patients in clinical remission for 3 years or more. A comparison was made of the titers of PV autoantibody in the IIF assay using monkey esophagus as substrate and the modified sensitive IB assay using preabsorbed normal human skin lysate and COLO-16 lysate as a substrate in the three groups of patients. The sensitivity of the Western blot was enhanced by modifications in the extraction procedure of the lysate, by absorption of lysate with normal human serum and by the use of an enzygraphic web. In group 1, comprising 23 PV patients with active generalized disease, the titers of the autoantibody in the IB assay were 2-4-fold higher than in the IIF assay. This difference was highly significant (P = 0.0001). In group 2, comprising 10 patients with limited or minimal PV who were positive on DIF and negative on IIF, all the patients were positive in the IB assay. In group 3, comprising 9 patients clinically free of disease and off all therapy for at least 3 years and negative in IIF assay, all the patients were positive in the IB assay.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

High sensitivity two-site immunoradiometric and immunochemiluminometric assays for rat growth hormone-releasing hormone. Development of an acridinium ester based chemiluminescence assay for the avidin-coated bead system.

High sensitivity two-site immunoradiometric and immunochemiluminometric assays were developed for the direct measurement of rat GHRH in perifusate of hypothalamic fragments and in hypophysial portal plasma. A solid phase immunoradiometric assay was developed initially using avidin-coated polystyrene beads and radioimmunoassay quality polyclonal antibodies to N-terminal and C-terminal fragments of GHRH. Immunoaffinity purification of the high affinity antibodies required a strong eluant, 1 M acetic acid with 10% dioxane. For high sensitivity, the affinity-purified C-terminal signal antibody was iodinated by the lactoperoxidase-glucose oxidase method and absorbed with avidin-BSA-IgG conjugated matrix. Conversion to an acridinium ester based immunochemiluminometric assay required substantial modifications to optimize performance. For both assays, the innovative modification of delayed addition of the bead enhanced assay performance by two-fold. By precision profile analysis the sensitivity of the immunochemiluminiometric assay (0.035 pg/tube) was two-fold better than the sensitivity of the immunoradiometric assay (0.066 pg/tube). These assays, 4-7-fold more sensitive than a highly optimized radioimmunoassay (0.26 pg/tube), were validated for direct unextracted measurement of GHRH in perifusate and in hypophysial portal plasma. They have significant advantages for biological measurements, reducing the number of hypothalami per perifusion chamber and enabling measurement of low concentrations of plasma GHRH not previously possible. The avidin-coated bead system can be flexibly used to develop high sensitivity two-site peptide assays using polyclonal antibodies. Further, this system has been extended to acridinium ester based immunochemiluminometric assays with improvement of assay performance.

Acridines↗