Sandwich Enzyme-linked Immunoadsorbent Assay Research Articles

Effect of emodin on gut microbiota of rats with acute kidney failure

The aim of this paper was to investigate the effect of emodin on gut microbiota in acute kidney injury rats( AKI). Rats were randomly divided into several groups: normal group,model group,low-dose of emodin group( 10 mg·kg~(-1)),medium-dose of emodin group( 25 mg·kg~(-1)),high-dose of emodin group( 50 mg·kg~(-1)) and control group( 5 mg·kg~(-1) of benazepril hydrochloride).The AKI model rats were established by intraperitoneal injection of small dose of gentamicin sulfate for 7 days. Two hours after intraperitoneal injection,except for the normal group and the model group,the other groups were given corresponding doses of drugs for 15 days. The serum levels of serum creatinine( SCr),urea nitrogen( BUN),plasma endotoxin level,24 h urinary protein and D-lactate in the plasma were determined by sarcosine oxidase,urease method,tal reagent method,bromo cresol chloroform method and double antibody sandwich enzyme-linked immunoadsorbent assay,respectively. Gut microbial communities were assayed by fluorescent quantitative PCR methods. HE staining was used to detect the pathological changes of the kidneys. Compared with the normal group,there were significant differences in body weight,urinary protein( UTP),bacterial endotoxin,urea nitrogen,creatinine,D-lactate in the plasma and four bacterial contents in the model group( P<0. 05). The urinary protein,urea nitrogen,D-lactate,creatinine and plasma bacterial endotoxin in control group and each emodin group were lower than those in model group,especially for high-dose of emodin( P<0. 01). Moreover,pathology resolution in high-dose emodin was better than other groups. Except for low-dose of emodin group,qRT-PCR data suggested that the amounts of Escherichia coli and Enterococcus in medication administration group were increased,while the amounts of Lactobacilli and Bifidobacterium were reduced compared with model group( P<0. 05),especially for high-dose of emodin( P<0. 01). There is a clear imbalance of gut microbiota in rats with AKI. Emodin could regulate the imbalance of gut microbiota,which might be one of the mechanisms of its effects on AKI rats.

Development of sandwich ELISA for detection and quantification of invertebrate major allergen tropomyosin by a monoclonal antibody

Muscle protein tropomyosins of invertebrates are major allergens responsible for wide spread allergic reactions against invertebrates such as shellfish and insects. In order to develop a sandwich enzyme-linked immunoadsorbent assay (ELISA) for detection and quantification of the invertebrate pan-allergen tropomyosin, a specific monoclonal antibody (MAb), CE7B2, was produced. We have successfully established a sandwich ELISA for measuring invertebrate tropomyosin concentrations in food and food materials. The sandwich ELISA system using the MAb CE7B2 is a useful tool to detect and quantify levels of tropomyosin in food. The method is also helpful to detect mite and cockroach tropomyosins, the important indoor allergens.

Development of sandwich ELISA for detection and quantification of human and murine a disintegrin and metalloproteinase17

ADAM17 (a disintegrin and metalloproteinase-containing protein 17) is a membrane-bound metalloproteinase, implicates in many physiological processes, including cell migration and proliferation. Of particular note, most of the studies so far are restricted on the analysis of ADAM17 mRNA levels. In this study we generated, utilizing hybridoma technology, three monoclonal antibodies (mAbs) (A 300, A 309 and A 318) against the extracellular domain of human ADAM17 to enable quantification of protein expression. The specificity of these mAbs against ADAM17 was tested by enzyme-linked immunoadsorbent assay (ELISA), flourescence-activated cell sorting (FACS) and western blotting. In order to quantify human and murine ADAM17 expression two pairs of these mAbs (biotinylated A 309 in combination with A 300 and biotinylated A 300 in combination with A 318), were used to develop sandwich ELISA. A panel of monoclonal antibodies was generated for first time to measure mouse ADAM17 with a sensitivty of 2ng/ml. Such systems provide a useful tool to quantify protein levels of ADAM17 and are valuable tools for diagnostic purposes.

Correlation between proinflammatory serum markers: high sensitivity C-reactive protein, interleukin-6 with disability score in acute ischemic stroke.

Stroke being the third leading cause of death and foremost cause of disability, if potential diagnostic utility of blood borne protein biomarkers in predicting acute stroke is established, it would be a substantial adjunct to computerized tomography and magnetic resonance imaging which have their own limitations. This study was done to correlate serum Interleukin 6, high sensitivity C reactive protein at the time of admission with neurological worsening assessed by NIHSS at the time of admission and 7days after admission. 46 Patients admitted in neurology department SAIMS, Indore with first ever ischemic stroke within 72h of onset were included in the study. All patients with history of stroke of more than 72h onset, Infection & peripartum stroke were excluded from the study. Disability scoring was done by NIHSS and their serum samples assayed for hsCRP, IL6 by commercially available quantitative sandwich enzyme-linked immunoadsorbent assay kits. Serum samples of 50 control cases which included healthy volunteers and staff from SAIMS were also analyzed for hsCRP, IL6 for comparative study. A significant correlation was observed between NIHSS scoring and serum hsCRP and IL6 at the time of admission. Patients with initial high serum IL6 and hsCRP also showed significant clinical deterioration as assessed by NIHSS scoring 7days after admission. Elevated hsCRP and IL6 within 72h of admission strongly correlated with functional disability in study population in India and may serve as useful adjunct to CT Scan in emergency setting.

Correlation of cytologic examination with ELISA assays for hyaluronan and soluble CD44v6 levels in evaluation of effusions

Hyaluronan (HA) and its major cell surface receptor, CD44, play an important role in tumor growth, proliferation, neovascularization, and invasion. CD44 is an integral transmembrane protein and exists in standard form (CD44s), as well as a myriad of CD44 variants isoforms (CD44v1-v10). Functional fragments of the CD44 can be released from the cell membrane by proteolytic cleavage of extracellular domain producing soluble CD44. Although studies have proposed the use of serum HA and soluble CD44, specifically soluble CD44v6 (sCD44v6) levels, as a tumor markers, its diagnostic utility in body fluid samples has not been clearly established. The purpose of this study was to correlate HA and sCD44v6 levels in effusions with the cytology diagnosis and to assess their usefulness in differentiating between malignant and nonmalignant effusions. In this retrospective study we evaluated HA and sCD44v6 contents in 20 effusions from cytologically positive samples and 10 effusions from cytologically negative samples. Corresponding cytopathology slides were reviewed to confirm the diagnoses. Malignant effusions included 18 cases of metastatic adenocarcinomas (9 ovarian, 3 breast, 3 pulmonary, 3 adenocarcinoma of unknown primary) and 2 cases of lymphomas. The level of HA and sCD44v6 were measured using a sandwich enzyme-linked immunoadsorbent assay. For HA, we used hyaluronic acid quantitative test kit (Corgenix, Denver, CO) and for sCD44v6 we used Human sCD44v6 Instant ELISA (Bender MedSystems, Vienna, Austria). HA concentrations (microg/mL) and sCD44v6 concentrations (ng/mL) were calculated and correlated with clinical data as well as cytodiagnosis. The mean concentration of HA (22.42 +/- 5 microg/mL) and sCD44v6 (70 +/- 42 ng/mL) in the cytologically positive samples was significantly higher than those in the cytologically negative samples for HA (5.5 +/- 5 microg/mL, P < 0.01) and sCD44V6 (17 +/- 10 ng/mL, P < 0.01). Using benign effusions as control and the upper limits of its mean levels for HA (10.5 microg/mL) as the positive boundary value, HA levels exceeded the boundary line in 17 out of 20 malignant effusions and 2 out of 10 benign effusions. Meanwhile, sCD44v6 exceeded the boundary line (27 ng/mL) in 18 out of 20 malignant effusions and 3 out of 10 benign effusions. The calculated sensitivity and specificity of this assay to the diagnosis of malignant effusions were 85 and 80% for HA and 90 and 70% for CD44v6, respectively. We conclude that the HA and sCD44v6 levels in body fluids correlate with the cytology diagnosis and could be used as an ancillary study in cytology to differentiate nonmalignant from malignant effusions.

Markers for endothelial activation during open heart surgery

Background Reliable markers for endothelial activation are needed when studying biocompatibility of cardiopulmonary bypass. Methods Blood samples from 21 patients undergoing combined valve and coronary artery bypass surgery were collected before anesthesia (T1), after re-transfusion of blood from the heart-lung machine (T2), and on the first postoperative morning (T3). Concentrations of soluble markers were determined using sandwich enzyme-linked immunoadsorbent assay for sICAM-1, sVCAM-1, and sE-selectin. The sera were also used to stimulate human umbilical vein endothelial cells (HUVEC) in culture for 6 hours, in which activation was measured using cell enzyme immunoassay for mICAM-1 and mVCAM-1. Results The concentrations of sICAM-1 and sVCAM-1 increased during both measurement intervals ( p < 0.05). The sICAM-1 T1 was 311.0 ng/mL (range, 271.0 to 350.7 ng/mL); the sICAM-1 T2 was 341.6 ng/mL (range, 322.0 to 422.0 ng/mL), and the sICAM-1 T3 was 400.2 ng/mL (range, 348.0 to 556.4 ng/mL; the sVCAM-1 T1 was 607.5 ng/mL (range, 497.8 to 813.8 ng/mL), the sVCAM-1 T2 was 755.3 ng/mL (range, 660.6 to 834.4 ng/mL), and the sVCAM-1 T3 was 1149.0 ng/mL (946.0 to 1406.0 ng/mL); whereas the sE-selectin increased from T1 to T3 ( p < 0.01). Both the mICAM-1 ( p < 0.002) and the mVCAM-1 ( p < 0.005) increased on the human umbilical vein endothelial cells in culture after stimulation with the patient sera. The amounts of soluble markers in vivo were not correlated with the degree of endothelial activation in vitro, but were correlated with various operative variables including age, medication, and time of aortic cross-clamping. Conclusions Endothelial cells were activated during cardiopulmonary bypass. The soluble adhesion molecules sICAM-1, sVCAM-1, and sE-selectin displayed different kinetics, rendering it difficult to determine a simple expression for the degree of endothelial cell activation. Clinically, sVCAM-1 seemed to be the best-suited marker for endothelial cell activation, because it was only associated with aortic cross-clamping and heparin and protamine doses, and it also showed the largest numerical changes.

Detection of VEGF levels in ascites and peritoneal fluid

Objective: The expression of vascular endothelial growth factor (VEGF) is correlated to the invasion and metastasis of tumor cells in many clinical carcinomas. In this study, we detected soluble VEGF levels in ascites and peritoneal fluid and explored its clinical significance. Methods: A total of 91 samples were collected and divided into 5 experimental groups: petitoneal fluid of patients with benign (n=10) and malignant disease (n=14), cirrhotic ascites (n=36), tuberculous ascites (n=8) and malignant ascites (n=23). Using a sandwich enzyme-linked immunoadsorbent assay, the concentration of soluble VEGF was measured in ascites (n=67) and petitoneal fluid (n=24). Results: VEGF levels in malignant ascites were 640.74±264.81 pg/ml, significantly higher than those in cirrhotic ascites, tuberculous ascites and peritoneal fluid of patients with benign and malignant disease (P<0.01, separately). However, the difference of VEGF levels among the latter 4 groups had no statistic significance (P>0.05), separately). Furthermore, VEGF levels in malignant ascites from patients with ovarian cancer were higher than those with gastric and colon cancer (P<0.01, respectively), while there was no significant difference between gastric cancer and colon cancer (P>0.05). Using VEGF level of 118.96 pg/ml as a minimum cutofflimit, the sensitivity and specificity of VEGF of this assay to diagnose malignant ascites were 91.3% and 73.9% respectively. Conclusion: The elevated levels of VEGF may be useful as an index in differential diagnosis of benign and malignant ascites. It appears that VEGF plays an important role in malignant ascites formation.

Association between plasma oxidized low-density lipoprotein and diabetic nephropathy

To investigate the association of oxidized low-density lipoprotein (ox-LDL) with the development of diabetic nephropathy, plasma levels of ox-LDL were measured in 70 patients with type 2 diabetes mellitus. A sandwich enzyme-linked immunoadsorbent assay (ELISA) using the mouse monoclonal antibody FOH1a/DLH3, which specifically recognizes oxidized phosphatidylcholine, and a horseradish peroxidase (HRP)-labeled goat anti-human apolipoprotein B IgG was used to measure ox-LDL levels. The mean age of the patients was 57.0±1 3.4 years, and the mean duration of diabetes was 13.4±8.5 years. Plasma ox-LDL levels were similar in patients with normoalbuminuria (13.7±3.9 U/ml), patients with microalbuminuria (12.8±3.9 U/ml), and normal controls (12.5±4.2 U/ml). However, the plasma ox-LDL level in patients with macroalbuminuria (16.8±7.5 U/ml) was significantly higher than those in the other groups ( P<0.05). Hemoglobin A1c (HbA1c) levels were similar in diabetic patients with normoalbuminuria (8.2±2.2%), microalbuminuria (7.8±1.3%), or macroalbuminuria (7.2±1.4%). There was no significant correlation between the ox-LDL level and the HbA1c level. The significantly elevated plasma ox-LDL levels in patients with macroalbuminuria suggest that ox-LDL may play an important role in the progression of diabetic nephropathy.

Development of a sandwich ELISA test for arginase measurement based on monoclonal antibodies.

Human arginase was purified from liver and two monoclonal antibodies (MAbs), HA1 and HA2, were produced by fusion of spleen cells from an arginase-immunized BALB/c mouse and the NS-1 myeloma cell line. Both MAbs were of the IgG3 subclass and contained the kappa light chain. HA1 inhibited arginase activity, suggesting that it binds to the arginase catalytic site. HA1 and a horseradish peroxidase-conjugated polyclonal rabbit anti-human arginase antibody were used to develop a sandwich enzyme-linked immunoadsorbent assay (ELISA) for the quantification of human arginase, which can be used in the 1 to 300 ng/mL range. Because of its sensitivity and specificity, this MAb can be successfully applied to the ELISA quantification of arginase in serum and culture supernatants.

Vascular endothelial growth factor in malignant and tuberculous pleural effusions

The purpose of this study is to assess the usefulness of soluble vascular endothelial growth factor (VEGF) in the effusions of patients with malignant and tuberculous diseases. Using a sandwich enzyme-linked immunoadsorbent assay, VEGF concentration was measured in malignant (n=17) and tuberculous (n=11) pleural effusions. Pleural biopsy, cytology or microbiological methods were used to make final diagnoses. Adenosine deaminase (ADA) levels in tuberculous pleural effusions were significantly higher than those in malignant pleural effusions. The median level of VEGF in patients with malignant effusions (median, 2418 pg/mL; range, 97-62103 pg/mL) was significantly higher than tuberculous effusions (median, 994 pg/mL; range, 44-3552 pg/mL). There were no significant differences in pleural VEGF levels in patients with different histological types of lung cancer. The VEGF level was not correlated with ADA, lactate dehydrogenase and total protein levels of pleural fluid. In conclusion, pleural VEGF levels in patients with malignant effusions were significantly higher than tuberculous effusions, and the measurement of pleural VEGF is helpful in discriminating between malignant and tuberculous effusions. Further studies are needed to determine the clinical value of VEGF as a tumor marker and a prognostic factor.

Vascular endothelial growth factor in the sera and effusions of patients with malignant and nonmalignant disease

Clinical data clearly indicate a correlation between tumor neovascularization, aggressiveness of tumor growth, and metastatic spread. One of the key factors capable of stimulating tumor angiogenesis is vascular endothelial growth factor (VEGF). Using an immunoassay for VEGF, we assessed the levels of soluble VEGF in the sera and effusions of patients with malignant and nonmalignant disease as well as in the sera of healthy controls. Using a sandwich enzyme-linked immunoadsorbent assay, the concentration of VEGF was measured in serum specimens (n=445) and effusions (n=56) collected from a total of 212 patients with various types of cancer, 88 patients with nonmalignant disease, and 145 healthy individuals. Low and rather stable levels of VEGF were detected in the serum of healthy individuals (median, 294 pg/mL; range, 30-1752 pg/mL; 95th percentile, 883 pg/mL). Compared with healthy individuals, serum levels in patients with acute infections were elevated (P=0.03), whereas patients with chronic cirrhosis had lower serum VEGF levels. Among patients with various types of neoplasia, VEGF serum levels in patients with ovarian or gastrointestinal carcinoma were significantly higher than in healthy individuals. Moreover, VEGF concentrations in sera from patients with metastatic disease were higher than in sera from patients with localized tumors. Maximum serum concentrations of VEGF (median, 1022 pg/mL; range, 349-7821 pg/mL) were found in patients with metastatic ovarian carcinoma. Median VEGF levels (and ranges) in malignant effusions were up to 10-fold higher than in matched serum samples: 5528 pg/mL (468-49269 pg/mL) in ovarian carcinoma, 885 pg/mL (77-14,337 pg/mL) in breast carcinoma, and 813 pg/mL (372-18,331 pg/mL) in gastrointestinal carcinoma. In contrast, ascitic fluid from patients with cirrhosis contained only 303 pg/mL (median, range 116-676 pg/mL) of VEGF, corresponding to the low serum levels in this patient group. Depending on the tumor type, elevated levels of VEGF are detectable in the serum of only 0-20% of patients with localized cancer but in 11-65% of patients with metastatic cancer. Of cytology-proven malignant ascites or peritoneal exudates from various malignancies, 46-96% show VEGF levels above the upper limit (95th percentile, 676 pg/mL) of nonmalignant ascites. Maximum VEGF concentrations in malignant effusions indicate abundant local release of VEGF within the pleural or peritoneal cavity. These results suggest that VEGF might play an important role in tumor progression and the formation of malignant effusions. Further studies are warranted to determine the clinical value of soluble VEGF as a tumor marker, a prognostic factor, and a surrogate indicator of tumor angiogenesis.

Plasma glutathioneS-transferase P1-1 levels in patients with head and neck squamous cell carcinoma

Many tumors contain high amounts of the detoxification enzyme glutathione S-transferase P1-1 (GSTP1-1). Elevated levels of GSTP1-1 have also been detected in serum and plasma from patients with gastrointestinal, lung, or head and neck tumors. The authors of this report evaluated the role of GSTP1-1 as a plasma tumor marker in patients with head and neck squamous cell carcinoma (HNSCC) of the larynx, hypopharynx, or oropharnyx and in patients with benign head and neck lesions (BHNL). GSTP1-1 levels were measured in EDTA plasma combined with ethylenediaminetetraacetic acid using a recently developed sensitive and specific sandwich enzyme-linked immunoadsorbent assay. A normal reference level with an upper limit of 21.8 microg GSTP1-1 per liter of plasma was calculated from results obtained with samples from 230 blood donors. Median GSTP1-1 levels in samples from 53 patients with oral/oropharyngeal SCC (10.6 microg/L; range, 3.7-46.1 microg/L), 12 patients with hypopharyngeal SCC (11.9 microg/L; range, 5.2-146.6 microg/L), and 28 patients with laryngeal SCC (14.4 microg/L; range, 6.4-141.5 microg/L) were significantly elevated when compared with plasma GSTP1-1 levels in samples from 45 patients with BHNL (8.1 microg/L; range, 3.3-32.3 microg/L; P < 0.0001, P < 0.01, and P < 0.0001, respectively). However, only 6 of 53 patients (11%) with oral/oropharyngeal SCC, 1 of 12 patients (8%) with hypopharyngeal SCC, and 6 of 28 patients (21%) with laryngeal SCC had plasma GSTP1-1 levels above the upper limit of the normal reference level. Thus, only 13 of 93 patients (14%) with HNSCC had elevated plasma GSTP1-1 levels overall. No significant relation between plasma GSTP1-1 levels and TNM classification of the tumors was observed. GSTP1-1 is not a suitable plasma tumor marker for HNSCC.

Measurement of glutathione S-transferase P1-1 in plasma: pitfalls and significance of screening and follow-up of patients with gastrointestinal carcinoma.

Gastrointestinal tumors often contain high amounts of the detoxification enzyme glutathione S-transferase P1-1 (GSTP1-1). Elevated levels of GSTP1-1 were found in serum and plasma from most patients with gastrointestinal tumors. The authors evaluated the role of GSTP1-1 as a plasma tumor marker in patients with gastrointestinal tumors. A sensitive and specific sandwich enzyme-linked immunoadsorbent assay for quantification of GSTP1-1 in human plasma was developed. GSTP1-1 levels in serum samples from 10 healthy controls were significantly (P < 0.0001) higher than in corresponding ethylenediaminetetraacetic acid (EDTA) plasma and varied with the type of blood collection tube used. Refrigeration or delayed centrifugation of blood collected in plain EDTA tubes resulted in spuriously high plasma GSTP1-1 levels. Therefore, all plasma samples were collected in silicone-coated EDTA tubes. The distribution of plasma GSTP1-1 levels in 230 blood donors was nearly normalized by logarithmic transformation and an upper normal reference level of 21.8 microg/L was calculated. Males had significantly higher (P < 0.0001) plasma GSTP1-1 levels than females and a significant increase (P < 0.004) in plasma GSTP1-1 with age was noted. In only 20 of 55 patients (36%) with gastrointestinal tumors was the plasma GSTP1-1 level above the upper normal reference limit. No significant decrease in plasma GSTP1-1 was noted in matched pairs of plasma samples collected from 17 patients before and at least 2 weeks after resection of the tumor. The GSTP1-1 level in serum and plasma depends on the materials and methods used to collect the samples. Only 36% of the patients with gastrointestinal tumors had elevated plasma GSTP1-1 levels that did not decrease after resection of the tumor. These findings argue against the use of GSTP1-1 as a serum or plasma marker for gastrointestinal tumors.

A new approach to fecal occult blood testing based on the detection of haptoglobin

Colorectal carcinoma is a common disease, occurring in 1 in 20 adults in Western society, and there is a compelling need for an effective early diagnostic test. Several serum tests, including carcinoembryonic antigen have been used, but none are sufficiently sensitive for the early diagnosis of the disease. In a novel approach using fecal extracts from patients with colorectal cancer as the antigen for immunization, several MoAbs were produced. One (FE14.1) was found to react with the feces from patients with colon cancer, but not with those from normal subjects. A sandwich enzyme-linked immunoadsorbent assay was developed, and its ability to diagnose colorectal carcinoma evaluated. Of the patients with colorectal carcinoma, 91.5% (43/46) were positive compared with 1.9% of normal individuals (4/211). Analysis of the N-terminal amino acid sequence of a subunit of the molecule detected by FE14.1 shows it to be the beta chain of haptoglobin. The assay developed in this study has several advantages compared with current fecal occult blood tests, including no requirement for dietary restriction and the ability to distinguish between upper and lower gastrointestinal bleeding, while retaining the sensitivity and specificity of the current tests. Furthermore, the sensitivity of the tests increases to 100% if the FE14.1 and HemeSelect are combined. In addition, the study shows the potential to produce anticancer agents by immunizing with fecal material.

Lymphokine release during co-culture of human lympho-mononuclear cells and fresh or cultured human, porcine and bovine pancreatic islets.

In this study we evaluated whether isolated human (HI), porcine (PI) and bovine (BI) islets, either fresh (Fr) or cultured for 4 weeks (4 w) affect cytokine release from human lymphomononuclear cells (LMC) differently. We prepared LMC from peripheral blood by density gradient purification and co-cultured 1 x 10(6) LMC for 24 h with 100 hand-picked islets, either within 48 h of isolation or after culture for 4 weeks. Soluble interleukin-2 receptor (IL-2R), interferon-gamma (IFN), interleukin-4 (IL-4) and interleukin-10 (IL-10) were measured by sandwich enzyme-linked immunoadsorbent assay. Compared with controls (Ctrl, LMC without islets), Fr-HI, Fr-PI and Fr-BI caused a similar increase of IL-2R and IFN release, whereas 4 w-HI and 4 w-BI did not lead to any significant production of these two cytokines. IL-10 concentrations increased with Fr-PI and Fr-BI, but not with Fr-HI, and no major effect of the 4-week culture was seen. IL-4 levels were below the detection limit of the method used in these experiments. Thus, fresh allo- and xeno-islets caused a similar increase of the release of cytokines known to be markers of Th1 activation, whereas the release of IL-10, a marker of Th2 activation, increased with xeno-, but not with allo-islets; culturing the islets for 4 weeks decreased Th1, but not Th2 activation.

Comparison of sandwich enzyme-linked immunoadsorbent assay and radioimmunoassay for determination of exogenous glucagon-like peptide-1(7–36)amide in plasma

A sensitive sandwich enzyme-linked immunoadsorbent assay (ELISA) for determination of exogenous glucagon-like peptide-1(7–36)amide (GLP-1(7–36)amide) in plasma samples from pharmacokinetic studies is described. The assay employs an N-terminally directed antibody and a C-terminally directed antibody. The ELISA has a working range from 10 to 500 pmol 1 −1, and can be applied to plasma samples from humans, dogs, pigs, minipigs, cats, rabbits, and rats. The assay was compared to a validated radioimmunoassay (RIA), employing an antibody directed against the mid-region of GLP-1. After s.c. administration of GLP-1(7–36)amide, the plasma immunoreactivity of GLP-1 (P-GLP-1-IR) measured by ELISA was markedly lower than P-GLP-1-IR measured by RIA. After HPLC fractionation of plasma samples with subsequent RIA and ELISA analyses of the fractions, this difference was shown to be due to cross reaction with biologically inactive fragments of GLP-1(7–36)amide in the RIA but not in the ELISA.

Serum levels of soluble CD30 are elevated in the majority of untreated patients with Hodgkin's disease and correlate with clinical features and prognosis.

To evaluate the serum levels of the soluble form of the CD30 molecule (sCD30) in patients with Hodgkin's disease (HD) to establish whether there is a correlation with clinical features at presentation and prognosis. The sCD30 serum levels of 117 patients were measured at diagnosis with a commercial sandwich enzyme-linked immunoadsorbent assay (ELISA) test kit, and in 78 of these patients the sCD30 levels were also recorded during the follow-up period. sCD30 levels at diagnosis were increased (> 20 U/mL) in a high proportion of patients (87.2%; mean +/- SD, 108 +/- 134 v 5.3 +/- 5.7 U/mL in controls, P < .0001) and correlated with stage (stages I + II, 73 +/- 97 U/mL; III + IV, 162 +/- 165 U/mL; P < .0001), with presence of B symptoms (stage A, 69 +/- 82 U/mL; stage B, 162 +/- 171 U/mL; P < .0001), and, to some extent, with tumor burden (bulky presentation, 141 +/- 129 U/mL; nonbulky, 91 +/- 133 U/mL; P = .058). Patients with sCD30 levels greater than 100 U/mL at diagnosis had a significantly higher rate of poor outcome in terms of failure to achieve a complete remission (CR) or disease relapse after CR achievement. In fact, the event-free survival (EFS) duration of patients with sCD30 levels greater than 100 U/mL was significantly worse (P = .0016). Using multivariate analysis, an sCD30 level greater than 100 U/mL retained its significance after adjustment for other prognostic parameters. sCD30 in HD at presentation strictly correlates with clinical features. Serum levels greater than 100 U/mL at diagnosis entail a significantly higher risk of treatment failure, a factor that is independent of other prognostic parameters.

Determination of fibronectin and its degradation products in bronchoalveolar lavage fluid

In order to estimate, in sarcoidosis, the extent of degradation and the availability of functionally active fibronectin (FN) in the alveolar space, native and total (= native + degraded) FN were determined in the bronchoalveolar lavage (BAL) fluid of 28 non-smoking patients with sarcoidosis, 12 healthy non-smokers and 24 asymptomatic smokers. The FN was measured in unconcentrated BAL fluid in the presence of the protease inhibitor aprotinin: total FN was assayed by a double-antibody sandwich enzyme-linked immunoadsorbent assay (ELISA) and native FN by a gelatin-antibody sandwich ELISA. Both in controls and in sarcoid patients, concentrations of native FN were lower than those of total FN, indicating that FN is degraded in the alveolar space. Compared to non-smoking controls, the sarcoid patients had significantly increased concentrations of both native and total FN (p less than 0.001 for both) in the BAL fluid. Native FN in percentage of total FN was similar in non-smoking sarcoids and controls, representing about 80% and 70%, respectively. In contrast, native FN corresponded to only 30% in the group of control smokers, indicating higher degradation in smokers. Thus, in smokers, less intact collagen-binding FN is available for forming an extracellular matrix, which may be necessary for repair. On the other hand, the elevation of FN, especially in its native form, in sarcoidosis implies that more of it may be available for repair but, if in excess, also for the build-up of fibrotic tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

A sandwich enzyme-linked immunoadsorbent assay for detection of murine macrophage colony-stimulating factor (CSF-1)

A simple three-layer sandwich enzyme-linked immunoadsorbent assay (sandwich-ELISA) has been developed for murine macrophage colony-stimulating factor-1 (CSF-1) using the two monoclonal antibodies on which we recently reported (J. Immunol. (1988) 141, 483). The anti-CSF-1 monoclonal antibodies used in this assay recognize different epitopes of the same antigen, thereby permitting the detection of low amounts of CSF-1. This assay is specific to murine CSF-1. Recombinant human macrophage colony-stimulating factor, murine GM-CSF, or IL-3, either alone or together with CSF-1, does not interfere with the assay. The advantage of this assay over other reported immunoassays for CSF-1 is that radiolabeled or large quantities of purified CSF-1 are not required. This sandwich-ELISA compares favorbly with other assays in its rapidity, simplicity, and sensitivity.

Soluble circulating Fcγ receptors in human serum: A new ELISA assay for specific and quantitative detection

We have developed a simple, rapid, and sensitive enzyme-linked immunoadsorbent assay (ELISA) to measure soluble cell-free human Fcγ receptor (FcγR) in serum. This assay is based on the use of two monoclonal antibodies directed against different epitopes expressed on the same low avidity human FcγR (CD16), which is present on polymorphonuclear leukocytes, macrophages and NK cells. This sandwich ELISA, which can measure 2 nM concentration of FcγR, has enabled us to demonstrate the presence and to measure the level of soluble cell-free human FcγR (CfH-FcγR) in normal human serum.