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PubMed2002

Quantification of T-cell receptor excision circle DNA using fluorescence resonance energy transfer and the LightCycler system.

T-cell receptor excision circles (TRECs) are circular, stable extrachromosomal DNA fragments and are generated during V(D)J gene recombination, a process responsible for diversity of the T-cell receptor repertoire. Here we describe a sensitive, rapid and easy to perform real-time PCR assay based on the LightCycler technique for the quantification of TRECs among peripheral blood cells and a comparison of this assay with conventional PCR-ELISA. By LightCycler, a sensitivity of 20 copies of plasmid deltaRec-psiJalpha Signal Joint TREC DNA was achieved whereas by PCR-ELISA, a detection limit of 2 copies was demonstrated. In blood samples from healthy individuals (n=52) a median TREC count of 1.6x10(4) copies [range 2x10(1) to 2x10(5)]/2x10(5) peripheral blood mononuclear cells (PBMNCs) and in cord blood, a median TREC count of 1.45x10(5) copies [range 1.2x10(5) to 1.6x10(5)]/2x10(5) PBMNCs) could be detected. No significant difference was found in 15 individuals when unfractionated PBMNCs (median count of 1.1x10(5) copies/2x10(5) PBMNCs) or magnetic associated cell sorter (MACS)-sorted CD45RA+ T-cells (median count of 2.5x10(5) copies/2x10(5) cells) were analyzed for TREC counts. In addition, we examined the number of deltaRec-psiJalpha Signal Joint TREC in paediatric (n=6) and adult patients (n=7) after allogeneic stem cell transplantation. In children, we observed a median TREC count of 5.7x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 5.6x10(4) copies/2x10(5) PBMNCs after 2 years, and in adults, a median count of 3.6x10(4) copies/2x10(5) PBMNCs after 1x10(4) and 1.1x10(4) copies/2x10(5) PBMNCs after 2 years. In conclusion, the LightCycler-based real-time PCR assay described offers a very sensitive and rapid tool for the quantification of TREC DNA.

Adolescent↗
PubMed1997

Quantification of apolipoprotein D by an immunoassay with time-resolved fluorescence spectroscopy.

Apolipoprotein D (apoD), also known as gross cystic disease fluid protein-24 (GCDFP-24), is a minor protein moiety of high-density lipoproteins in human plasma. ApoD is expressed in a subset of breast carcinomas and has been proposed as a tumor marker and prognostic indicator for breast cancer progression. Here we describe a new sensitive time-resolved fluorimetric immunoassay for quantification of human apoD in biological specimens using affinity-purified polyclonal anti-human apoD rabbit antibodies and Eu3+ as a specific probe. Both purified apoD and normal human pool-serum served as reliable primary and secondary standards in the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). Plasma apoD concentrations measured by the DELFIA were 99.6 +/- 32 microg/ml. The detection limit of the DELFIA procedure was 0.5 ng/ml after sample dilution of 1/8000. The intra-assay coefficient of variation averaged 3.5%, whereas the inter-assay coefficient of variation averaged 6.9%. The concentration of apoD in breast cyst fluids ranged from 6.82 to 28.37 mg/ml. Based on the low detection limit and the high specificity of the DELFIA procedure, we have applied this technique for the measurement of apoD in breast cancer cell supernatants. In estrogen-receptor positive cells, i.e., T-47D and ZR-75-1 cells, 42.6 +/- 1.4 and 2.7 +/- 0.2 ng apoD/ml supernatant after 4 days in culture without induction of apoD synthesis were measured. A comparison of the direct sandwich DELFIA procedure with an electroimmunoassay commonly used to assay apoD revealed correlation coefficients of 0.986 (serum) and 0.975 (cyst fluids). The present findings indicate that the direct sandwich DELFIA is appropriate for apoD quantification in plasma and breast cyst fluids. Furthermore, the technique should permit studies on the induction of apoD synthesis in the low picomolar range in different carcinoma cells to gain insight into the expression of this atypical apolipoprotein.

Apolipoproteins↗
PubMed1999

Comparison of different immunochemical methods for the detection and quantification of hazelnut proteins in food products.

Hazelnuts are widely used in the food industry owing to their nutritive value and taste. The amount of hazelnut present in a recipe is usually considered as a mark of quality. On the other hand, contamination of foods that normally do not contain hazelnuts is a threat for patients with a hazelnut allergy. For this reason, the availability of a method for the detection and quantification of hazelnuts in foods would be desirable. The objective of this study was to develop a method for the detection and quantification of minor amounts of hazelnut protein in food products that is potentially applicable for the food industry. Several immunochemical methods, e.g., immunoblotting and enzyme-linked immunosorbent assay (ELISA), were developed with antibodies from both hazelnut-sensitized patient sera and the sera of rabbits hyperimmunized with hazelnut protein. Immunoblotting appeared to be non-specific when the sera of patients were used as a source of antibodies. Using immunopurified antibodies from rabbits immunized with hazelnuts, immunoblotting became specific, but the sensitivity of this method was limited. Inhibition of IgE binding is a generally used test in clinical laboratories to establish contamination with hazelnuts. This approach is sensitive and specific, but not readily accessible for the food industry since patient serum is needed. Similar results in terms of sensitivity and specificity were obtained with a sandwich ELISA constructed with an immunopurified antibody from rabbits sensitized to hazelnuts. No substantial cross-reactivity with other nuts, legumes or other food constituents was observed, and concentrations as low as 5 ng/ml, corresponding to 1 ppm in food products, were detected. In a field test, several consumer products regarded to be free of hazelnuts were shown to contain traces of hazelnut. This sandwich ELISA constructed with immunopurified antibodies from rabbits sensitized with hazelnut protein is a sensitive and specific method to detect and quantify hazelnut and is useful in detecting trace contamination with hazelnut in various consumer products. Since this test does not require serum from patients, it is appropriate for use in the food industry.

Allergens↗
PubMed1985

Experimental catecholamine-induced myocardial necrosis. I. Morphology, quantification and regional distribution of acute contraction band lesions.

Acute histologic and ultrastructural changes, quantification and regional distribution (transmural, circumferential, transaxial) of damaged myocells from anesthetized, open-chested dogs following one hour of intravenous infusions of saline or increasing doses of isoproterenol (0.1, 1.0, 2.5 micrograms/kg/min) or norepinephrine (4.0 micrograms/kg/min) were investigated. Two predominant subsets of acute contraction band lesions were produced: 'paradiscal' involving aggregation of less than 15 sarcomeres adjacent to the intercalated disc and 'holocytic' involving coagulation of groups of adjoining sarcomeres into transverse bands interspersed with areas of myofibrillar rhexis throughout the cell. Both lesions were distributed as isolated cells or as small foci of myocells surrounded by normal myocardium. Quantification of 'paradiscal' and 'holocytic' contraction band lesions/mm2 of area was used as an index of the severity of catecholamine-induced necrosis. Numbers of 'paradiscal' myocells increased with increasing doses of isoproterenol, while 'holocytic' myocells were not present in any significant numbers until 1.0 microgram/kg/min) and increased further at 2.5 micrograms/kg/min. 'Paradiscal' myocells with both isoproterenol (2.5 micrograms/kg/min) and norepinephrine were distributed with the greatest number in the inner third of the free wall. This gradient was not significant for 'holocytic' lesions. There was, generally, no significant difference in distribution of either type of lesion around the circumference. However, transaxially there was a higher frequency of 'paradiscal' lesions at the apex with norepinephrine. The lesions were identical and present in comparable numbers at both the highest dose of isoproterenol and with norepinephrine. Thus, these two catecholamines result in a similar cardiotoxicity, each with two predominant subsets of lesions, despite their hemodynamic diversities.

Animals↗
PubMed2006

Pelvic organ support and prevalence by Pelvic Organ Prolapse-Quantification (POP-Q) in Korean women.

PURPOSE: We evaluated pelvic organ support and the prevalence of pelvic organ prolapse in Korean women using the Pelvic Organ Prolapse-Quantification system as the assessment tool. MATERIALS AND METHODS: The study population consisted of 713 women 18 to 72 years old who were seen for annual Papanicolaou testing and pelvic examinations. Pregnant patients and patients who had delivered within the previous 6 weeks were not recruited. All pelvic examinations were performed by a single examiner. The patient was examined in the dorsal lithotomy position in a pelvic examination chair positioned at a 15-degree angle. All 9 measurements except total vaginal length were taken with the patient performing the maximal Valsalva maneuver. RESULTS: Mean patient age was 41.6 years (range 18 to 72), mean weight +/- SD was 55.8 +/- 7.4 kg (range 40 to 83), mean height was 158.7 +/- 5.4 cm (range 138 to 177), mean body mass index was 22.3 +/- 8.1 kg/m2 (range 15.7 to 32) and median parity was 2 (range 0 to 6). Mean scores for the position of the cervix and posterior fornix, and total vaginal length were -5.0, -6.6 and 7.0 cm, respectively. In the 713 women with a uterus the incidence of anterior vaginal, uterine and posterior vaginal prolapse was 27.6%, 2.0% and 25.4%, respectively. The overall distribution of pelvic organ prolapse quantification system stage was stages 0 to 4 in 68.3%, 19.9%, 11.2%, 0.6% and 0.0% of patients, respectively. CONCLUSIONS: Vaginal size in Korean women differs from that in Western women. The prevalence of any degree of prolapse was approximately 31.7%. Korean women were at relatively higher risk for anterior and posterior vaginal prolapse than for uterine prolapse.

Adolescent↗
PubMed1985

Quantification of deposition of neutrophilic granulocytes on vascular grafts in dogs with 111In-labeled granulocytes.

A new radioisotopic technique has been developed for quantification of deposition of neutrophilic granulocytes on vascular grafts. Nine healthy mongrel dogs underwent bilateral femoral artery resection and reconstruction with grafts of femoral vein and Gore-Tex. Pure granulocytes that had been separated from whole blood by centrifugal elutriation were labeled with 111In-tropolone in plasma. The granulocyte harvesting efficiency was 25 +/- 12%, and the labeling efficiency was 87 +/- 7%. Three hours after injection of labeled granulocytes and 2 hours after reperfusion, the grafts were harvested and cut into several segments for study of areas of anastomoses and midsections. On the basis of the radioactivity in the blood and in anastomotic and graft sections, the area of graft sections, and the neutrophilic granulocyte and differential leukocyte counts, the number of neutrophilic granulocytes adherent to a unit area and the total number of neutrophilic granulocytes on graft sections were calculated. These quantifications of the deposition of neutrophilic granulocytes indicated that the midsections of Gore-Tex grafts retained more neutrophilic granulocytes than did the midsections of vein grafts. Although the anastomotic areas retained more neutrophilic granulocytes than did the midsections of vein grafts, the opposite finding prevailed for the Gore-Tex grafts. A major fraction of neutrophilic granulocytes on Gore-Tex grafts was incorporated into thrombus. Semiquantitative information obtained by scintigraphy of the deposition of neutrophilic granulocytes on vascular grafts also confirmed this observation.

Animals↗
PubMed1997

The use of benzo[a]pyrene diolepoxide-modified DNA standards for adduct quantification in 32P-postlabelling to assess exposure to polycyclic aromatic hydrocarbons: application in a biomonitoring study.

The 32P-postlabelling assay is one of the most sensitive methods for detection of DNA adducts induced by exposure to genotoxic chemicals. Under optimal conditions, detection limits of one adduct per 10(9)-10(10) nucleotides have been reported. This sensitivity now allows monitoring of occupational and even environmental exposure of humans to certain classes of chemicals, mainly polycyclic aromatic hydrocarbons (PAH). Despite its widespread use, 3P-postlabelling is still not a standardized method. Rigorous interlaboratory comparisons are scarce, and those that have been undertaken often show rather different results, both in relative and in absolute values, for the amounts of DNA adducts in the same samples. Furthermore, the optimization of many steps in the procedure has still not been given adequate attention. This paper deals with some technical aspects of detection of PAH-DNA adducts by 32P-postlabelling, in particular with assay calibration and adduct quantification. For this purpose, benzo[a]pyrene (BP)-modified DNA standards were prepared, the adduct contents of which were determined by use of an independent fluorometric method, viz. synchronous fluorescence spectrophotometry (SFS). These BP-DNA standards are processed along with the test samples throughout the entire 32P-postlabelling procedure, from the enzymic digestion up to and including the determination of radioactivity in adduct spots on the chromatogram. As such, these reference samples can be considered as external standards for inter-assay calibration. This method for adduct quantification was compared with the commonly used relative adduct labelling (RAL) and comparative dAMP labelling, which appeared to give rise to an underestimation of adduct levels. The method was applied in a biomonitoring study among workers in a carbon-electrode manufacturing plant, exposed to PAH. Although DNA adduct levels in peripheral blood lymphocytes of exposed workers, as determined by 32P-postlabelling, were not significantly different from those of controls, a significant difference was seen when smokers and non-smokers were compared.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
PubMed2001

Repeatability and validity of a fluorimetric HPLC method in the quantification of yessotoxin in blue mussels (Mytilus edulis) related to the mouse bioassay.

Repeatability and validity of a fluorimetric HPLC method in quantification of yessotoxin (YTX) in mussels related to the mouse bioassay was studied. Blue mussels (Mytilus edulis) from the Sognefjord, Norway were sampled from March to November, 1997, and October to December, 1998. A total of 75 samples were analysed for YTX by HPLC using 4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3, 4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3, 5-dione (DMEQ-TAD) as a fluorimetric derivatization agent. Among these, 28 of the samples were analysed by HPLC in duplicate. All samples were analysed by the mouse bioassay using both chloroform and ether in the final step of extraction. The duplicate measurements using HPLC was found equal and the method repeatable (p<0.05). The absolute difference between the two measurements was found to increase with increasing level of measurements. This significant positive correlation (p<0.05) was mainly due to concentrations of YTX higher than 200 microg/100g mussel meat. However the precision of the results obtained was not found to be less in the upper level than in the lower level. Based on the internal correlation analysis including the mouse bioassay and the HPLC method a cut-off value of < or =10 microg YTX/5 g digestive gland was found preferable. The mouse bioassay of ether extracts often failed to detect high levels of YTX, and as demonstrated by the low kappa-values, the agreement between the mouse bioassay of ether extracts and the HPLC method was very weak. The HPLC method was found to give repeatable results and thereby found to be reliable. Consequently, the HPLC method seems to the method of choice for detection and quantification of YTX in mussels when compared with the mouse bioassay.

Animals↗
PubMed1985

Non-Hodgkin's lymphoma classification: ultrastructural morphometric studies for the quantification of nuclear compartments in situ.

The condensed chromatin distribution in the nuclei of lymphocytes in non-Hodgkin's lymphoma (NHL) is a key element, along with nuclear size and shape, in the classification of this disease for therapeutic and prognostic purposes. This report describes the ultrastructural comparative quantification of the condensed chromatin and the interchromatinic (nuclear matrix or euchromatin) region in the nuclei of mitogen-stimulated human peripheral T lymphocytes and mouse spleen B lymphocytes, human germinal center lymphocytes, and lymphocytes in ten cases of NHL of a variety of subtypes. The sequential morphologic nuclear changes induced in lymphocytes by mitogens are reflected in human germinal center lymphocyte populations. The common features include the changes in the distribution and volume of condensed chromatin aggregates, as well as the fact that the major increments in nuclear volume during lymphocyte transformation result from increases in the volume of the interchromatinic region. In all subtypes of NHL analyzed morphometrically, subpopulations of lymphocytes were identified in which mean nuclear, condensed chromatin, and interchromatinic volumes were more or less equivalent to those of normal lymphocyte subsets in germinal centers in reactive hyperplasia. However, in NHL the abnormal cytologic characteristics of the nucleus result, at least in part, from a complex interplay of condensed chromatin distribution and amount, and the size of the interchromatinic region. Further complexity is introduced by the fact that in NHL these two nuclear compartments can independently be normal, increased, or reduced in size. Morphometric quantification of lymphocytes in NHL indicates that the interchromatinic (matrix) region of the nucleus is the key element in establishing the nuclear volume of neoplastic lymphocytes. The structural and functional, ribonucleoprotein interchromatinic region of the nucleus was visualized in normal and neoplastic lymphocytes by regressive uranyl-EDTA staining. Quantitative morphometric analysis indicates that the cytologic appearance of neoplastic lymphocytes, even within subtypes of NHL, is heterogeneous and that condensed chromatin quantity and distribution may be more critical than nuclear size in distinguishing between certain subtypes of NHL. Improvements in the classification of NHL will occur only with understanding of the alterations in the biologic mechanisms controlling gross nuclear organization and the morphologic events of the various differentiation pathways available to antigen-stimulated lymphocytes.

Animals↗
PubMed1999

CA-1 method, a novel assay for quantification of normal prothrombin using a Ca2+ -dependent prothrombin activator, carinactivase-1.

We established a novel prothrombin assay, designated CA-1 method, for quantification of normal prothrombin in application of a Ca2+ -dependent prothrombin activator, carinactivase-1 (CA-1), found in the venom of Echis carinatus leucogaster. On microplate, thrombin converted from normal prothrombin in plasma sample by CA-1 cleaves a thrombin specific chromogenic substrate, t-butoxy-Val-Pro-Arg-p-nitroanilide and liberates p-nitroaniline. Then, the normal prothrombin level is decided by measuring the velocity of p-nitroaniline liberation. Normal prothrombin levels in plasma from warfarin-treated individuals were highly correlated with coagulant activities assayed by both prothrombin time and thrombotest. CA-1 method is not only a rapid and highly sensitive chromogenic microplate assay for quantification of normal prothrombin in the range of 10-200 ng/100 microl in plasma samples but also suitable for analyses of many samples in a short time. In addition, normal prothrombin levels obtained by CA-1 method are not inhibited by EDTA and heparin, which reduce prothrombin time and thrombotest activities. CA-1 method is a novel assay for monitoring coagulant activity in warfarin-treated individuals.

Aniline Compounds↗
PubMed1993

A comparative study of methods for demonstration and quantification of capillaries in skeletal muscle.

Histological, histochemical, immunohistochemical, and microangiographic methods were compared for the demonstration of capillaries in skeletal muscle. Microangiography visualized the arterioles and venules, but was unsuitable for the quantification of capillaries. The histological and enzyme histochemical methods gave variable staining reactions for capillaries. The most reliable and reproducible enzyme histochemical reactions were those for alkaline phosphatase (Gomori) and ATPase (after preincubation at pH = 4.2 or 4.6 or 7.2) as well as PAS staining after amylase preincubation. Among the immunohistochemical reactions, type V collagen and fibronectin were suitable for the demonstration and quantification of capillaries in skeletal muscle.

Adenosine Triphosphatases↗
PubMed1983

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines.

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.

Animals↗
PubMed1998

Quantification of somatic and spermatogenic cell proliferation in the testes of ruminants, using a proliferation marker and flow cytometry analysis.

We compared 2 methods for the quantification of proliferation in somatic and spermatogenic compartments of post mortem-collected testes in cattle and roe deer. Proliferation was evaluated by estimation of the tissue polypeptid specific antigen (TPS) using an ELISA. This proliferation-specific marker was detected in homogenized cells after selective enrichment of different cell types by density gradient centrifugation. The haploid, diploid and tetraploid cells were monitored by one-parameter flow cytometry and analyzed for mitotic cell cycle. Somatic and spermatogenic cells were discriminated by dual-parameter flow cytometry after DNA staining with propidium iodide and selective labelling of stromatic cells with a vimentin antibody. The TPS was related to the ploidy of cells and their somatic or spermatogenic type. High concentrations of TPS were found in both species. The TPS values varied with different contents of spermatogenic and somatic cells in the fractions of the density gradient. The TPS was positively correlated with spermatogenic cells in the G2/M phase of mitotic cycle (r = 0.474; P < 0.01) and negatively correlated with somatic cells (r = -0.676; P < 0.0001) in roe deer (n = 40). Discrimination of germinative and stromatic cells in the G2-M phase showed their varying proliferation during the annual cycle in roe deer. The quantification of tetraploid spermatogenic cells allowed the calculation of an exact meiotic transformation (ratio haploid:tetraploid cells). In conclusion, TPS indicates proliferation in the germinative compartment of the testes. However, this marker provides only relative values, without information on the number and type of proliferating cells. Dual-parameter flow cytometry using specific staining for vimentin proves to be a better method for studying changing mitotic and meiotic steps during the involution and recrudescence of testes in seasonally breeding ruminants, as it relates proliferative processes directly to both spermatogenic and somatic cells.

Animals↗
PubMed2002

Software-supported image quantification of angiogenesis in an in vitro culture system: application to studies of biocompatibility.

Healing of soft tissue trauma and bone discontinuities following implantation involves acute inflammatory reactions and the formation of blood vessels (angiogenesis). During angiogenesis new capillary vessels arise from the existing vasculature. Endothelial cells (EC) are the major cell type involved in angiogenesis. Corrosion of orthopaedic metallic implant materials (e.g. CoCr alloys) can cause locally high concentrations of heavy metal ions in the peri-implant tissues. Some divalent metal ions (Co2+, Ni2+, Zn2+) lead to the activation of EC in vitro. Upon exposure to these ions. EC release cytokines and chemokines and increase the expression of cell surface adhesion molecules, which represents the pro-inflammatory phenotype. In this study we have examined whether metal ions influence the other endothelial aspect of wound healing, the angiogenic response. Therefore, we utilized an in vitro model of angiogenesis and examined the effects of divalent cobalt ions on the in vitro vessel formation. The quantification of the cobalt/ion-exerted effects on angiogenesis in vitro was performed using a contrast-rich vital staining and analysed by software-supported image quantification.

Alloys↗
PubMed2003

Quantification of ligand surface concentration of bulk-modified biomimetic hydrogels.

This study describes a method for the quantification of active ligand surface concentration for bulk-modified hydrogels. Two poly(propylene fumarate-co-ethylene glycol) (P(PF-co-EG)) block copolymers were synthesized with terminal poly(ethylene glycol) (PEG) chains of number average molecular weight 1960 and 5190 g/mol. Hydrogels were synthesized with bulk-modified biotin as a model ligand, making use of a PEG spacer arm with a molecular weight of 3400 g/mol. Bulk concentration of biotin was calculated from the initial concentration of biotin, sol fraction, equilibrium water content, and relative incorporation of the polymers to the hydrogel. Surface concentration of biotin bulk-modified hydrogels was quantified with an enzyme linked immunosorbent assay using mouse monoclonal anti-biotin antibody (IgG), horseradish peroxidase-conjugated anti-mouse IgG, and a chemiluminescent substrate. The larger size of the IgG relative to the mesh size of the hydrogels allowed for the quantification of the active biotin at the surface of the hydrogels. Luminescent imaging was used to qualitatively show the isolation of the horseradish peroxidase-conjugated antibodies to the surface of the bulk-modified hydrogel. The active biotin ligands at the surface of hydrogels synthesized with terminal PEG chains of 1960 g/mol were at the top 7.2 nm while for those synthesized with terminal PEG chains of 5190 g/mol were at the top 4.4 nm of the bulk-modified hydrogel. The relationship between bulk ligand concentration and the active ligand concentration at the surface was dependent on the hydrogel composition. The relative magnitude of the PEG spacer arm of the ligand compared to the PEG block length of the copolymer affected the surface availability of the ligand. The results suggest that steric hindrances caused by mobile PEG chains of the copolymer of molecular weight greater than that of the PEG spacer arm contributed to the decreased surface concentration of ligand. This work relates the bulk concentration of a ligand to its surface concentration, an important parameter for the adhesion, migration, and function of anchorage dependent cells.

Biomimetic Materials↗
PubMed1997

Quantification of P-glycoprotein in chronic lymphocytic leukemia by flow cytometry.

The P-glycoprotein (P-gp) was investigated in 40 patients with chronic lymphocytic leukemia by immunological, functional and quantitative assays. The proportion of positive cases with the anti-Pgp McAb UIC2 was 30% and increased to 64% after neuraminidase treatment (p = 0.002). Fifty-six per cent of cases were positive with the functional test with rhodamine 123 and verapamil. A negative correlation was found between the number of cells stained with the McAb and the functional test (p = 0.006). The mean of P-gp molecules was 2509 +/- 2805 molecules per cell; these values were higher than in the control K562 cell line in the majority of cases. The number of positive cases and P-gp molecules were higher in treated than in untreated patients (p = 0.01 and 0.07). There were no significant differences with respect to response to treatment, but a higher number of P-gp molecules was found in non-responders. When the results of the functional test were put together with the quantification assay this allowed the detection of 71% non-responders. Our findings suggest that quantification of the P-gp could be of value in the assessment of possible drug resistance in CLL.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Direct on air sampling filter quantification of cat allergen.

A direct on sampling filter in solution (DOSIS) method for quantification of airborne cat allergens has been developed. In this method, the allergens firmly adsorbed to a porous polytetrafluoroethylene filter are reacted with specific antibodies conjugated to alkaline phosphatase, generating a matrix-bound allergen-antibody-phosphatase complex. The treated filter is subsequently floated on a commercially available chemiluminescent phosphatase substrate solution. Aliquots of this solution are removed and analyzed luminometrically. The light intensity of the product is linearly related to the amount of allergen over a large mass range, 0-100 SQ units (1 SQ unit is about 146 pg of the allergenic protein Fel d 1). DOSIS demonstrated intra- and interassay precisions of 9% and 8% and 14% and 21% for the levels 4 and 20 SQ units per filter, respectively. The limit of quantification was estimated to 0.4 SQ units (58 pg Fel d 1) of cat allergen per filter. Application of DOSIS to analysis of cat allergen concentrations of indoor air in homes with and without cats revealed, on average, a six times higher concentration in the former (142 SQ units/m(3)) as compared to the latter (24 SQ units/m(3)). The recorded concentrations for airborne cat allergen in homes with cats are in accordance with previously reported figures. Allergen-specifically stained sampling filters revealed the particulate nature of airborne cat allergen which seemed predominantly to be carried by numerous large dust particles.

Air↗
PubMed2000

Miniaturized direct on air sampling filter quantification of pollen allergens.

A recently in this journal reported luminescence immunoassay for the direct quantification of birch and grass pollen allergens on air sampling filters. DOSIS, has been miniaturized. By means of a commercially available chlorinated analogue of the previously used 1,2 dioxetane phosphate derivative as enzyme substrate, the air sampling filter diameter could be reduced from 25 mm to 13 mm. The procedure leads to a more than twenty times reduction of the previously reported limit of quantification for the grass pollen allergen.

Air↗