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Biomedical subjects

H Wolf

Publications and source records attributed to H Wolf.

At least 19 recordsLinked to original sources

Human urinary bladder carcinoma glycoconjugates expressing T-(Gal beta(1-3)GalNAc alpha 1-O-R) and T-like antigens: a comparative study using peanut agglutinin and poly- and monoclonal antibodies.

T- and T-like antigens on glycoproteins and glycolipids were examined in extracts of human urinary bladder tumors and normal tissue by Western blot analysis and reagent binding to thin layer chromatograms. Three different anti-T-reagents were used: peanut (Arachis hypogaea) lectin (PNA) and mono- and polyclonal antibodies specific for T-antigen (Gal beta(1-3)GalNAc alpha 1-O-R). Immunodetection with the T-specific reagents in nitrocellulose replicas of bladder tumor glycoproteins, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, demonstrated tumor-specific T-antigen-bearing glycoproteins compared to normal urothelial glycoproteins. In addition, a remarkable difference in binding was found between the immunological reagents and PNA lectin. PNA showed major reactivity to a 28-kD glycoprotein extracted from tumors. Monoclonal anti-T-antibody (clone HH8) showed major reactivity with an M(r) 34,000 glycoprotein, and polyclonal anti-T-antibody showed major reactivity with an M(r) 36,000 glycoprotein. PNA agarose column affinity-purified tumor glycoproteins did not bind the antibodies. Glycoproteins, M(r) 28,000 and 34,000, were shown to be O-linked by stepwise deglycosylation. In solid phase monosaccharide inhibition tests, galactose followed by N-acetyl-galactosamine were the most potent monosaccharides inhibiting binding to immobilized bladder tumor glycoproteins. None of the anti-T-reagents reacted with glycolipids extracted from tumor tissue. It is concluded that PNA lectin, in addition to the T-disaccharide, reacts with other protein-anchored carbohydrate structures in carcinomas.

Antibodies, Monoclonal

Determination of placental ferritin (PLF)-positive lymphocytes in women in early stages of breast cancer.

Previous studies have proved that a certain acidic isoform of ferritin is specifically synthesized by the placenta and breast-cancer tissue. In this context it has been further reported that the determination of this so-called placental isoferritin (PLF) on the surface of a subset of peripheral lymphocytes is highly specific and sensitive for early stage breast cancer. By use of the monoclonal antibody CM-H-9 and flow cytometry, the levels of placental ferritin (PLF)-positive cells were determined in 133 female patients undergoing surgical excision of a controversial or highly suspicious lesion of the breast detected by mammography. In addition, 61 healthy blood donors served as controls. Values of PLF-positive cells in breast cancer patients differed significantly from those found in women with benign diseases and healthy controls (3.87% vs. 1.55% and 2.02, respectively; p less than 0.00001). Analysis of prognostic factors in breast cancer patients (tumor size, lymph-node status, menopausal status, estrogen receptor status, histologic grading and grading subfactors) revealed significantly higher levels of PLF-positive cells in lymph-node-negative patients compared with node-positive patients (p less than 0.00001). Furthermore, levels of PLF-positive cells showed a significantly negative correlation with tumor size and nuclear polymorphism. In 15 patients who underwent a guide-wire-directed surgical biopsy for a non-palpable, mammographically suspect lesion, 4 cases of cancer correlated with high values of PLF-positive lymphocytes while only 1 patient with a benign histologic result exhibited more than 4% positive cells.

Adult

Increased ELISA sensitivity using a modified method for conjugating horseradish peroxidase to monoclonal antibodies.

We investigated the importance of monoclonal antibody (MCA) purity and the input molar ratio of horseradish peroxidase (HRPO)/IgG used for MCA conjugation on various immunoenzymometric assay (IEMA) parameters. The sensitivity of IEMAs for human follicle stimulating hormone (hFSH), human chorionic gonadotropin (hCG) and the free alpha subunit of hCG (hCG alpha) could be increased up to 6-fold, whereas non-specific binding remained within tolerable limits (E less than 0.1), when MCAs purified by high performance liquid chromatography (HPLC) using a hydroxylapatite column (HPHT) were conjugated with an input molar HRPO/IgG ratio of four instead of the usual ratio of two.

Animals

Lipid utilization by human lymphocytes is correlated with high-density-lipoprotein binding site activity.

The nature and physiological importance of high-density lipoprotein (HDL) binding sites on unstimulated (resting) and mitogen-activated (blast) human peripheral blood lymphocytes were investigated. Specific HDL binding on resting and blast T-lymphocytes was saturable at 50 micrograms of 125I-HDL/ml and of high affinity, with Kd values of 8.1 x 10(-8) M and 6.5 x 10(-8) M, respectively, and Bmax. values of 79 ng and 180 ng/mg of cell protein respectively at 4 degrees C. Binding of HDL double-labelled with fluorescent dioctadecylindocarbocyanine (Dil) and isotope (125I) as well as of single fluorescence- or isotope-labelled HDL was inhibited competitively by HDL apoproteins. Studies of the cholesterol flux between the cells and HDL showed that HDL, low-density lipoprotein (LDL) or BSA at a concentration of 100 micrograms/ml in the tissue culture medium did not result in a significant difference in exogenous [3H]cholesterol efflux from the cell membrane at 37 degrees C. Proliferating T-blasts incorporated more cholesterol from HDL or LDL than did resting lymphocytes. When the cells were pulsed with 125I-HDL and chased in fresh lipid-free medium, up to 80% of the radioactivity released was not precipitable with trichloroacetic acid. This percentage decreased in a competitive manner when unlabelled HDL was present in the chase incubation medium. Finally, cultivation of lymphocytes with conditioned medium from macrophages increased Dil-HDL binding/uptake, while it was decreased by mevinolin-induced inhibition of hydroxymethylglutaryl-coA reductase. In conclusion, human lymphocytes possess a HDL binding site (receptor) responsible for lipid binding/uptake and concomitant internalization and degradation of apoproteins from HDL, but not for reverse cell membrane cholesterol transport. The activity of the binding site is up-regulated during cell proliferation and down-regulated during cell growth suppression.

Adult

Low molecular weight heparin and prevention of postoperative thrombosis in abdominal surgery.

In a prospective, double-blind, randomized multicenter trial the efficacy and safety of low molecular weight heparin and unfractionated heparin were compared for the prevention of postoperative deep vein thrombosis in patients undergoing abdominal surgery. Six hundred and seventy-three patients were randomly allocated to the two prophylaxis groups; 20 of these, however, did not undergo surgery and did not receive any prophylaxis. Of the remaining 653 patients 323 received one subcutaneous injection of 3,000 anti-Xa units of low molecular weight heparin and 330 received subcutaneously 5,000 U heparin three times a day. Treatment was initiated 2 h preoperatively and continued for 7 to 10 days. The occurrence of DVT was determined by the 125I-labelled fibrinogen uptake test and phlebography. Venous thrombosis was diagnosed in 24 of 323 patients (7.4%) treated with low molecular weight heparin and in 26 of 330 patients (7.9%) treated with low-dose heparin. DVT of proximal veins was detected in four patients of the low molecular weight heparin group and in three patients of the low-dose heparin group. During the observation period three pulmonary emboli - one fatal and two non-fatal - occurred in patients receiving prophylaxis with low-dose heparin. No pulmonary embolism was found in patients treated with low molecular weight heparin. Both prophylactic schemes were well tolerated. Intra- and postoperative blood loss, incidence of wound hematoma, frequency and volume of intra- and postoperative blood transfusion were similar in both groups with a slight advantage for the low molecular weight heparin group.(ABSTRACT TRUNCATED AT 250 WORDS)

Abdomen

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.

Animals

Nuclear volume and expression of T-antigen, sialosyl-Tn-antigen, and Tn-antigen in carcinoma of the human bladder. Relation to tumor recurrence and progression.

The T-antigen system and the mean nuclear volume have been proposed as risk variables in bladder tumors. This study includes 34 patients with initially noninvasive (Ta) transitional cell carcinomas who experienced different courses of disease. Tissue specimens of primary tumors were analyzed for the expression of T-antigen, Tn-antigen, and sialosyl-Tn-antigen using monoclonal antibodies (MoAb) and the lectin peanut agglutinin (PNA) in an indirect immunoperoxidase method. In addition, the mean nuclear volume was estimated by morphometry. Tissue from 7 of 13 patients (54%) who had invasive disease during a follow-up period of 5 years expressed T-antigen, as defined by MoAb HH8 in the primary tumor, whereas tissue of only 3 of 21 patients who did not have invasive disease expressed the antigen (P less than 0.02). No association was found between tumor progression to invasion and the expression of Tn-antigen or sialosyl-Tn-antigen. Tn-antigen expression was partially lost in invasive tumors (P less than 0.03) when compared with the expression in primary noninvasive tumors. A high mean nuclear volume in tissue specimens of primary tumors correlated with a progression to invasive disease (P less than 0.01). A significantly (P less than 0.003) higher mean nuclear volume was found in tumor areas that were positive for PNA compared with areas that were negative for PNA in primary tumors. A significantly lower mean nuclear volume was found in Tn-antigen-positive invasive Grade 3 tumor areas than in Tn-antigen-negative areas (P less than 0.005). The combined use of T-antigen expression and mean nuclear volume is of potential clinical interest for determining patients who are at high risk of disease progression.

Aged

Multicenter dose-finding trial for thrombolysis with urokinase preactivated pro-urokinase (TCL 598) in acute myocardial infarction. German Preactivated Pro-Urokinase Study Group.

In a multicenter dose-finding study, the thrombolytic potency of urokinase preactivated pro-urokinase was evaluated. Sixty-two patients were randomly assigned to receive 250,000 U of urokinase plus either 4.5 mega U (group I: n = 33) or 6.5 mega U (group II: n = 29) of pro-urokinase. Patency rates were 36.4% (20.4-54.9%) vs. 54.5% (36.3-71.9%) (n = 27) at 60 minutes and 55.6% (32.5-70.6%) vs. 62.1% (42.3-79.3%) at 90 min into thrombolysis (n.s.). In a third group of 12 patients treated with 500,000 U of urokinase plus 6.5 mega U of pro-urokinase patency was achieved in 33.3% (9.9-65.1%) and 41.7% (15.2-72.3%) at 60 and 90 min, respectively. Patency rates at 24 hr follow-up angiography (n = 35) were 78.6% (49.2-95.3%), 85.7% (57.2-98.2%), and 85.7% (42.1-99.6%). Coagulation analysis in 37 patients revealed similar alterations in the three treatment groups with minor decreases in fibrinogen levels, moderate drops in plasminogen and alpha-2-antiplasmin levels, and moderate increases in the concentrations of the total fibrinogen/fibrin degradation products, the differences between the groups not being significant. Bleeding complications were observed in 12.9%, 13.8%, and 25% of patients in groups I, II, and III, respectively, mainly related to catheter sites. Hence, the safety profile of urokinase preactivated pro-urokinase seems comparable to other thrombolytic regimens. Reopening of occluded coronary arteries, however, is achieved relatively slowly. Thus, in its use for thrombolysis in myocardial infarction, urokinase preactivated pro-urokinase does not seem to offer superior advantages.

Coronary Angiography

Assessment of major histocompatibility complex class I interaction with Epstein-Barr virus and human immunodeficiency virus peptides by elevation of membrane H-2 and HLA in peptide loading-deficient cells.

Earlier findings indicate that peptides can affect the expression of major histocompatibility complex (MHC) class I molecules on the surface of cells with defective peptide loading mechanism. We have used peptide induced increase of class I antigen expression to assess peptide interaction with MHC class I molecules. A panel of 41 overlapping synthetic peptides derived from the human immunodeficiency virus-1 (HIV-1) gag protein and 33 nonoverlapping peptides from Epstein-Barr virus (EBV) proteins EBNA-1, 2, 3, 4, 5, 6, LMP, BZLF2, BILF2, BSLF2, BALF4 and BcLF1 was assessed for the ability to enhance the expression of HLA-A2.1, H-2Db, Kb and Dd on the murine RMA-S and human 721.174/T2 (.174/T2) lines by indirect immunofluorescence. Considering doubling of the fluorescence intensity in the peptide-treated samples as positivity, 6 of 39 HIV and 1 of 32 EBV peptides were found to bind to A2.1, 6 of 39 HIV gag and 7 of 16 EBV peptides to Db, 8 of 39 HIV gag and 5 of 16 EBV peptides to Kb and 2 of 39 HIV gag and 1 of 17 EBV peptides to Dd. The sensitivity of the method is comparable to the in vitro class I assembly assay with conformation-dependent monoclonal antibody and is more discriminating than the solid-phase assay. Due to its simplicity this method can also serve for testing large peptide panels for binding capacity to various class I molecules. Moreover, the method provides information about the relevance of in vitro tests for class I assembly in living cells.

Amino Acid Sequence

Fine specificity of the humoral immune response to HIV-1 GP160 in HIV-1 infected individuals from Tanzania.

A total of 160 sera from HIV-1 infected individuals from Tanzania were examined for their fine specificity characteristics relative to 9 synthetic peptides that define HIV-1 gp160 epitopes. Immunorecessive and immunodominant epitopes were identified in both gp120 and gp41 based on serologic reactivity of these HIV-1 infected sera. A significant difference in fine specificity among HIV-1 infected individuals from Tanzania and the United States was observed for an immunodominant gp41 epitope. No significant differences in reactivity among asymptomatic vs. symptomatic HIV-1 infected individuals were detected for the selected HIV-1 gp160 epitopes defined by these peptides. The majority of sera from HIV-1 infected Tanzanians contained antibodies that recognized a peptide corresponding to the V3 region of gp120 from the HIV-1 MN isolate. These data suggest that regional isolates of HIV-1 may exist in Tanzania that differ from HIV-1 isolated in the United States. However, based on serology, HIV-1 isolates exhibiting sequences with HIV-1 MN V3 similarity may also be prevalent in Tanzania. The results of this study may be useful for the design of more effective AIDS diagnostic and therapeutic products for use worldwide.

Amino Acid Sequence

Nutritional control of nikkomycin and juglomycin production by Streptomyces tendae in continuous culture.

Continuous cultures with Streptomyces tendae revealed some interesting facts. In a continuous culture running for more than 2500 h the production of either nikkomycines or juglomycins could be selected by varying the feed composition. Decreasing the phosphate supply in the feed broth from the initial concentration of 2.5 mM to 1.0 mM enhanced the productivity of nikkomycins and decreased the productivity of juglomycins. When switching back to the initial conditions of the experiment after 2000 h nearly the same production behaviour as at the beginning of the fermentation could be observed. This indicated a stable behaviour of the population with regard to nikkomycin productivity. The long continuous fermentation showed the ability of S. tendae Tü 901/8c to produce nikkomycin at a high level for at least 1500 h. In a second continuous culture it was shown that the productivity of the nikkomycins and juglomycins decreased and increased, respectively, with increasing dilution rate. Comparing batch cultures with continuous fermentations, higher juglomycin productivity was found in the latter. These facts indicate that the strain responds to complex interacting physiological controls, by producing either nikkomycins or juglomycins in a higher amount.

Aminoglycosides

Prevention of deep vein thrombosis in neurosurgical patients: a prospective double-blind comparison of two prophylactic regimen.

In a prospective, randomized, double-blind investigation of anticoagulant agents for prevention of deep vein thrombosis in patients undergoing operations at the lumbar-vertebral disc, 179 patients were randomly allocated to two groups. 87 patients received a fixed combination of low-molecular weight heparin 1,500 U-aPTT plus dihydroergotamine 0.5 mg (LMWH/DHE) once a day and additionally one injection of placebo per day, 92 patients received a fixed combination of sodium heparin 5,000 U plus dihydroergotamine 0.5 mg (HDHE) twice a day. Treatment was initiated two hours preoperatively in both groups and continued for at least seven days. Deep vein thrombosis (DVT), detected by the 125Iodine-labelled fibrinogen uptake-test, occurred in four patients treated with LMWH/DHE and in three patients with HDHE. In all seven patients phlebography was performed, confirming the diagnosis of DVT in one patient of the LMWH/DHE group and in two patients of the HDHE group, only. No increased bleeding was found in either group. Especially no neurological complications caused by epidural bleeding were observed. We therefore recommended to treat routineously all patients undergoing operations at the vertebral disc with antithrombotic agents. The advantages of the once daily regimen with low-molecular weight heparin include better patients' acceptance and less nursing time.

Adult

Studies on processing, particle formation, and immunogenicity of the HIV-1 gag gene product: a possible component of a HIV vaccine.

Antigens in a particulate conformation were shown to be highly immunogenic in mammals. For this reason, the particle forming capacity of derivatives of the HIV-1 group specific core antigen p55 gag was assayed and compared dependent on various expression systems: recombinant bacteria, vaccinia- and baculoviruses were established encoding the entire core protein p55 either in its authentic sequence or lacking the myristylation consensus signal. Moreover, p55 gag was expressed in combination with the protease (p55-PR) or with the entire polymerase (p55-pol), respectively. Budding of 100-160 nm p55 core particles, resembling immature HIV-virions, was observed in the eucaryotic expression systems only. In comparison to the vaccinia virus driven expression of p55 in mammalian cells, considerably higher yields of particulate core antigen were obtained by infection of Spodoptera frugiperda (Sf9) insect cells with the recombinant Autographa californica nuclear polyhedrosis (AcMNPV) baculovirus. Mutation of the NH2-terminal myristylation signal sequence prevented budding of the immature core particles. Expression of the HIV p55-PR gene construct by recombinant baculovirus resulted in complete processing of the p55 gag precursor molecule in this system. The introduction of an artificial frameshift near the natural frameshift site resulted in constitutive expression of the viral protease and complete processing of p55, both in Escherichia coli and in vaccinia virus infected cells. Interestingly, significant processing of p55 resembling that of HIV infected H9 cells could also be achieved in the vaccinia system by fusing the entire pol gene to the gag gene. Moreover, processing was not found to be dependent on amino-terminal myristylation of the gag procursor molecule, which is in contrast to observations with type C and type D retrovirus. However, complete processing of p55 into p24, p17, p9 and p6 abolished particle formation. Purified immature HIV-virus like particles were highly immunogenic in rabbits, leading to a strong humoral immune response after immunization. Empty immature p55 gag particles represent a noninfectious and attractive candidate for a basic vaccine component.

AIDS Vaccines

Immunological reactivity of a human immunodeficiency virus type I derived peptide representing a consensus sequence of the GP120 major neutralizing region V3.

To reduce the opportunities for human immunodeficiency virus type 1 (HIV-1) to evade vaccine induced immunity, the development of subunit vaccines must focus on the characterization of immunogenic epitopes, which are major targets for the immune system. The most dominant site for elicitation of neutralising immune response is located on the external envelope glycoprotein gp120 within the third variable domain (V3). To overcome virus type specificity of antibodies directed to the V3-domain we designed a 36 amino acids long gp120/V3-consensus peptide (V3-C36) based on published biological data and sequence comparisons of various HIV-1 virus isolates. This peptide contains a conserved core sequence which is suggested to form a surface-exposed beta-turn. This peptide also includes T-cell epitopes defined in mice and humans, an ADCC-epitope and two highly conserved cysteine residues which were oxidized to form a cystine derivate, thus allowing correct peptide folding. In ELISA-tests, this peptide reacts with at least 90% of randomly selected sera of European and African patients infected with HIV-1 and is recognized by three different HIV-1/V3 "type-specific" antisera (MN, RF, IIIB-strain). Using this peptide as immunogen in rabbits, antisera could be raised with highly cross-reactive and HIV-1/IIIB strain neutralizing properties. Moreover, HTLV/HIV-1/IIIB specific cytotoxic T-lymphocytes (CTLs) of BALB/c mice infected with a gp120 recombinant vaccinia virus recognized the central 16- and 12-mer peptides of the V3-C36 consensus peptide in cytolytic assays, indicating perfect compatibility of the consensus peptide with the IIIB-primed CTLs. The DNA-sequence encoding the V3-consensus loop region might be an important component in newly designed recombinant subunit vaccines. In addition, due to its broad serological reactivity, the V3-consensus peptide might play an important role in special diagnostic purposes.

AIDS Vaccines

Identification of a protein encoded in the EB-viral open reading frame BMRF2.

Using monospecific rabbit sera against a peptide derived from a potential antigenic region of the Epstein-Barr viral amino acid sequence encoded in the open reading frame BMRF2 we could identify a protein-complex of 53/55 kDa in chemically induced B95-8, P3HR1 and Raji cell lines. This protein could be shown to be membrane-associated, as predicted by previous computer analysis of the secondary structure and hydrophilicity pattern, and may be a member of EBV-induced membrane proteins in lytically infected cells.

Amino Acid Sequence

Bromocriptine lessens the incidence of mortality in L-dopa-treated parkinsonian patients: prado-study discontinued.

L-Dopa supplemented by a peripheral decarboxylase inhibitor is considered the most potent therapeutic regimen prolonging active life in Parkinsonian patients. The long-term benefit of therapy is limited by adverse effects, such as dyskinesia and on-off phenomena, which can be mitigated by the concomitant administration of dopamine agonists, such as bromocriptine. In order to quantify the beneficial impact of early combination therapy, a controlled clinical trial (PRADO: PRA videl1 + DO pa) in patients with early Parkinson's disease was carried out, whereby L-Dopa monotherapy (in a fixed combination with benserazide (DoBe) was being compared with the same combination plus bromocriptine (DoBeBro). Patients were recruited and treated by 101 practising neurologists in the Federal Republic of Germany and in Hungary. Twenty seven clinical university centers cross-checked the patients at regular intervals. The trial started with 3 months of DoBe monotherapy (median dose of 375 mg L-Dopa for both randomized groups) followed by gradual substitution of DoBe by bromocriptine over 3 months in one of the groups (250 mg L-Dopa/10 mg bromocriptine). The target medication was maintained from study months 6 to 54. Parkinsonian symptoms were classified according to the Webster rating scale, the Hoehn and Yahr scale and the Zung Self-Rating Depression Scale. Adverse events and life status were checked at regular intervals. Special emphasis was given to motor performance tests. 587 patients (302 in the DoBe group and 285 in the DoBeBro group) were available for intention-to-treat analysis. Both groups were homogeneous at baseline in all observed parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Aged

A quartz crystal biosensor for measurement in liquids.

The detection of anti-human immunodeficiency virus (HIV) antibodies by means of synthetic HIV peptide immobilized on a piezoelectric quartz sensor is demonstrated. The measurement set-up consists of an oscillator circuit, a suitably modified AT-cut thickness-shear-mode quartz crystal with gold electrodes, which is housed in a special reaction vessel, and a computer-controlled frequency counter for the registration of the measured frequency values. The quartz crystal is adapted for a steady operation in liquids at a frequency of 20 MHz. In phosphate-buffered saline solution the oscillator reaches a stability of about 0.5 Hz within a few seconds, of about 2 Hz within 10 min and about 30 Hz within 1 h. The frequency shift due to the adsorption of various proteins to the uncoated sensor surface has been investigated. It can be shown that a stable adsorptive binding of proteins to an oscillating gold surface is feasible and can be used for the immobilization of a receptor layer (e.g. HIV peptide). Specific binding of the anti-HIV monoclonal antibody to the HIV peptide immobilized on the quartz sensor is demonstrated. Control experiments show, however, additional unspecific binding. According to the experiments, the Sauerbrey formula gives a sufficiently accurate value for the decrease of the resonant frequency due to adsorption or binding of macromolecular proteins on the quartz crystal surface.

Biosensing Techniques