Number Of Antibody Binding Sites Research Articles

Improved estimation of the ratio of detection efficiencies of excited acceptors and donors for FRET measurements.

Förster resonance energy transfer (FRET) is a radiationless interaction between a donor and an acceptor whose distance dependence makes it a sensitive tool for studying the oligomerization and the structure of proteins. When FRET is determined by measuring the sensitized emission of the acceptor, a parameter characterizing the ratio of detection efficiencies of an excited acceptor versus an excited donor is invariably involved in the formalism. For FRET measurements involving fluorescent antibodies or other external labels, this parameter, designated by α, is usually determined by comparing the intensity of a known number of donors and acceptors in two independent samples leading to a large statistical variability if the sample size is small. Here, we present a method that improves precision by applying microbeads with a calibrated number of antibody binding sites and a donor-acceptor mixture in which donors and acceptors are present in a certain, experimentally determined ratio. A formalism is developed for determining α and the superior reproducibility of the proposed method compared to the conventional approach is demonstrated. Since the novel methodology does not require sophisticated calibration samples or special instrumentation, it can be widely applied for the quantification of FRET experiments in biological research.

Everolimus TDM using Thermo Fisher QMS immunoassay on Indiko, Beckman DxC, AU680, and AU5800 analyzers

ObjectivesEverolimus (EVR), a mTOR inhibitor immunosuppressant approved for renal, liver and cardiac transplants. This study established the appropriate TDM performances of the Thermo Scientific QMS EVR Assay by using the Beckman DxC, followed by comparison to the Thermo Scientific Indiko and a previously published LC-MS/MS assay, and Beckman AU680 and AU5800 analyzers. Design and methodsThe study initially established acceptable linearity, precision and accuracy of the QMS EVR turbidimetric assay. Sample preparation was initiated by mixing patient whole blood with methanol and a precipitation reagent. Supernatant was transferred to sample cups. Drug in the supernatant and drug coated on microparticle compete for the limited number of antibody binding sites. If EVR is absent in the sample supernatant, EVR coated microparticles are agglutinated in the presence of antibody reagent. If EVR is present, agglutination is partially inhibited, depending on EVR concentration. Calibrators range was from 1.5 to 20ng/mL. Comparison studies data were analyzed by Deming Regression and Bland-Altman plots. ResultsPrecision studies showed the following mean concentrations 4.00–4.72, 7.70–8.20 and 14.80–15.56ng/mL, and CVs of 3.1–8.7%, 3.4–8.9% and 2.6–4.4% respectively. Comparison of analyses of 107 de-identified transplant samples by three analyzers - Indiko, DxC and AU680 showed: EVR concentrations from <1.5 to 13.6ng/mL, slopes 1.000 to 1.076, intercepts −0.053 to 0.462, and R 0.945 to 0.981. Another series of comparison studies (n=104) of Indiko and AU680 with LC-MS/MS showed the following: slopes 1.035 to 1.086, intercepts –0.019 to −0.265, and R 0.924 to 0.980. 2013–2016 CAP survey results were acceptable. ConclusionsBased on the experience of the past 3.5years, Thermo Scientific QMS EVR Immunoassay using four different analyzers offered adequate limit of detection and acceptable accuracy and precision, suitable for monitoring renal and liver transplant recipients.

Development of Sensor Surfaces Using Poly-(N-vinylformamide) for Sensitive Detection of 2,4,6-Trinitrotoluene by Displacement Method on a Surface Plasmon Resonance Sensor

To detect 2,4,6-trinitrotoluene (TNT) using a displacement method over a surface plasmon resonance immunosensor, we developed sensor surfaces modified with a poly-(N-vinylformamideco-vinylamine), which was synthesized from a monomer, N-vinylformamide, and was hydrolyzed to produce amine groups, and TNT analogs. Poly-(N-vinylformamide-co-vinylamine) that reacted with succinic acid was used as a matrix of a sensor surface for increasing the number of antibody binding sites. 2,4-Dinitrophenyl glycine, 3-nitrophenyl acetic acid, and 4-nitrophenyl acetic acid were immobilized on the polymer-modified sensor surfaces. We evaluated the suppression ability of the sensor surface for nonspecific adsorption and determined the limit of detection (LOD) for TNT. As a result, a sensor chip suppressed nonspecific adsorption for 1 mg/mL bovine serum albumin and lysozyme. TNT was measured by the displacement method, and the LOD achieved over a 3-nitrophenyl acetic acid-modified sensor chip was 0.9 ng/mL (ppb).

Development of a detector-free semiquantitative immunochromatographic assay with major aflatoxins as target analytes

Recent trend toward semiquantitative strip tests has been driven by a strong demand for rapid screening of toxic or harmful substances in food. Here, a detector-free semiquantitative immunochromatographic assay (ICA) has been developed. In the assay, the test zone is formed by dispensing three test lines [TLs (TL-I, TL-II and TL-III)] with the same capture reagent onto a nitrocellulose membrane. The competition between the migrating analyte and the immobilized capture reagent on three TLs for the limited number of antibody binding sites causes less nanogold–Ab probe to accumulate on TLs, which provides an easily visible color owing to the red color itself. When analyte concentration is up to the threshold level, no color appears on the TL. That's to say, the appearing number of color bands after a reaction indicates the content (range) of analyte. Analyte concentration levels can thus be determined by observing the number of TLs developed in the test zone. Taking major aflatoxins as target analytes, the threshold levels for TL-I–III and the visual detection limit of the detector-free semiquantitative strip (DFQ-strip) assay were determined, respectively. Results show that the DFQ-strip assay is superior to traditional ICAs because it offers multiple dynamic detection ranges and semiquantitative analytical capability.

A naked-eye based strategy for semiquantitative immunochromatographic assay

It is critical to develop a cost-effective quantitative/semiquantitative assay for rapid diagnosis and on-site detection of toxic or harmful substances. Here, a naked-eye based semiquantitative immunochromatographic strip (NSI-strip) was developed, on which three test lines (TLs, TL-I, TL-II and TL-III) were dispensed on a nitrocellulose membrane to form the test zone. Similar as the traditional strip assay for small molecule, the NSI-strip assay was also based on the competitive theory, difference was that the analyte competed three times with the capture reagent for the limited number of antibody binding sites. After the assay, the number of TLs developed in the test zone was inversely proportional to the analyte concentration, thus analyte content levels could be determined by observing the appeared number of TLs. Taking aflatoxin B1 as the model analyte, visual detection limit of the NSI-strip was 0.06ngmL−1 and threshold concentrations for TL-I–III were 0.125, 0.5, and 2.0ngmL−1, respectively. Therefore, according to the appeared number of TLs, the following concentration ranges would be detectable by visual examination: 0–0.06ngmL−1 (negative samples), and 0.06–0.125ngmL−1, 0.125–0.5ngmL−1, 0.5–2.0ngmL−1 and >2.0ngmL−1 (positive samples). That was to say, compared to traditional strips the NSI-strip could offer more parameter information of the target analyte content. In this way, the NSI-strip improved the qualitative presence/absence detection of traditional strips by measuring the content (range) of target analytes semiquantitatively.

Toward quantitative fluorescence measurements with multicolor flow cytometry

A procedure is presented for calibrating the output of a multicolor flow cytometer in units of antibodies bound per cell (ABC). The procedure involves two steps. First, each of the fluorescence channels of the flow cytometer is calibrated using Ultra Rainbow beads with assigned values of equivalent number of reference fluorophores (ERF). The objective of this step is to establish a linear relation between the fluorescence signal in a given fluorescence channel of multicolor flow cytometers and the value of ERF. The second step involves a biological standard such as a lymphocyte with a known number of antibody binding sites (e.g., CD4 binding sites). The biological standard is incubated with antibodies labeled with one type of fluorophores for a particular fluorescence channel and serves to translate the ERF scale to an ABC scale. A significant part of the two-step calibration procedure involves the assignment of ERF values to the different populations of Ultra Rainbow beads. The assignment of ERF values quantifies the relative amount of embedded fluorophore mixture in each bead population. It is crucial to insure that the fluorescence signal in a given range of fluorescence emission wavelengths is related linearly to the assigned values of ERF. The biological standard has to poses a known number of binding sites for a given antibody. In addition, this antibody has to be amenable to labeling with different types of fluorophores associated with various fluorescence channels. The present work suggests that all of the requirements for a successful calibration of a multicolor flow cytometer in terms of ABC values can be fulfilled. The calibration procedure is based on firm scientific foundations so that it is easy to envision future improvements in accuracy and ease of implementation.

Kinetic studies on the interactions between glycolipid biosurfactant assembled monolayers and various classes of immunoglobulins using surface plasmon resonance

Kinetic studies on the interactions between self-assembled monolayers of mannosylerythritol lipids (MELs), which are glycolipid biosurfactants abundantly produced by microorganisms, and various classes of immunoglobulins including human IgG, IgA, and IgM were performed using surface plasmon resonance (SPR). The effect of the MEL structure on the binding behavior of HIgG was examined. Assembled monolayers of MEL-A having two acetyl groups on the headgroup gave a high affinity ( K d = 1.7 × 10 −6 M) toward HIgG, while those of MEL-B or MEL-C having only one acetyl group at C-6′ or C-4′ position gave little affinity. Our kinetic analysis revealed that the binding manner of HIgG, HIgA ( K d = 2.4 × 10 −7 M), and HIgM ( K d = 2.2 × 10 −7 M) to the assembled monolayers of MEL-A is not the monovalent mode but the bivalent mode, and both the first and second rate association constants ( k a1, k a2) increase with an increase in the number of antibody binding sites, while those for dissociation ( k d1, k d2) changed little. Moreover, we succeeded in directly observing great amounts of HIgG, HIgA, and HIgM bound to MEL-A monolayers using atomic force microscopy (AFM). Finally, we found that MEL-A assembled monolayer binds toward various IgG derived from mouse, pig, rabbit, horse, goat, rat, and bovine as well as human IgG (HIgG), and the only exception was sheep IgG. These results clearly demonstrate that MEL-A assembled monolayers would be useful as noble affinity ligand system for various immunoglobulins.

Radial Mass Density, Charge, and Epitope Distribution in theCryptococcus neoformansCapsule

Exposure of Cryptococcus neoformans cells to gamma radiation results in a gradual release of capsular polysaccharide, in a dose-dependent manner. This method allows the systematic exploration of different capsular regions. Using this methodology, capsule density was determined to change according to the radial distribution of glucuronoxylomannan and total polysaccharide, becoming denser at the inner regions of the capsule. Scanning electron microscopy of cells following gamma radiation treatment confirmed this finding. The zeta potential of the capsule also increased as the capsule size decreased. However, neither charge nor density differences were correlated with any change in sugar composition (xylose, mannose, and glucuronic acid) in the different capsular regions, since the proportions of these sugars remained constant throughout the capsule. Analysis of the capsular antigenic properties by monoclonal antibody binding and Scatchard analysis revealed fluctuations in the binding affinity within the capsule but not in the number of antibody binding sites, suggesting that the spatial organization of high- and low-affinity epitopes within the capsule changed according to radial position. Finally, evidence is presented that the structure of the capsule changes with capsule age, since the capsule of older cells became more resistant to gamma radiation-induced ablation. In summary, the capsule of C. neoformans is heterogeneous in its spatial distribution and changes with age. Furthermore, our results suggest several mechanisms by which the capsule may protect the fungal cell against exogenous environmental factors.

Variability of PECAM-1 Expression on Blood Cells.

PECAM-1 is a 130 kDa member of the Immunoglobulin gene superfamily that is expressed on circulating platelets, neutrophils, monocytes, and certain lymphocyte subpopulations. Deficiency of PECAM-1 in mice results in hyper-reactive platelets, B cells, and mast cells, compromised vascular integrity, and endothelial cells that are more susceptible to radiation-induced apoptosis in vivo. Whether or not PECAM-1 expression levels vary in the human population, and whether variable PECAM-1 expression renders one more or less susceptible to cardiovascular disease, autoimmune disorders, or sepsis has not been investigated to date. As a first step in examining these questions, we have developed a quantitative flow cytometry-based assay to determine the number of PECAM-1 molecules expressed on the surface of blood and vascular cells. Whole blood samples obtained from anonymous normal volunteer blood donors were stained with saturating amounts of fluorochrome-conjugated anti-PECAM-1 antibody, and relative fluorescence intensities were determined for platelets, neutrophils, monocytes, and lymphocytes by flow cytometry. Polystyrene microbeads coated with calibrated numbers of antibody binding sites were saturated with the same fluorochrome-conjugated anti-PECAM-1 antibody and used to create a standard curve that allowed us to relate relative fluorescence intensities to known numbers of antibody binding sites. By plotting the fluorescence intensity of anti-PECAM-1 antibody binding to each cell population against this standard curve, we were able to quantify the number of PECAM-1 molecules on the surface of each cell population. We found that PECAM-1 expression levels most often ranged between 5,000–12,000 molecules/platelet, 6,000–10,000/lymphocyte, 30,000–40,000/neutrophil, and 50,000–80,000/monocyte. Some individuals, however, expressed 2–5 times more PECAM-1 on a given blood cell subtype. Interestingly, individuals who expressed high levels of platelet PECAM-1 did not necessarily also express high levels of leukocyte PECAM-1, and vice versa. Since a number of naturally-occurring alleles of the PECAM-1 gene have recently been associated with the progression of atherosclerosis, myocardial infarction, and/or coronary artery disease, correlation of genetic variation within the PECAM-1 locus with its variable expression on the surface of blood and vascular cells may permit addition of PECAM-1 to the growing list of cell adhesion and signaling receptors whose expression is linked to bleeding and clotting disorders in humans.

Paramagnetic bead based enzyme electrochemiluminescence immunoassay for TNT

We have developed an electrochemiluminescence immunoassay system for TNT (2,4,6-trinitrotoluene) in which enzyme labelled antibodies bound to paramagnetic beads are concentrated on an electrode magnetically, and light emission is triggered electrochemically. Full details of the instrumentation used to carry out these immunoassays are described. The paramagnetic beads are coated with haptenylated dextrans prepared by substituting biotin and analogues of TNT into aminodextrans. Three methods for synthesizing aminodextrans are compared and a method for substituting haptens and biotin into aminodextrans is described. Antibodies to TNT were labelled with glucose oxidase, and used for immunoassays in which TNT and haptenylated dextran competed for a limited number of antibody binding sites. The haptenylated dextran was bound to streptavidin coated paramagnetic beads, and the antibodies bound to the dextran were separated from the sample by concentrating them on an electrode magnetically. Bound antibodies were quantified by integrating the light emitted when enzymatically generated H 2O 2 reacted with electrochemically-oxidized luminol. The time taken for the detection step was 80 s compared with 20 min for colorimetric ELISAs using the same reagents, and the limit of detection for TNT was 31 ppb (3×S.D. zero calibrator; n=8).

Quantitative Flow Cytometric Analysis of Membrane Antigen Expression

Immunological analysis for cell antigens has been performed by flow cytometry in a qualitative fashion for over thirty years. During that time it has become increasingly apparent that quantitative measurements such as number of antigens per cell provide unique and useful information. This unit on quantitative flow cytometry (QFCM) describes the most commonly used protocols, both direct and indirect, and the major methods of analysis for the number of antibody binding sites on a cell or particle. Practical applications include detection of antigen under- or overexpression in hematological malignancies, distinguishing between B cell lymphoproliferative disorders, and precise diagnosis of certain rare diseases.

Development of a flow-injection liposome immunoanalysis system for fumonisin B1

Fumonisins are a group of naturally occurring mycotoxins produced by several species of the fungus Fusarium (mainly moniliforme) and occur worldwide on corn intended for human and animal consumption. Contamination of food and feed with fumonisins has been implicated in and associated with a number of diseases in both livestock as well as human beings. In this study, a Flow-Injection Liposome ImmunoAnalysis (FILIA) system was developed for the heterogeneous quantitative determination of fumonisin B1 (FmB1). The immunoassay is based on the competition between FmB1 and sulforhodamine B (SRB) dye-encapsulating, FmB1-tagged liposomes for a limited number of antibody binding sites. The antibodies are immobilized via protein A in a capillary immunoreactor column, and 35% MeOH is used for the regeneration of antibody binding sites after each measurement, which allows the immunoreactor to be used for up to 70 sequential sample injections without any loss of reactivity. Sample preconcentration is not needed and a single assay can be performed in less than 11 min. The calibration curve for FmB1 in Tris-buffered saline (TBS) solution had a working range of 1–1000 ng/ml.

Stoichiometry of a Ligand-gated Ion Channel Determined by Fluorescence Energy Transfer

We have developed a method to determine the stoichiometry of subunits within an oligomeric cell surface receptor using fluorescently tagged antibodies to the individual subunits and measuring energy transfer between them. Anti-c-Myc monoclonal antibody (mAb 9-E10) derivatized with a fluorophore (europium cryptate, EuK) was used to individually label c-Myc-tagged alpha1-, beta2-, or gamma2-subunits of the hetero-oligomeric gamma-aminobutyric acid (GABAA) receptor in intact cells. The maximal fluorescent signal derived from the alpha1(c-Myc)beta2gamma2 and the alpha1beta2(c-Myc)gamma2 receptors was twice that obtained with alpha1beta2gamma2(c-Myc), suggesting that there are 2x alpha-, 2x beta-, and 1x gamma-subunits in a receptor monomer. This observation was extended using fluorescence energy transfer. Receptors were half-maximally saturated with EuK-anti-c-Myc mAb, and the remaining alpha1(c-Myc) subunits were labeled with excess anti-c-Myc mAb derivatized with the fluorescence energy acceptor, XL665. On exposure to laser light, energy transfer from EuK to XL665 occurred with alpha1(c-Myc)beta2gamma2 and alpha1beta2(c-Myc)gamma2, but no significant energy transfer was observed with alpha1beta2gamma2(c-Myc) receptors, indicating the absence of a second gamma-subunit in a receptor monomer. We confirm that the GABAA receptor subtype, alpha1beta2gamma2, is composed of two copies each of the alpha- and beta-subunits and one copy of the gamma-subunit (i.e. (alpha1)2(beta2)2(gamma2)1) and conclude that this method would have general applicability to other multisubunit cell surface proteins.

Capillary electrochemical enzyme immunoassay (CEEI) for phenobarbital in serum

A competitive heterogeneous capillary enzyme immunoassay with electrochemical detection has been developed for phenobarbital in serum. The oxidized primary antibody was attached covalently to the modified interior surface of a microcapillary (22 μl). The competition between analyte phenobarbital and alkaline phosphatase labeled phenobarbital for a limited number of antibody binding sites was complete in 1.5 h. The enzymatic product ( p-aminophenol) from the catalytic conversion of the substrate ( p-aminophenyl phosphate) was detected by amperometric flow injection analysis. The calibration curve for phenobarbital had a detection limit of 30 μg l −1 (2.8 pmoles or 0.65 ng) and a range of 30–3000 μg l −1. The assay could be used to determine the phenobarbital serum concentration in a 4 μl clinical serum sample without pretreatment.

The HepG2 Extracellular Matrix Contains Separate Heparinase- and Lipid-releasable Pools of ApoE: IMPLICATIONS FOR HEPATIC LIPOPROTEIN METABOLISM

We have examined the association of apoE with the extracellular matrix (ECM) of HepG2 cells. Comparison of ECM prepared by previously published methods demonstrated that cytochalasin B-prepared material yielded the highest endogenous apoE, representing 23.6% of that in cell monolayers. ECM prepared with EDTA or Triton X-100 exhibited decreased levels of apoE, 3 and 6%, respectively. ECM bound very low density lipoprotein poorly (5-6% of the monolayer capacity); however, these incubations dramatically increased the apoE content of the ECM. Heparinase or suramin decreased apoE of the ECM by 19.6 and 37.3%, respectively, suggesting association with heparin sulfate proteoglycans. EDTA or EGTA also displaced 35% of the apoE, suggesting a Ca2+-dependent association. Incubation with phosphatidylcholine vesicles (PCV) displaced 30% of the apoE, suggesting that lipid content affects association of apoE with the ECM. Data derived from sequential incubations with combinations of suramin, EGTA, and PCV were consistent with the presence of two distinct pools of apoE on the HepG2 ECM, one releasable with suramin and EGTA and the other releasable with lipids. Exogenously applied lipid-free apoE readily bound to the ECM; however, increasing the lipid content decreased its association. Lipid-free apoE could be equally displaced from the ECM with PCV or suramin. When lipid-free apoE adsorbed to microtiter wells was incubated with a triglyceride emulsion or palmitoyloleyl phosphatidylcholine micelles, the immunoreactivity of 3H1 (but not other antibodies), a monoclonal antibody against an epitope in the C-terminal domain of apoE, increased about 4-fold. In a similar manner, incubation of ECM with lipid dramatically increased the immunoreactivity of 3H1, indicating that apoE of the ECM exists in a lipid-poor form. Scatchard analysis demonstrated that the increased immunoreactivity was due to an increase in the number of antibody binding sites. In conclusion, the ECM contains two pools of lipid-poor apoE. One pool associates with the ECM through heparin sulfate proteoglycans- and Ca2+-dependent interactions. A second pool of apoE dissociates from the ECM upon lipidation. The lipid-sensitive pool of apoE may participate in secretion or efflux of lipids or in the capture of lipoproteins by providing the apoE needed for receptor-mediated uptake.

Determination of Imazethapyr Using Capillary Column Flow Injection Liposome Immunoanalysis

A sensitive immunoanalysis system was developed for the quantitation of imazethapyr, the active ingredient in PURSUIT herbicide. Imazethapyr [5-ethyl-2-(4-isopropyl-4-methyl-5-oxo-2-imidazolin-2-yl)nicotinic acid] is one of the imidazolinone class of herbicides. The assay was based on sequential competitive binding of imazethapyr and liposomes for a limited number of antibody binding sites. A capillary tube (20 cm × 0.53 mm i.d.) with immobilized antibody was used as the immunoreactor column. Liposomes that entrap fluorescent molecules as the detectable label provide instantaneous, rather than time-dependent, enhancement, common with enzyme immunoassays. In this study, liposomes encapsulated carboxyfluorescein dye and were made antigenic by incorporating in the bilayer a phospholipid that had the analyte conjugated to its polar head group. The calibration curve for imazethapyr in Tris-buffered saline solution had a working range of 0.1−100 ng/mL. In the range between 1 and 100 ng/mL, recoveries from fortified tap and pond water samples ranged from 93 to 114%. Filtration was the only step needed for sample cleanup, and an assay could be performed in <10 min. Keywords: Imazethapyr; FILIA (flow injection liposome immunoanalysis); capillary immunocolumn; immunoassay; herbicide; liposomes

Competitive immunosorbent assays using ligand-enzyme conjugates bifunctional liposomes: theory and experiment.

Two models of immunoadsorbent assays are developed that describe the competitive adsorption of labeled antigen and unlabeled analyte to antibody binding sites immobilized on a solid surface. In the first model, a small labeled antigen and a small unlabeled analyte compete with only binding site limitations and no steric limitations. A multicomponent langmuir isotherm results that is sufficient to quantify competitive adsorption. This model can describe, with no adjustable parameters, the data of competitive assays for biotin using biotinylated horseradish peroxidase (B-HRP) over a wide range of anti-biotin antibody (ABA) surface densities. In the second model, the small unlabeled analyte competes with a large colloidal particle containing many antigens and enzyme labels attached to its surface. This model quantifies the steric interference that large particles can experience upon binding (large ligand effect) due to the lower probability of finding an available area of the right size to accommodate the larger adsorbent. This large ligand model also takes into account the increased probability of binding a large particle due to the larger number of antibody binding sites covered per collision. The resulting model is used to analyze the competitive assay data of biotin competing with liposomes to which many biotin and HRP molecules have been conjugated. This analysis is of interest because previous work has shown that these bifunctional liposomes can reduce the detection limit for antigens in bulk solution relative to assays performed with conventional small labeled antigens.

Alloimmune neonatal neutropenia resulting from immunization to a high‐ frequency antigen on the granulocyte Fc gamma receptor III

Alloimmune neonatal neutropenia is mainly caused by NA- or NB1-specific alloantibodies. An antibody in the serum of a Turkish mother who had given birth to an infant with alloimmune neonatal neutropenia showed no NA or NB specificity and was therefore investigated further. The number of antibody-binding sites was calculated by determination of elutable IgG from granulocytes using a quantitative sandwich enzyme-linked immunosorbent assay. Complement activation was tested by immunofluorescence (C3d) and cytotoxicity tests. The antigen was identified using the antigen-capture assay, monoclonal antibody-specific immobilization of granulocyte antigens, and a modified immunoprecipitation method based upon biotinylation of proteins and visualization by luminescence (luminoimmunoprecipitation). Family study and determination of antigen frequency were done by immunofluorescence and agglutination tests. A noncytotoxic, granulocyte-specific alloantibody that recognized the Fc gamma receptor III, independent of the NA phenotype, was detected, and 242,000 binding sites per cell were calculated. Of granulocytes from 150 randomly selected German blood donors, the alloantibody bound to all. The maternal cells were typed NA1/NA2- and NB1-positive. These data reveal the presence of a previously unrecognized, high-frequency epitope on the granulocyte Fc gamma receptor III. Luminoimmunoprecipitation proved to be a simple, nonradioactive technique that was useful in identifying the molecule involved.

CEDIA in vitro diagnostics with a novel homogeneous immunoassay technique: Current status and future prospects

CEDIA assays represent a state of the art technique utilizing two genetically engineered, enzymatically inactive fragments of β-galactosidase as the basis for a homogeneous enzyme immunoassay. The smaller, amino-terminal polypeptide, designated the enzyme donor (ED), can recombine spontaneously with the large residual fragment, called the enzyme acceptor (EA), to form active β-galactosidase, in a process called complementation. ED have been designed in such a way that a ligand, such as a hormone or drug, can be chemically attached to a specific amino acid residue without affecting the enzyme complementation. However, the binding of a ligand-specific antibody to the ED-ligand conjugate will inhibit complementation. If a sample containing ligand is added to the reaction mixture, the ligand will compete with the ED-ligand conjugate for the limited number of antibody binding sites. Thus, the ligand concentration in the sample will modulate enzymatic activity by influencing the amount of free ED-ligand conjugate available for complementation. The basic technology of CEDIA assays has a number of inherent advantages, the most important of these being a linear calibration curve with high precision over the whole assay range, lack of endogeneous enzyme activity and minimal serum interference, chemically defined conjugates and flexibility in assay design. These provide significant advantages in comparison to other homogeneous immunoassay techniques. As a result, CEDIA assays have been successfully developed for high concentration drugs such as theophylline, phenobarbital and phenytoin as well as for very low concentration analytes such as digoxin, B12 and folate. In a modified assay format, even the determination of binding proteins has been accomplished, an example being thyroxine binding proteins in the CEDIA T-uptake assay. More recently, the methodology has been extended to the measurement of high molecular weight analytes like ferritin.

Drug effects on CA125 antigen expression and antibody binding to cancer cells.

CA125 is a high-molecular weight glycoprotein expressed on most serous-type ovarian cancer and some lung adenocarcinoma tissues. The effects of various drugs on the release of CA125 antigen into culture medium and on monoclonal antibody (MAb) binding to cancer cells were studied using 8 human cancer cell lines, all of which expressed CA125 on their cell surfaces. The effect of dexamethasone was seen at as low a concentration as 10(-9) M dexamethasone, and the release of CA125 and the binding of radiolabelled anti-CA125 antibody were completely inhibited after exposure to 10(-7) M dexamethasone. The number of antibody binding sites markedly decreased. In contrast, sodium butyrate increased CA125 expression. These findings were clearly detected in only 3 cancer cell lines and a significant effect was not seen in the 5 other cancer cell lines. Interferon-gamma, examined in 3 cell lines, suppressed in a dose-dependent manner in 2 CA125 expression cell lines, but enhanced it in one line. No apparent effects were seen after exposure to tissue necrosis factor or interleukin-2. These results suggest that drugs may regulate the CA125 antigen expression in some, but not all, cancer cells and may affect the biodistribution of radiolabelled MAbs.