A 3-dimensional tumor growth inhibition assay for testing monoclonal antibody cytotoxicity

Summary A 3-dimensional tumor growth inhibition assay [18] has been adapted to test the cytotoxic activity of a panel of monoclonal antibodies directed to various antigenic determinants on the surface of mouse mammary tumor cells. Target cells can be prepared from either cultured cells or from pieces of fresh tumor. Antibody and complement are added when cells are growing actively and cell growth can be measured, non destructively, over a 7–10-day period. Effective diffusion of antibody through collagen gel and binding to target cells embedded in the gel is demonstrated by indirect immunofluorescent staining. The specificity of monoclonal antibody AMT 101 cytotoxicity for mouse mammary tumor cells is the same in trypan blue exclusion assays of single-cell suspensions as in collagen gel assays, with complete killing seen in the collagen gel assay only. The collagen gel assay allows the testing of repeated treatments in vitro, as well as combined treatment with multiple antibodies. It also allows cell-cell interaction and preserves all cell components in the tumor. The collagen gel assay has potential as a method of predicting the outcome of monoclonal antibody treatment of solid tumors.     

A solid-phase assay for antiactin antibody and actin using protein-A.

A method has been developed for the detection of an immunocomplex between an agaroselinked antigen and its antibody using ¹²⁵I-labeled protein-A. This procedure has been used to follow the appearance of antiactin antibody in rabbits injected with 200 μg of sodium dodecyl sulfate (SDS)-denatured and electrophoretically purified murine skeletal muscle actin on Days 0 and 20. Significant immunoglobulin G (IgG) binding to agarose-actin was detected at about Day 50 and the titer was maximum at about Day 100. The antibody binding reaction is not affected by bovine serum albumin, ovalbumin, or a bacterial extract, but can be blocked by soluble actin. Murine skeletal muscle actin (0.1 mg/ml) prevents about 80–90% of the binding, with 50% inhibition observed at about 10 μg/ml. Using an SDS extract of neuroblastoma cells, only about 60–70% inhibition was obtained, suggesting that the nonmuscle actin in this preparation does not react with all the antibodies which bind to muscle actin. The specificity and general usefulness of this assay are also suggested by the fact that antiactin antibodies do not bind to agarose-BSA, agarose-ovalbumin, or agarose-uterine myosin, while antibodies to the two latter proteins bind preferentially to their respective antigens. Thus, this method can potentially be used to assay any antigen immunologically active in the solid phase, the antibody to that antigen, and also crossreacting antigens.     

A radiolabeled monoclonal antibody binding assay for cytoskeletal tubulin in cultured cells

To detect changes in the extent of tubulin polymerization in cultured cells, we have developed a radioactive antibody binding assay that can be used to quantitate total cytoskeletal tubulin or specific antigenic subsets of polymerized tubulin. Fibroblastic cells, grown to confluence in multiwell plates, were permeabilized and extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer. These extracted cytoskeletons were then fixed and incubated with translationally radiolabeled monoclonal antitubulin antibody (Ab 1-1.1), an IgM antibody specific for the beta subunit of tubulin. Specific binding of Ab 1-1.1 to the cytoskeletons was saturable and of a single apparent affinity. All specific binding was blocked by preincubation of the radiolabeled antibody with excess purified brain tubulin. Specific Ab 1-1.1 binding appeared to represent binding to cytoskeletal tubulin inasmuch as: pretreatment of cells with colchicine decreased Ab 1-1.1 binding in a dose-dependent manner which correlated with the amount of polymerized tubulin visualized in parallel cultures by indirect immunofluorescence, taxol pretreatment alone caused an increase in Ab 1-1.1 binding and prevented in a dose-dependent manner the colchicine-induced decrease in antibody binding, in cells pretreated with colcemid and returned to fresh medium, Ab 1-1.1 binding decreased and recovered in parallel with the depolymerization and regrowth of microtubules in these cells, and comparison of maximal antibody binding per cell between primary mouse embryo, 3T3, and human foreskin fibroblasts correlated with immunofluorescence visualization of microtubules in these cells. Thus, this assay can be used to measure relative changes in the level of polymerized cytoskeletal tubulin. Moreover, by Scatchard-type analysis of the binding data it is possible to estimate the total number of antibody binding sites per cell. Therefore, depending on the stoichiometry of antibody binding, this type of assay may be used for quantitating total cytoskeletal tubulin, specific antigenic subsets of cytoskeletal tubulin, or other cytoskeletal proteins.     

A differential cell capture assay for evaluating antibody interactions with cell surface targets

Many biological and biomedical laboratory assays require the use of antibodies and antibody fragments that strongly bind to their cell surface targets. Conventional binding assays, such as the enzyme-linked immunosorbent assay (ELISA) and flow cytometry, have many challenges, including capital equipment requirements, labor intensiveness, and large reagent and sample consumption. Although these techniques are successful in mainstream biology, there is an unmet need for a tool to quickly ascertain the relative binding capabilities of antibodies/antibody fragments to cell surface targets on the benchtop at low cost. We describe a novel cell capture assay that enables several candidate antibodies to be evaluated quickly as to their relative binding efficacies to their cell surface targets. We used chimeric rituximab and murine anti-CD20 monoclonal antibodies as cell capture agents on a functionalized microscope slide surface to assess their relative binding affinities based on how well they capture CD20-expressing mammalian cells. We found that these antibodies’ concentration-dependent cell capture profiles correlate with their relative binding affinities. A key observation of this assay involved understanding how differences in capture surfaces affect the assay results. This approach can find utility when an antibody or antibody fragment against a known cell line needs to be selected for targeting studies.     

An attempt to analyze various thyroid stimulators by the receptor assay using hTSH radioimmunoassay.

In an attempt to analyze thyroid stimulators in serum we developed an assay procedure using hTSH radioimmunoassay (RIA) in combination with receptor competition. The principle of this method is the determination by RIA of hTSH displaced by other thyroid stimulators from a thyroidal receptor preparation which previously bound unlabelled hTSH. Practically 4 microunits of hTSH were bound with human or bovine receptor, and then hTSH displaced by addition of test serum (0.1 ml) or samples dissolved in serum (0.1 ml) was measured by RIA. This assay can determine the thyroid stimulators other than hTSH in serum that has the displacement activity of 0.5-4.0 microunits of hTSH in the useful range, such as mU/ml level of bovine TSH or rat TSH. Cholera toxin that has the thyroid stimulating activity like TSH also showed the displacement of the bound hTSH. This assay is not applicable for the human serum with more than 5 microunits/ml of TSH, because the assay value is over estimated by the free hTSH derived from the test serum. On the other hand, eighteen sera with high LATS activity and 42 sera with negative LATS activity from patients with untreated hyperthyroidism did not show any displacement. This might be due to the lower binding activity of LATS with hTSH receptor or the lower sensitivity of this assay method. Although it is difficult to use this assay clinically because of its low sensitivity, increased TSH in animal serum can be determined by this assay. The principle of this method may be also useful for examining the receptor binding of other peptide hormone that can be determined by an RIA method.     

Selective binding of a monoclonal antibody to Aspergillus niger glucose oxidase by formaldehyde fixed human polymorphonuclear leukocytes

BACKGROUND: Many of the procedures used in handling neutrophils may affect the expression of surface antigens, and hence their quantitation by flow cytometry. METHODS: Because the enzyme glucose oxidase of Aspergillus niger is absent in human tissues, an IgM against it (mAb GO) was used as negative control in a study involving the normal expression of neutrophil specific BH2-Ag in different age groups. RESULTS: When peripheral blood leukocytes (PBL) were freshly prepared, processed and stained with FITC-mAb GO without fixation or when the cells were stained with FITC-mAb GO prior to fixation with 2% formaldehyde, both median fluorescent intensity (MFI) and per cent of positively stained polymorphonuclear leukocytes (PMN) were similar to that obtained with a background sample without any antibody. However, when PBL were fixed after isolation with different concentrations of formaldehyde and for varying durations, MFI and per cent of positively stained PMN but not of monocytes or lymphocytes with FITC-mAb GO increased in a time and concentration dependent manner. Saturation was achieved at a finite concentration of the antibody. In a competition assay unlabelled mAb GO reduced binding of FITC-mAb GO to PMN by 79% and 95% at concentrations 100 and 200 times that of FITC labelled antibody, respectively. CONCLUSIONS: These observations strongly suggest that formaldehyde fixation causes the expression or accessibility of an epitope on PMN that is specifically recognized by the mAb GO.     

A high-throughput hybridoma selection method using fluorometric microvolume assay technology

Monoclonal antibodies (mAb) are not only useful reagents but also represent a promising type of therapeutics due to their high affinity and exquisite specificity for their antigens. A critical step in mAb generation is to identify antigen-specific antibodies. Although enzyme-linked immunosorbent assay (ELISA) has been broadly applied for antibody selection against secreted antigens, an inherent disadvantage for ELISA is the difficulty in identifying antibodies that recognize the native conformation of cell surface antigens. To overcome this drawback, the authors have developed a high-throughput cell-based antibody binding assay using fluorometric microvolume assay technology (FMAT). This method offers a homogeneous assay for detection of antibody binding to its antigen on the cell surface. To distinguish antibodies that bind to antigen on the cell surface from those that bind nonspecifically to cells, the binding is assessed using both antigen-expressing cells and related cells devoid of the antigen expression. This assay can detect antibodies at a concentration as low as 5 ng/mL and cell surface antigen as low as 9000 copies per cell. Results demonstrate that the FMAT method provides a sensitive and homogeneous assay to detect antibody binding to cell surface antigens and is amenable for high-throughput hybridoma selection.     

Use of monoclonal antibodies in the assay of hepatitis B core antigen and antibody

Hybridoma cells secreting antibody against hepatitis B core antigen (HBc Ag) were prepared. BALB/c mice were immunized with 0.2 ml of purified HBc Ag, and their spleen cells were fused with mouse myeloma (P3U1) cells by means of polyethylene glycol 1000. Activities of antibodies against HBc Ag (anti-HBc) were tested by the immune adherence hemagglutination (IAHA) and reverse passive hemagglutination inhibition (RPHI) techniques. Hybridoma cells found to contain antibodies accounted for 26.5% by IAHA and 52.1% by RPHI, respectively. Among 32 monoclonal anti-HBc antibodies, 18 were found to be positive by both IAHA and RPHI, and the remaining 14 positive by RPHI only. After cloning, they were injected intraperitoneally into ascitic mice. The highest anti-HBc activity with an IAHA titer of 1:4 X 10(6) and with an RPHI titer of 1:1 X 10(5) was detected in this ascitic fluid. Enzyme immunoassay (EIA) and RPHI with monoclonal antibody containing the highest anti-HBc activity were developed. All the sera in which anti-HBc was detected by IAHA and RPHI with polyclonal antibody were positive in EIA. RPHI titers obtained with monoclonal antibody were in good agreement with usual IAHA and RPHI titers obtained with polyclonal antibody. These results indicate that monoclonal antibody can be used in the HBc Ag and anti-HBc assay system.     

Identification of a high-affinity anti-phosphoserine antibody for the development of a homogeneous fluorescence polarization assay of protein kinase C

In the last few years, fluorescence polarization (FP) has been applied to the development of robust, homogeneous, high throughput assays in molecular recognition research, such as ligand-protein interactions. Recently, this technology has been applied to the development of homogeneous tyrosine kinase assays, since there are high-affinity anti-phosphotyrosine antibodies available. Unlike tyrosine kinases, application of FP to assay development for serine/threonine kinases has been impeded because of lack of high-affinity anti-phosphoserine/threonine antibodies. In the present study, we report the discovery of a high-affinity, monoclonal anti-phosphoserine antibody, 2B9, with a Kd of 250 +/- 34 pM for a phosphoserine-containing peptide tracer, fluorescein-RFARKGS(PO(4))LRQKNV. Our data suggest that 2B9 is selective for fluorescein-RFARKGS(PO(4))LRQKNV. The antibody and tracer have been used for the development of a competitive FP assay for protein kinase C (PKC) in 384-well plates. Phosphatidylserine, which enhances the kinase activity of PKC in a Ca(2+)-dependent manner and has a structure similar to that of phosphoserine, did not interfere with binding of the peptide tracer to the antibody in the FP assay. The data indicate that the FP assay is more sensitive and robust than the scintillation proximity assay for PKC. The FP assay developed here can be used for rapid screening of hundreds of thousands of compounds for discovery of therapeutic leads for PKC-related diseases.     

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

BACKGROUND: Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. METHODS: Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. RESULTS: Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. CONCLUSIONS: The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.     

ELISA-based assay for scatchard analysis of ligand-receptor interactions

A simple, nonradioactive method is presented that can be used for performing large numbers of binding assays of cell membrane receptors with their ligands. The method adopts the simple membrane preparation and biotin-based quantitation methods of the semi-intact cell endocytosis assays. After binding of the biotinylated ligand to its receptors on the semi-intact cell membranes, a rapid centrifugation step separates the membranes from unbound ligand. Bound ligand is subsequently released by detergent, captured by a specific antibody coated on the surface of microwells, and quantitated with peroxidase-conjugated streptavidin in a colorimetric assay. Using this assay, Scatchard analysis was performed on the data for the specific binding of iron-loaded transferrin to its receptors on mouse fibroblasts and yieldedK _d values similar to those obtained with other published methods. The assay is sensitive, rapid, and also convenient, because aliquots of semi-intact cells can be stored frozen. The perforated plasma membrane of the cells offers the additional possibility of screening factors that interact with the cytoplasmic domain of the receptors for their possible effects on the parameters of the extracellular ligand-receptor interaction.     

Solid-phase peptide quantitation assay using labeled monoclonal antibody and glutaraldehyde fixation

A solid-phase radioimmunoassay utilizing iodinated peptide-specific monoclonal antibody as a detection system instead of labeled peptide has been developed. Regional specific monoclonal antibodies to either gastrin-releasing peptide or gastrin were used as models to validate the general application of our modified assay. Conditions for radioactive labeling of the monoclonal antibody were determined to minimize oxidant damage, which compromises the sensitivity of other reported peptide quantitation assays. Pretreatment of 96-well polyvinyl chloride test plates with a 5% glutaraldehyde solution resulted in consistent retention of sufficient target peptide on the solid-phase matrix to allow precise quantitation. This quantitative method is completed within 1 h of peptide solid phasing. Pretreatment of assay plates with glutaraldehyde increased binding of target peptide and maximized antibody binding by optimizing antigen presentation. The hypothesis that glutaraldehyde affects both peptide binding to the plate and orientation of the peptide was confirmed by analysis of several peptide analogs. These studies indicate that peptide binding was mediated through a free amino group leaving the carboxy-terminal portion of the target peptide accessible for antibody binding. It was observed that the length of the peptide also affects the amount of monoclonal antibody that will bind. Under the optimal conditions, results from quantitation of gastrin-releasing peptide in relevant samples agree well with those from previously reported techniques. Thus, we report here a modified microplate assay which may be generally applied for the rapid and sensitive quantitation of peptide hormones.     

A humanized monoclonal antibody constructed from intronless expression vectors targets human hepatocellular carcinoma cells

An anti-human hepatocellular carcinoma (HCC) monoclonal antibody, hHP-1, was genetically humanized from a murine monoclonal antibody. In this study, a concept of positional template approach was applied to design the amino acid sequence of hHP-1's variable region, and synthetic DNA fragments for protein expression were produced through overlapping PCR from single strand oligonucleotides. Synthetic DNA fragments and human antibody constant region cDNA were used to construct two CMV promotor-based expression vectors for the antibody light and heavy chains, in which the variable region was connected directly to the constant region without an intron sequence. Completely assembled humanized antibody was successfully expressed in mammalian cells as IgG1 kappa molecules and purified using protein A affinity column. The immunogenicity of the hHP1 was estimated by the amino acid sequence and determined through a HAMA (human anti-murine antibody) serum reaction assay. Results indicated that the immunogenicity of hHP-1 was significantly reduced. In vitro binding activity assay showed that the hHP-1 had retained its binding function to a human HCC SMMC-7721 cell-line, without cross binding to other human normal tissues. Immunofluorescence staining showed that hHP-1 had a strong binding activity to SMMC cells. A competitive binding assay showed that the relative binding activity of hHP-1 was approximately 25% binding activity of the original murine antibody. Our results indicate that a humanized antibody could be produced using intronless vectors and expressed as a complete IgG1 kappa antibody. Hence we believe that hHP-1 could be a potential candidate for HCC treatment.     

An ultrahigh-throughput screening assay for estrogen receptor ligands.

Members of the steroid and thyroid hormone receptor superfamily (nuclear receptors) play diverse roles in mammalian physiology, in both normal and pathological states. For this reason, and because nuclear receptors are natural receptors for lipophilic small molecules, they are important therapeutic targets for the pharmaceutical industry. Here we describe a method for screening for ligands for one of these receptors, the estrogen receptor. The method is rapid, robust, and reliable, and has been used in an ultrahigh-throughput robotic screen. The receptor is crosslinked to a scintillant-containing solid support (FlashPlate) via a receptor-specific antibody. Test compounds are assayed for competition with a radiolabeled estrogen for binding to the immobilized receptor. Receptor-ligand complexes are allowed to form and receptor-bound radioactivity is detected in a scintillation counter. The assay has been designed for both isoforms of the estrogen receptor, alpha and beta, using separate antibodies for each. Given a radioactive tracer and an appropriate antibody, many of which are now commercially available, the assay could be established for any nuclear receptor.     

Fluorescence microplate-based assay for tumor necrosis factor activity using SYTOX Green stain

We have developed a simple, sensitive, fluorescence microplate-based assay for tumor necrosis factor (TNF) biological activity. The assay employs SYTOX Green nucleic acid stain to detect TNF-induced cell necrosis in actinomycin D sensitized cultured cell lines. SYTOX Green stain is a cationic unsymmetrical cyanine dye that is excluded from live cells but can readily penetrate cells with compromised cell membranes. Upon binding to cellular nucleic acids, the dye exhibits a large enhancement in fluorescence, which is monitored at fluorescein wavelengths. We detected 2.5 pg/mL and quantitated 25-500 pg/mL recombinant murine (rm) and recombinant human (rh) TNF-alpha, using mouse fibroblast-derived WEHI 164, WEHI 13var, and L929 cell lines. The procedure can also be used to detect agents that modulate TNF activity. We demonstrated complete inhibition of rhTNF-alpha using monoclonal anti-human TNF-alpha antibody and determined that approximately 20 ng/mL antibody was sufficient to neutralize 50% of the biological activity of 250 pg/mL rhTNF-alpha in these cell lines. Reagents are added in a single step, followed by a 6- to 8-h incubation period, during which the cytokine exhibits its effects. There are no wash steps, and the assay is readily amenable to automation and high-throughput screening procedures.     

A new competition assay for the solubilized hepatic galactosyl receptor

The present method of quantitating soluble asialoglycoprotein (galactosyl) receptor activity relies on the selective precipitation of receptor-ligand complexes to allow separation from free ligand. To provide an alternative to selective precipitation procedures, a simple and rapid method to assay for detergent-solubilized galactosyl receptor activity has been developed which uses permeabilized, fixed cells as a source of immobilized solid-phase receptors. Isolated rat hepatocytes were treated with digitonin to make available the internal as well as the external receptors. The permeable cells were also treated with glutaraldehyde to prevent further protein loss during subsequent exposure to detergents such as Triton X-100. The permeable/fixed cells, which retained about 70% of their total 125I-asialo-orosomucoid (125I-ASOR)-binding activity, with 89% specific binding, were insoluble even in 0.5% Triton X-100 and were easily pelleted. The permeable/fixed cells can be prepared in advance and stored frozen for months. A detergent extract of receptor is mixed with a constant amount of both 125I-ASOR and permeable/fixed cells. Soluble active receptors compete with immobilized receptors on the treated cell for binding of the 125I-ASOR. The assay is reproducible, linear over a broad range of soluble receptor concentration, and can quantitate receptor activity from as few as 10(5) hepatocytes. A modified purification procedure for the rat hepatic galactosyl receptor using this competition assay is also described.     

Monoclonal antibody to the human glucose transporter that differentiates between the glucose and nucleoside transporters

A monoclonal antibody to the glucose transporter has been prepared with band 4.5 (Mr 45,000-65,000) from human erythrocyte ghosts as antigen. This antibody, designated 7F7.5, is of the IgG2b type. The antibody bound exclusively to proteins in the band 4.5 region of immunoblots of human erythrocyte ghosts separated on sodium dodecyl sulfate-polyacrylamide gels. Immobilized 7F7.5 antibody removed glucose transport activity from solubilized alkaline-treated ghosts. The material that was eluted from the immobilized antibody matrix migrated primarily in the band 4.5 region of electrophoretic gels and bound the antibody in immunoblots. To test the specificity of the antibody, glucose and nucleoside transporters in alkaline-treated human erythrocyte ghosts were affinity labeled with [3H]cytochalasin B and [3H]-S-(nitrobenzyl)thioinosine (NBMPR), respectively. Both of these transporters are band 4.5 proteins and "copurify" by DEAE-cellulose chromatography. A filter paper assay was developed to assess the presence of the labeled transporters. Immobilized 7F7.5 antibody bound 99% of the labeled glucose transporter. In contrast, only 3% of the specifically labeled nucleoside transporter bound to the immobilized antibody. Furthermore, the antibody did not remove nucleoside transport or NBMPR binding activities from detergent solution. The antibody recognized two tryptic fragments, Mr 23,000 and 18,000, which contain the cytochalasin B binding site of the glucose transporter. By immunoblot, the monoclonal antibody recognized the glucose transporter in cultured human IM9 lymphocytes, synovial cells, and HBL 100 mammary cells but not cells of murine or rat origin. These results indicate that the glucose and nucleoside transporters are distinct proteins which can be distinguished by monoclonal antibody 7F7.5. The method developed to quantitate covalently labeled glucose and nucleoside transporters should have broad applicability as a rapid and easy method for determining the recovery of affinity-labeled membrane proteins in detergent solution during purification. Because of the location of the epitope, the antibody itself should prove to be a valuable tool in establishing the molecular basis for the function and regulation of the glucose transporter.     

A novel assay to detect low-titred antibodies to interferon beta in multiple sclerosis patients

Neutralizing antibodies (NAbs) may compromise interferon (IFN) clinical efficacy in patients with multiple sclerosis (MS) receiving IFN-beta treatment. When bioassays are used for anti-IFN-beta antibody detection, they are unable to discriminate between NAbs or other interfering substances with anti-IFN activity. Here we report the development of an anti-IFN-beta Western blot method that facilitates the detection of IFN low-titred antibodies and characterizes such low neutralizing activity as specifically due to the presence of particular IFN antibodies. The assay was characterized using serum samples from patients with MS treated with IFN-beta. It was developed by adding anti-IFN-positive antibody sera to Dynabeads M-280 tosylactivated followed by Western blot analysis. All sera samples from MS patients with IFN-betala NAbs (< or = 50 t1/10) proved to be antibody-positive using this new method and, more importantly, four of 27 binding antibody-negative sera samples were scored as IFN antibody-positive. The method was found to be rapid, specific and sensitive and consistent with respect to well-established antiviral neutralization or commercial enzyme-linked immunosorbent assays.     

A bovine monoclonal antibody detecting a class I BoLA antigen.

The bovine IgG1 monoclonal antibody (mAb) ILA70 was made by immunizing a calf with peripheral blood mononuclear cells (PBM) from a BoLA-w10 homozygous heifer and subsequently fusing lymphocytes from the local lymph-node with the heterohybridoma 53B3. Family and population studies, antibody binding inhibition and immunoprecipitation of the target antigen all indicate that ILA70 detects a polymorphic epitope on a bovine class I major histocompatibility complex (MHC) molecule. The antibody is complement fixing and so may be used in a standard cytotoxicity assay. Ascitic fluid with antibody activity many times greater than that of the tissue-culture supernatant has been prepared in nude mice. The antibody-producing heterohybridoma has been subcloned three times and appears to be stable. Such heterohybridomas may prove to be a valuable source of particularly discriminating and informative mAbs for the serological analysis of the products of the bovine MHC.     

A fluorescence lifetime based binding assay to characterize kinase inhibitors

The authors present a fluorescence lifetime-based kinase binding assay that identifies and characterizes compounds that bind to the adenosine triphosphate (ATP)-binding pocket of a range of tyrosine and serine/threonine kinases. The assay is based on displacement of an Alexa Fluor 647 conjugate of staurosporine from the ATP-binding site of a kinase, which is detected by a change in the fluorescence lifetime of the probe between the free (displaced) and kinase-bound states. The authors screened 257 kinases for specific binding and displacement of the Alexa Fluor 647-staurosporine probe and found that approximately half of the kinases tested could potentially be assayed with this method. They present inhibitor binding data against 4 selected serine/threonine kinases and 4 selected tyrosine kinases, using 6 commonly used kinase inhibitors. Two of these kinases were chosen for further studies, in which inhibitor binding data were compared to inhibition of kinase activity using 2 separate activity assay formats. Rank-order potencies of compounds were similar, but not identical, between the binding and activity assays. It was postulated that these differences could be caused by the fact that the assays are measuring distinct phenomena, namely, activity versus binding, and in a purified recombinant kinase preparation, there can exist a mixture of active and nonactivated kinases. To explore this possibility, the authors compared binding affinity for the probe using 2 kinases in their respective nonactivated and activated (phosphorylated) forms and found a kinase-dependent difference between the 2 forms. This assay format therefore represents a simple method for the identification and characterization of small-molecule kinase inhibitors that may be useful in screening a wide range of kinases and may be useful in identifying small molecules that bind to kinases in their active or nonactivated states.