Continuous monitoring of analyte concentrations.

We have investigated the application of a modified, heterogeneous, competitive enzyme immunoassay for the continuous measurement of small analytes in a medium stream. The analytical system contains two antibodies that are immobilized on spatially separated areas, one binding the analyte (Ab1) and the other binding the enzyme (Ab2). An analyte-enzyme conjugate serves as signal generator. The analyte-enzyme conjugate functions as a heterobifunctional shuttle that can bind to either antibody. A semipermeable membrane retains the enzyme shuttle in the internal volume of the sensor but permits the passage of small analytes from the medium stream. The amount of enzyme bound to Ab1 is inversely proportional and the amount of enzyme bound to Ab2 is directly proportional to the analyte concentration. We have demonstrated that this analytical system (1) can provide a larger total signal; (2) has a sensitivity comparable with conventional competitive immunoassays; (3) does not require the separation of bound from free antigens; and (4) is therefore suitable for the continuous measurement of analytes in a medium stream. With a model system, an increase from 0 ng ml-1 to 20 ng ml-1 of the steroid hormone progesterone and the subsequent fall to 0 ng ml-1 could be monitored.     

Modeling of immunosensors under nonequilibrium conditions. I. Mathematic modeling of performance characteristics.

Immunosensors for the detection of small analytes that use analyte-enzyme conjugates as signal generators require special attention if operated under nonequilibrium conditions. If the size of the analyte and the analyte-enzyme conjugate differ substantially, the two antigens do not diffuse at the same rate. This can cause time-dependent shifts in the sensitivity of competitive immunoassays. Therefore, immunosensors operating at short incubation times require precise timing that meets closely the specifications for which the sensors were calibrated. As an example, we have analyzed kinetic binding curves for the quantitative determination of progesterone with an immobilized monoclonal antibody and a conjugate between horseradish peroxidase and progesterone as signal generator. Mathematical paradigms have been developed to simulate the diffusion, antigen-antibody complex formation, and competitive binding processes in this analytical system. Dose-response curves obtained under nonequilibrium conditions can vary substantially from those obtained at equilibrium of antigen-antibody interaction. The degree of this variation depends on the performance characteristics of the major components of the immunosensor. The developed mathematical solutions reflect experimental results and can be used to model optimal conditions for immunosensors operating under nonequilibrium conditions. In this paper (Part I), we report on the mathematical modeling of the interaction between analyte, analyte-enzyme conjugate, and an immobilized antibody. In Part II (W. Schramm and S.-H. Paek (1991) Anal. Biochem. 196), we present experimental results and compare them with the theoretical models.     

Irreversible enzyme-shuttle immunoassay

The concept of a competitive enzyme immunoassay that utilizes simultaneously the bound and the free analyte-enzyme conjugate (heterobifunctional conjugate) for signal generation in response to varying analyte concentrations in samples has been investigated. Two antigenic sites of the heterobifunctional conjugate are used in the assay for binding to immunoglobulins: the analyte derivative binds to an immobilized antibody, Ab(1), and the enzyme component binds to a spatially separated immobilized antibody, Ab(2). The analytical system is set up such that in the absence of analyte, the conjugate is predominantly bound in the compartment that contains Ab(1). With increasing concentration of native analyte in samples, an increasing concentration of native analyte in samples, an increasing amount of conjugate migrates to the second compartment that contains Ab(2). The enzyme bound in each compartment is used for signal generation. Mathematical models have been developed to determine the optimal conditions and to predict the performance of such dual-antibody systems. The theoretical predictions are supported by experimental results. The dual-antibody system has been compared with a conventional competitive enzyme immunoassay using the same reagents.     

A chemiluminescence flow immunosensor based on a porous monolithic metacrylate and polyethylene composite disc modified with protein G

A generic, fast, sensitive and new type of flow immunosensor has been developed. The basis is a monolithic porous poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) polymer disc modified with protein G, placed in a fountain type flow cell compartment, in close proximity to a photomultiplier tube (PMT). Analyte and HRP labelled analyte derivative (tracer) compete for anti-analyte antibody binding sites. The mixture is then injected into the flow immunosensor system where the formed analyte- and tracer-antibody complexes are trapped by the monolithic protein G disc. The amount of bound tracer, inversely related to the concentration of analyte in the sample, is determined in a second step by injection of luminol, p-iodophenol and H2O2, generating enhanced chemiluminescence (CL) with horseradish peroxidase (HRP). A third and final step is need for regeneration of the protein G disc so that a new analysis cycle can take place. The performance of the disc immunosensor system was compared with a one step continuous flow injection immunoassay (FIIA) system, using the same reagents and a protein G column, in terms of assay sensitivity and influence of matrix effects from various water samples (millipore-, tap- and surface water). The detection limit for the analyte atrazine in PBS and surface water (SW) was 0.208 +/- 0.004 microg l(-1) (PBS) and 0.59 +/- 0.120 microg l(-1) (SW) for the FIIA and 0.033 +/- 0.003 microg l(-1) (PBS) and 0.038+/-0.003 microg l(-1) (SW) for the disc immunosensor. Statistical comparison of the two systems shows that the disc immunosensor results were significantly less influenced by the sample matrix, which is explained by the fact that the sample in the FIIA arrives simultaneously with the matrix to the detector, whereas these are separated in time in the disc immunosensor system.     

Amperometric immunosensor for nonylphenol determination based on peroxidase indicating reaction

Novel immunosensor for nonylphenol (NP) determination has been developed by immobilization of specific antibodies together with horseradish peroxidase on the surface of carbon screen-printed electrode. The signal of the immunosensor is generated by the involvement of NP accumulated in the peroxidase oxidation of mediator (Methylene Blue, hydroquinone or iodide). This results in the increase of the signal recorded by linear-sweep voltammetry. The sensitivity of the detection depends on the nature of mediator, its concentration and incubation period. Cross-selectivity of the response toward readily oxidized phenolic compounds has been determined. The immunosensor developed makes it possible to detect from 20 microgL(-1) to 44 mgL(-1) of NP with detection limit 10 microgL(-1) of NP.     

A highly sensitive flow-through amperometric immunosensor based on the Peroxidase chip and enzyme-channeling principle

A concept based on the Peroxidase-chip (P-chip), antibody co-immobilization, competitive and enzyme-channeling principle was exploited to develop an integrated flow-through amperometric biosensor for detection of environmental pollutants such as s-triazine herbicides. In this concept, recombinant peroxidase is immobilized on the gold electrode (P-chip) in such a way that direct electron transfer is achieved. The recognition and quantitation the target analyte is realized through the competition between the simazine-glucose oxidase (GOD) conjugate and free simazine for the binding sites of the monoclonal antibody co-immobilized with peroxidase on the gold electrode. The arrangement allows to generate a specific signal in the presence of glucose through the channeling of H2O2 produced by GOD conjugate bound to the antibody. The immunosensor exhibited 50% signal decrease (IC50 value) at approximately 0.02 microg l(-1). A concentration of 0.1 ng l(-1) gave a signal clearly distinguishable from the blank whereas the ELISA using the same antibody had a typical detection limit of about 1 microg l(-1), which is four orders of magnitude higher compared to the presented biosensor system. The results demonstrated that gene engineering biomolecules, in this case recombinant peroxidase, might be attractive reagents for the development of electrochemical immunosensors.     

基于“酶-邻氨基苯酚”体系的电化学免疫传感方法及其应用

本研究建立了一种新的电化学免疫传感方法,并将其应用于实际样本的检测。采用辣根过氧化物酶(horseradish peroxidase,HRP)的新型底物邻-氨基苯酚(O-aminophenol,O-AP)构建了基于"酶-邻氨基苯酚"体系的电化学免疫传感方法,并用于血吸虫抗体检测。邻-氨基苯酚在辣根过氧化物酶催化双氧水(H2O2)时被氧化,生成的产物3-氨基吩噁嗪能通过电化学方法检测。实验结果验证了基于"酶-邻氨基苯酚"的新型蛋白检测体系用于检测人血清中血吸虫抗体的可操作性,实现了实际样本的检测。并且,采用方波伏安法作为最终检测方法,具有更高的灵敏性。     

Magnetic beads-based electrochemical immunosensor for monitoring allergenic food proteins

Screen-printed platinum electrodes as transducer and magnetic beads as solid phase were combined to develop a particle-based electrochemical immunosensor for monitoring the serious food allergen ovalbumin. The standard arrangement of enzyme-linked immunosorbent assay became the basis for designing the immunosensor. A sandwich-type immunocomplex was formed between magnetic particles functionalized with specific anti-ovalbumin immunoglobulin G and captured ovalbumin molecules, and secondary anti-ovalbumin antibodies conjugated with the enzyme horseradish peroxidase were subsequently added as label tag. The electrochemical signal proportional to the enzymatic reaction of horseradish peroxidase during the reduction of hydrogen peroxide with thionine as electron mediator was measured by linear sweep voltammetry. The newly established method of ovalbumin detection exhibits high sensitivity suitable for quantification in the range of 11 to 222 nM and a detection limit of 5 nM. Magnetic beads-based assay format using external magnets for rapid and simple separation has been proven to be an excellent basis for electrochemical detection and quantification of food allergens in highly complex sample matrices.     

Electrochemical enzyme immunoassay for detection of toxic substances.

Sensors that provide reliable, rapid measurement of toxic substances are needed to solve significant human health and safety problems. We developed a new biosensor design that combines the advantages of immunoassay with electrochemical response. We established that this enzyme-linked immunosensor measures toxic substances in biological samples. The biosensor consists of two major elements: (1) an electrical conducting layer having immobilized enzyme, polyclonal or monoclonal antibodies, and other necessary reagents, and (2) the electronic components used in the signal readout. The result is an amperometric immunoassay based on coupling the immunochemical reaction to the enzyme electrode response by using a soluble, electrochemically active mediator. The specific question addressed was: Does the system's immunochemical detection reliably respond at sufficiently low analyte concentrations? We present our results in these areas: (1) enzyme immobilization on colloidal gold; (2) colloidal gold-enzyme deposition on the electrode surface; (3) mediator-antigen conjugate synthesis; (4) antibody incorporation at the electrode surface; (5) bioelectrode characterization and optimization; and (6) immunosensor demonstration to detect antigen. Sensors that employ immunochemical detection will have broad applicability to detect/diagnose toxic substances in biological samples such as blood and urine and in environmental samples such as wastewater and drinking water.     

Simultaneous trichromatic fluorescence detection of proteins on Western blots using an amine-reactive dye in combination with alkaline phosphatase- and horseradish peroxidase-antibody conjugates

Three-color fluorescence detection methods are described based upon covalently coupling the dye 2-methoxy-2,4-diphenyl-2(2H)-furanone (MDPF) to proteins immobilized on poly(vinylidene difluoride) (PVDF) membranes, followed by detection of target proteins using alkaline-phosphatase-conjugated reporter molecules in combination with the fluorogenic substrate 9H-(1,3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO-phosphate) as well as horseradish peroxidase-conjugated reporter molecules in combination with the new fluorogenic substrate Amplex Gold reagent. This results in all proteins in the profile being visualized as fluorescent blue signal, those detected specifically with the alkaline phosphatase conjugate appearing as fluorescent red signal and those detected specifically with the horseradish peroxidase conjugate appearing as fluorescent yellow signal. Using conventional secondary antibodies, two different targets may be identified as long as primary antibodies generated from two different species are used in the analysis. However, Zenon antibody labeling technology eliminates this restriction, permitting the simultaneous use of two different mouse monoclonal antibodies or two different rabbit polyclonal antibodies in the same electroblotting experiment. The trichromatic detection system is broadly compatible with UV epi-illuminators combined with photographic or charge-coupled device (CCD) cameras, and xenon-arc sources equipped with appropriate excitation/emission filters. Alternatively, the enzyme conjugates may be detected using a laser-based gel scanner. The trichromatic method permits detection of low nanogram amounts of protein and allows for unambiguous identification of two different target proteins relative to the entire protein profile on a single electroblot, precluding any requirement for running replicate gels that would otherwise require separate visualization of total proteins and subsequent alignment with multiple chemiluminescent or colorimetric signals generated on different electroblots.     

Modeling of immunosensors under nonequilibrium conditions. II. Experimental determination of performance characteristics.

In an attempt to optimize immunosensors operating with an immobilized antibody as binding protein and an analyte-enzyme conjugate as signal generator that is significantly larger in molecular size than the analyte, in a previous communication (Part I) (S.-H. Paek and W. Schramm (1991) Anal. Biochem. 196) we developed mathematical models for the prediction of performance characteristics. These models are compared in this contribution with experimentally obtained results. As an example, a monoclonal antibody to the steroid hormone progesterone has been used as binding protein, an 125I-progesterone derivative, and a progesterone-horseradish peroxidase derivative as tracers for signal generation. A minimum of parameters needs to be experimentally determined to calculate the performance: the amount of immobilized antibody, the diffusion coefficient of antigens, the thickness of the penetration layer, and the on- and off-rates for binding of the antigen to the antibody. We have described simple methods to obtain these data for the labeled antigen and for the unlabeled analyte that does not provide a signal per se. Kinetic binding curves for antigen-antibody complex formation obtained with the mathematical models correlated well with experimentally obtained results for antigens of different sizes. Although equilibrium of the antigen-antibody complex for the enzyme-labeled analyte conjugate requires about 4 h in the absence of free analyte, dose-response curves can be obtained after 5 min and the relative position of these curves does not change significantly after 30 min. Using a total volume of 200 microliters for the analytical procedure in microtiter wells, agitation as a means to accelerate convective diffusion during an incubation period of 30 min is not necessary with the analyte-enzyme conjugate. However, immunosensors using large analyte-enzyme conjugates as signal generators for the detection of small analytes require strict control of the incubation time if operated within short periods of time (less than 30 min).     

Development of a membrane strip immunosensor utilizing ruthenium as an electro-chemiluminescent signal generator

A photometric immunosensor that can be used for on-site diagnosis has been constructed. The sensor system was assembled by partially superimposing a nitrocellulose membrane strip (the lower) containing an immobilized antigen on the surface with a glass fiber membrane strip (the upper) including two electrodes on the opposite surfaces. To amplify the signal, we introduced a liposome, containing ruthenium molecules trapped in the core, chemically coupled to an antibody specific to the analyte (e.g. Legionella antigen). In the presence of the analyte, immune complexes were formed by antigen-antibody reactions upon addition of the immuno-liposome into a sample. This mixture was then absorbed by the capillary action from the bottom of the membrane strip. The liposome particles in the complexes were carried by a medium through the antigen pad without interaction, while free immuno-liposome was trapped by immune reactions on the pad surfaces. The aqueous medium influx into the glass pad dissolved a detergent pre-located within the compartment and the liposome rupture thereby released ruthenium molecules into the solution. The molecules were oxidized on the electrode surfaces and produced an electro-chemiluminescence (ECL) in proportion to the analyte concentration. The signal generation based on ECL resulted in an exponential dose-response pattern and the analyte detection limit of 2 ng/ml was approximately 10-fold more sensitive than that obtained from a conventional system.     

Double immunocytochemical staining in the study of antibody-producing cells in vivo. Simultaneous detection of cells producing monospecific antibodies and cells producing cross-reacting antibodies in the spleen of rabbits after injection of two related antigens

Rabbits were injected simultaneously with both human gamma globulin (HGG) and bovine gamma globulin (BGG). Sections of spleen tissue were prepared from spleen biopsies taken during the primary or secondary immune response, and incubated simultaneously with horseradish peroxidase (HRP)-HGG conjugate and alkaline phosphatase (AP)-BGG conjugate in order to detect cells containing specific antibodies against one or both of the antigens. After both HRP and AP cytochemistry, cells with a red-stained cytoplasm, cells with a blue-stained cytoplasm, and cells with a violet-stained cytoplasm were detected in the spleen. The red-stained cells had bound the HRP-HGG conjugate, indicating that these cells contained anti-HGG antibodies. The blue-stained cells had bound the AP-BGG conjugate, indicating that these cells contained anti-BGG antibodies. The violet-stained cells had obviously bound both the HRP-HGG conjugate and the AP-BGG conjugate, indicating that these cells contained antibodies cross-reacting with both antigens. Results are compared with earlier studies on the antigenic similarities and differences between HGG and BGG when used as antigens in rabbits.     

A disposable electrochemical immunosensor based on carbon screen-printed electrodes for the detection of prostate specific antigen

A novel screen-printed electrode (SPEs) on sheets of vegetable parchment was prepared. The obtained SPEs were stable, convenient, inexpensive and suitable for large-area screen-printing. With these SPEs, we explored the fabrication of a novel, disposable and highly sensitive electro-analytical immunosensor using graphene nanosheets (GS) and horseradish peroxidase (HRP)-labeled signal antibody functionalized with gold nanoparticles (HRP-Ab(2)/Au NPs). GS was used to increase the conductivity and stability of this immunosensor due to its fast electron transportation and good biocompatibility. Au NPs could not only provide a large surface area for the immobilization of HRP-Ab(2) but also enhance the electroreduction between HRP and H(2)O(2) to amplify the electrochemical signal on the sandwich immuno-complexes modified SPEs. The proposed SPEs were characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and electrochemical methods involving cyclic voltammetry (CV), and electrochemical impedence method. Using prostate specific antigen (PSA) as a model analyte, this immunosensor showed a wide linear range over 6 orders of magnitude with the minimum value down to 2pgmL(-1). In addition, this immunosensor could avoid the need of deoxygenation for the electrochemical immunoassay. Thus, it provided a promising potential in clinical applications.     

Core alpha1,3-fucose is a key part of the epitope recognized by antibodies reacting against plant N-linked oligosaccharides and is present in a wide variety of plant extracts

Carbohydrates have been suggested to account for some IgE cross- reactions between various plant, insect, and mollusk extracts, while some IgG antibodies have been successfully raised against plant glycoproteins. A rat monoclonal antibody raised against elderberry abscission tissue (YZ1/2.23) and rabbit polyclonal antiserum against horseradish peroxidase were screened for reactivity in enzyme-linked immunosorbent assay against a range of plant glycoproteins and extracts as well as neoglycoproteins, bee venom phospholipase, and several animal glycoproteins. Of the oligosaccharides tested, Man3XylFucGlcNAc2(MMXF3) derived from horseradish peroxidase was the most potent inhibitor of the reactivity of both YZ1/2.23 and anti- horseradish peroxidase to native horseradish peroxidase glycoprotein. The reactivity of YZ1/2. 23 and anti-horseradish peroxidase against Sophora japonica lectin was most inhibited by a neoglycoconjugate of bromelain glycopeptide cross-linked to bovine serum albumin, while the defucosylated form of this conjugate was inactive as an inhibitor. A wide range of plant extracts was found to react against YZ1/2.23 and anti-horseradish peroxidase, with particularly high reactivities recorded for grass pollen and nut extracts. All these reactivities were inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate. Bee venom phospholipase and whole bee venom reacted weakly with YZ1/2.23 but more strongly with anti-horseradish peroxidase in a manner inhibitable with the bromelain glycopeptide/bovine serum albumin conjugate, while hemocyanin from Helix pomatia reacted poorly with YZ1/2.23 but did react with anti-horseradish peroxidase. It is concluded that the alpha1, 3-fucose residue linked to the chitobiose core of plant glycoproteins is the most important residue in the epitope recognized by the two antibodies studied, but that the polyclonal anti-horseradish peroxidase antiserum also contains antibody populations that recognize the xylose linked to the core mannose of many plant and gastropod N-linked oligosaccharides.      

Evaluation of progesterone content in saliva using magnetic particle-based immuno supported liquid membrane assay (m-ISLMA)

Progesterone in saliva was monitored using a new method called magnetic particle-based immuno supported liquid membrane assay (m-ISLMA) in a sequential injection (SI) setup, allowing automatic sample cleanup, analyte enrichment, and detection in a single analysis unit. Progesterone (Ag) diffuses from a continuous flowing sample - the donor - into a supported organic liquid membrane (SLM), based on analyte partitioning (solubility) between the aqueous donor and the organic phase. The Ag is re-extracted from the SLM into a second stagnant aqueous acceptor, containing antibodies (Ab) immobilized on magnetic beads, held at the bottom of the acceptor by a magnet. Due to the formation of strong Ag-Ab-bead complexes and a large excess of Ab-beads, the Ag is accumulated and selectively enriched in the acceptor. The extracted progesterone was quantified by injecting into the acceptor a horseradish peroxidase (HRP) labeled analyte tracer, the substrate (luminol, H(2)O(2), and p-iodophenol), and finally detection of the generated chemiluminescence by a photomultiplier tube. After optimization of experimental parameters (e.g., sample flow rate, extraction time, type of organic solvent and antibody-bead concentration in the acceptor), a detection limit of 8.50+/-0.17 fgL(-1) and a dynamic range between 35 fgL(-1) and 10 pgL(-1) was reached. The progesterone level of saliva for three subjects (women in different period of ovarian cycle) was investigated, and the corresponding progesterone concentrations detected with m-ISLMA coincided well with the expected values.     

Primary enzyme quantitation using substrates labeled with a second indicator enzyme. I. Elastase determination using peroxidase-labeled elastin

Quantitation of proteolytic enzyme concentration can be accomplished by measuring the release, due to primary enzyme catalysis, of a second enzyme bound to a particulate substrate. As the primary enzyme acts on the substrate, release of the indicator enzyme into the surrounding medium occurs, which in turn can be quantitated colorimetrically, and under suitable reaction conditions the amount of indicator enzyme released is directly proportional to the amount of primary enzyme present. A specific example of such an assay is that for elastolytic activity using powdered elastin labeled with horseradish peroxidase. The detection sensitivity of the system described is 1 ng/ml of pancreatic elastase, and the dynamic range of the assay is 2 orders of magnitude. The reaction time for optimal elastase detection sensitivity is 3 h. For the assay, horseradish peroxidase is coupled to insoluble elastin. Labeled elastin is incubated with varying amounts of pancreatic elastase. The elastase in the test sample solubilizes the elastin and the horseradish peroxidase bound to it. The amount of peroxidase released is then quantified using the colorimetic reaction produced by catalysis of 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonate)-H₂O₂. For a fixed, nonsaturating concentration of elastase, the amount of peroxidase released is proportional to the elastase concentration.     

Peroxidase and fluorescein isothiocyanate as antibody markers. A quantitative comparison of two peroxidase conjugates prepared with glutaraldehyde or periodate anda fluorescein conjugate.

Batches of rabbit anti-human immunoglobulin G antibodies were labeled either with horseradish peroxidase, using the two-step glutaraldehyde method or the periodate method, or with fluorescein isothiocyanate (FITC). The peroxidase conjugates were isolated by chromatography using two different gel types. The five types of conjugates thus obtained were standardized to the same amount of rabbit immunoglobulin G. The antibody activity, as estimated by means of single radial immunodiffusion and passive hemagglutination, and the enzyme activity, determined with orthodianisidine, were compared. The ultimate dilutions and absolute amounts of the five conjugates giving positive reactions were determined in direct and indirect immunohistochemical tests, using both cryostat sections of skin and the agarose bead model system. It appeared that during the peroxidase conjugation procedures there was a considerable loss of abtibody and enzyme activity, whereas in the FITC conjugation procedure the antibody activity remained intact. Neverthe less, peroxidase conjugates prepared with glutaraldehyde still gave positive staining reactions in equal or somewhat higher dilutions than the fluorescin conjugate did. The peroxidase conjugates prepared with periodate could not be diluted to the same extent. For the detection of antibodies by indirect immunohistochemical methods, the peroxidase conjugate, prepared with glutaraldehyde, was comparable to the FITC conjugate. The peroxidase conjugate, prepared with periodate, was less effective.     

Regional distribution of prostaglandin endoperoxide synthase studied by enzyme-linked immunoassay using monoclonal antibodies

Prostaglandin endoperoxide synthase transforms arachidonic acid to prostaglandin H2 via prostaglandin G2. The enzyme purified from bovine vesicular gland was given to mice as antigen, and monoclonal antibodies were raised by the hybridoma technique. Two species of the monoclonal antibody recognizing different sites of the enzyme were utilized to establish a peroxidase-linked immunoassay of prostaglandin endoperoxide synthase. Fab' fragment of one of the antibodies was prepared and conjugated to horseradish peroxidase. The conjugate was then bound to prostaglandin endoperoxide synthase, and the labeled enzyme was precipitated by the addition of the other antibody. The peroxidase activity of the immunoprecipitate correlated linearly with the amount of prostaglandin endoperoxide synthase. This sensitive and convenient method to determine the enzyme amount rather than the enzyme activity was utilized to extensively screen the amount of prostaglandin endoperoxide synthase in various bovine tissues. In addition to vesicular gland, platelets and kidney medulla previously known as rich enzyme sources, the immunoenzymometric assay demonstrated a high content of the enzyme in various parts of alimentary tract and a low but significant amount of enzyme in some parts of brain.     

Proteolytic processing in a non-lysosomal compartment is required for transcytosis of protein-polylysine conjugates in cultured Madin-Darby canine kidney cells

The transcytosis of horseradish peroxidase, as well as its poly(L-lys) and poly(D-lys) thioether conjugates, was investigated in Strain I Madin-Darby canine kidney (MDCK) cell monolayers grown on 0.4 microns pore size polycarbonate membranes in Costar Transwells. The 3 types of HRP had almost identical rates of transport during the first 2 hr of incubation. However, a significant increase of basal-to-apical transport was detected beginning at 3 hr only in Transwells containing the poly(L-lys) conjugate. This increase was inhibited by colchicine (2 microM) and by the Bowman-Birk protease inhibitor (0.1 mg/ml), but not by NH4Cl (10 mM) or chloroquine (0.1 mM). The increase was abolished either by prior trypsinization of the conjugate or by incubation at 4 degrees C. Ultrafiltration studies indicated that the transcytosed poly(L-lys) conjugate was smaller in size than the original conjugate. These results indicate that the conjugate was processed during transcytosis in a non-lysosomal proteolytic compartment, where its poly(L-lys) moiety was selectively degraded, allowing active peroxidase to be released into the apical medium.