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Differentiated mammalian cell lines can be established by introducing viral oncogenes into primary cells. Such lines can retain their original specialised functions while being adapted to prolonged life in culture; but most transformed cell lines obtained in this way characteristically show altered properties compared with the primary cells. The result of these changes is that transformed cell lines no longer provide a good model of the original tissue, and indeed often resemble other transformed lines more than the initial cell type. In our laboratory three murine peritoneal macrophage-like cell lines have been isolated by transforming primary cells with SV40 origin-deleted DNA. These lines have been in continuous culture for approximately 1 year and have been shown to express many macrophage-specific properties throughout this time, including Fc receptors and staining for non-specific esterase. The cell lines phagocytosed IgG-coated particles, they were positive for the murine macrophage-specific marker F4/80 and they showed antigen-presentation function. Lysozyme, acid phosphatase, plasminogen activator, collagenase, prostaglandin E2 and 5'-nucleotidase activities have also been detected in these lines. In this paper the method of DNA transformation will be described as well as some of the assays used for the characterization of the three immortalized cell lines.

Members of the mononuclear phagocyte system use their extensive repertoire of cell surface receptors to interact with their external environment. A number of different assays are available for the study of these molecules and their many functions. In this paper we describe how monoclonal antibodies may be generated against macrophage molecules, and discuss how screening strategies aimed at producing functionally active reagents may be devised. Using the macrophage mannose and scavenger receptors as examples, we describe assays for determining macrophage adhesion to culture plastic surfaces, endocytosis of soluble ligands and phagocytosis of particles in vitro. These assays may be used in the production of inhibitory monoclonal antibodies, as well as in studies of the regulation of macrophage phenotype and activity.

This paper reviews the main immunohistochemical techniques that can be used for studying mononuclear phagocytes in tissue sections.

This paper describes a defined serum-free medium (SFM), designated A-X, which supports in vitro proliferation of human primary T cells stimulated with mitogens, two-way mixed leukocyte reactions, anti-CD3, and a superantigen. A-X is a 1:1 mixture of two commercially available SFM, AIM V and EX-CELL 300. In each assay tested it supported uptake of [3H]thymidine as well as or better than the standard culture medium, namely RPMI 1640 supplemented with 10% fetal calf serum. A-X also allowed the detection of interleukin-2 by ELISA at levels comparable to those produced in serum-supplemented medium. A-X will likely be useful in further studies as it eliminates many of the problems usually associated with the use of serum-supplemented media.

The technique of V region PCR to clone antibody V regions from hybridomas has been extensively used. However, in addition to, or even instead of, cloning the V regions with the desired specificity, myeloma cell derived V regions, V regions which are the result of non-productive rearrangements, and also V regions which are productive but which do not recognise the antigen of interest, may be isolated. In this paper we describe a comparison of the use of V region PCR and a modification of the RACE technique to clone the V region of the anti-NGF hybridoma, alpha D11. This hybridoma has heavy and light chain V regions which are refractory to amplification with V region primers, but which are easily amplified using RACE, a PCR based procedure which is independent of the variability within the V regions.

alpha-Naphthyl acetate esterase (alpha-NAE) is primarily found in mononuclear phagocytes and may be used to distinguish them from other leucocytes. Conventional cytochemical techniques are subjective and may be difficult to interpret, especially with cells which express only low levels of activity. This has caused difficulties in the classification of non-lymphoblastic leukaemias. This paper describes the adaptation of a cytochemical assay for use with the flow cytometer. The alpha-NAE activity of peripheral blood mononuclear cells was examined and found to be associated with the expression of the surface antigen CD14. The reaction could be inhibited by sodium fluoride. A series of human cell lines were also compared for alpha-NAE activity. Distinct differences in staining observed between the cell lines correlated with the number of cell-associated granules observed under the microscope.

Most therapeutic proteins induce an unwanted immune response. Antibodies elicited by these therapeutic proteins may significantly alter drug safety and efficacy, highlighting the need for the strategic assessment of immunogenicity at various stages of clinical development. Immunogenicity testing is generally conducted by a multi-tiered approach whereby patient samples are initially screened for the presence of anti-drug antibodies in a screening assay. The screening assay cut point is statistically determined by evaluation of drug-naive samples and is typically chosen to correspond to a false positive rate of 5%. While various statistical approaches for determination of this screening cut point have been commonly adopted and described in the immunogenicity literature, the performance of these approaches has not been fully evaluated. This paper reviews various statistical approaches for cut point calculation, evaluates the impact of sampling design and variability on the performance of each statistical approach, and highlights the difference between an 'average' or 'confidence-level' cut point in order to develop more specific recommendations regarding the statistical calculation of immunogenicity screening cut points.

Many research projects in cell biology now use flow cytometry for analysis or for isolation of specific cell types. In such studies, cell viability is obviously a crucial issue. However, many studies appear to rely upon light-scattering characteristics to identify and gate out non-viable cells, despite the fact that reliable identification of such cells can only be achieved through staining with impermeable fluorescent nuclear dyes such as propidium iodide or 7-amino actinomycin. In this paper we apply mathematical analysis to the theoretical problem of quantifying cell sub-populations labeled with two or more fluorescent markers, comparing situations in which dead cells have been identified with those in which cell viability has not been assessed. We demonstrate that in all cases in which dead cells are present within the population, percentages of live sub-populations in different subsets are mis-estimated. In cases where the pattern of marker expression differs greatly between live and dead cells, or where the proportion of dead cells is high, this mis-estimation will be aggravated; the subsets pattern will therefore be biased in a population selected only on the basis of light-scatter behavior. The importance of accurately detecting and gating out dead cells is illustrated by an experimental example accompanying the mathematical analysis. To conclude, identification of dead cells by means of viability stains should be an absolute routine in practical flow cytometry, so as to avoid mis-estimation in sorting or analysis.

This paper describes a colorimetric assay for the rapid quantification of chemotaxis in multiple samples. In this assay, cells that have migrated through polycarbonate membrane filters are collected onto the bottom wells of a chemotaxis chamber after centrifugation then the number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT). The degree of MTT reduction, which corresponds to the relative cell number, is measured automatically with an ELISA reader. The MTT method of quantitation is as sensitive as the standard manual method, is especially useful for large numbers of samples and requires no specialized laboratory equipment.

Locomotion of lymphocytes and other leukocytes is an essential feature of the immune system, and therefore the evaluation of the locomotor behaviour of a lymphocyte population is part of its functional analysis. Paradoxically, the locomotor status of leukocytes is usually assessed on the basis of static information, by counting the number of spherical versus non-spherical cells. In this paper we describe two methods for the measurement of shape changes in microscopic images of lymphoid cells. First we computed a simple shape change factor, coined incongruence factor, based on the degree of non-overlap of the contours of the cell at the beginning and at the end of a 1 min time interval. Second we have used Fourier analysis of the cell outline: a function describing the undulations of the cell outline is broken down into sinusoidal ‘waves’ of increasing frequency, each with its corresponding amplitude. The amplitude values for the first ten frequencies produced a satisfactory mathematical description of lymphoid cell shapes, and the change of these amplitudes over a 1 minute time interval produced a quantitative description of the shape alterations of the cells. We have used five approaches to evaluate the shape and shape changes in the following populations of mouse lymphoma cells: a constitutively low-motile T lymphoma cell line (BW5147), a high-motile hybridoma (BW-O-Li1) either on plastic or on a precultured fibroblast-like monolayer, BW-O-Li1 cells after penetration through the monolayer, and BW-O-Li1 cells after treatment with cytochalasin B. We compare the results from direct visual evaluation of cell shape, from computer assisted assessment of sphericity and from Fourier analysis of cell shape at one moment, with the two methods for quantitative shape change analysis. All approaches revealed a clear distinction between spherical low-motile populations, and non-spherical high-motile cells. Moreover, the incongruence factor proved to be a reliable single parameter of active cell deformation. In addition, the Fourier analysis of cell outlines produced useful measures of static shape and of dynamic shape change, at any user-defined level of accuracy.

This paper describes the development of quantitative immunoassays utilizing mouse monoclonal antibodies which are monospecific to type II collagen. The monoclonal antibodies were characterized and tested extensively for reactivity against a panel of antigens including actin, myoglobin, thyroglobulin, ssDNA, tetanus toxin and different types of collagens including their CnBr-derived peptides. Four monoclonal antibodies having strict monospecificity to type II collagen were selected. Quantitative immunoassays developed with these antibodies can measure either type II collagen in its native conformation or type II collagen-derived cyanogen bromide peptides. The assay conditions such as coating concentration of antigen, monoclonal antibody concentration, second antibody concentration and incubation times were optimized to obtain maximum possible sensitivity. These quantitative immunoassays can be employed to measure type II collagen or type II collagen-derived peptides in low amounts ranging from 20 to 100 ng/ml. The assays can be applied to chondrocyte cultures without interference from serum components or other collagen types.

In order to induce an optimal plaque-forming cell response to sheep red blood cells (anti-SRBC PFC response) in vitro, murine splenocytes have been cultured in RPMI 1640 medium containing 10% fetal calf serum (FCS). In this paper, we report that 10% intact FCS in such a conventional medium can be replaced by 2% modified FCS which has been exposed to insoluble polymer beta-cyclodextrin (CD). The most efficient procedure of treatment of FCS was an incubation of 1/50 diluted FCS with 20 mg/ml polymer beta-CD for 3.5 h at 20 degrees C. The PFC response supported by 2% polymer beta-CD-treated FCS, was the same as that obtained when mixing 10% FCS, and was completely dependent on the existence of antigen (SRBC) and enhanced by 2-mercaptoethanol. The time-course profiles of the PFC responses induced in both cultures were almost the same. The 2% polymer beta-CD-treated FCS-containing medium was also effective in inducing both a primary PFC response to the T cell-independent antigen (trinitrophenyl-lipopolysaccharide) and a polyclonal PFC response stimulated by lipopolysaccharide. Inactive FCS lots (so called deficient lots) also became fully supportive at 2% after treatment with polymer beta-CD. These results indicate that polymer beta-CD-treated FCS is not economical for supporting antibody production, but also useful for those experiments on isolation and analysis of factors derived from cultured lymphocytes.

This review paper describes a new technology, mass cytometry, that addresses applications typically run by flow cytometer analyzers, but extends the capability to highly multiparametric analysis. The detection technology is based on atomic mass spectrometry. It offers quantitation, specificity and dynamic range of mass spectrometry in a format that is familiar to flow cytometry practitioners. The mass cytometer does not require compensation, allowing the application of statistical techniques; this has been impossible given the constraints of fluorescence noise with traditional cytometry instruments. Instead of "colors" the mass cytometer "reads" the stable isotope tags attached to antibodies using metal-chelating labeling reagents. Because there are many available stable isotopes, and the mass spectrometer provides exquisite resolution between detection channels, many parameters can be measured as easily as one. For example, in a single tube the technique allows for the ready detection and characterization of the major cell subsets in blood or bone marrow. Here we describe mass cytometric immunophenotyping of human leukemia cell lines and leukemia patient samples, differential cell analysis of normal peripheral and umbilical cord blood; intracellular protein identification and metal-encoded bead arrays.

Interleukin-1 (IL-1) is an important mediator in inflammation and immunological processes. The findings of native IL-1 inhibitors suggest a negative feedback mechanism to down-regulate IL-1 mediated acute inflammation. IL-1 inhibitors were also found elevated in disease states associated with high IL-1 levels. We have previously described one such IL-1 inhibitor derived from the human M20 myelomonocytic cell line. In this paper we present several biological and biochemical characteristics of the M20 IL-1 inhibitor. Various in vitro activities of the inhibitor are described and its IL-1 specificity in these assays is demonstrated. Purification of the inhibitor was performed by DEAE-high performance liquid chromatography, isoelectric focusing, gel filtration and dye ligand chromatography column. This protein factor has a MW of 52 ± 4 kDa and a pI of 4.15 ± 0.1. The inhibitor has no cross-reactivity against a panel of known cytokines (IL-1α, IL-1β, IL-2, sIL-2R, IL-6, tumor necrosis factor (TNF), interferon-γ (IFN-γ)) and is distinct from the IL-1 receptor antagonist (IL-1ra). The purified IL-1 inhibitor was destroyed by trypsin, 2-mercaptoethanol, sodium dodecyl sulfate and extremes in pH and in temperature. Only IL-1 induced (but not the IL-2, IL-6, or TNF induced) thymocyte proliferation and PGE2 production by fibroblasts were inhibited by the inhibitor, thus showing specifity to IL-1 in these assays.

Heterogeneous immunoassays require wash steps in order to separate bound from free constituents. In this paper we demonstrate that in microplate assays the washing process includes two separate physical processes: (1) a rapid and wash volume-dependent direct dilution of the droplet-shaped residual volume, and (2) a diffusion-limited and strongly time-dependent dilution of a residual layer of liquid, which necessitates the use of time-consuming soak times in the immunoassay. We have shown that optimizing the motion of the wash fluid effectively reduces the residual layer thickness that results in extended soak times. This results not only in improved washing efficiency and reduced background variance in the immunoassay, it also yields a significantly improved immunoassay sensitivity.

The stability and recovery of six human recombinant cytokines (tumour necrosis factor (TNF), interferon-alpha (IFN-alpha), IFN-gamma, interleukin-1 alpha (IL-1 alpha), IL-1 beta, and IL-6) from whole blood was investigated with a view to optimizing blood collection and storage procedures prior to performing immunoassays. Blood from healthy volunteers was subjected to various processing and storage procedures. Blood samples were treated with either: ethylenediamine tetraacetic acid (EDTA) (1.5 mg/ml blood) (E); EDTA/Trasylol (1.5 mg and 1000 KIU/ml blood) (ET); heparin (30 IU/ml) (H) or allowed to clot (serum). The bloods were spiked with individual cytokines, split into aliquots and kept at 4 degrees C or RT. In the first instance spiked bloods from healthy volunteers (n = 5 per cytokine) were processed using sterile and non-pyrogenic materials and procedures. At regular time intervals, samples were cold spun, separated, flash frozen and assayed for the appropriate cytokine using RIA/IRMA methods. In a further study, timed separation was repeated with spiked blood from healthy volunteers (n = 5 per cytokine) using normal commercially available blood collection materials and procedures. In a third study, spiked blood from healthy volunteers (n = 3 per cytokine) was processed under sterile and non-pyrogenic conditions, and the blood samples separated, aliquoted and flash frozen within half hour of collection. These were then subjected to repeated cycles of freeze thawing at 4 degrees C or RT before assaying. In general, the stability of cytokines in whole blood was improved by storage at 4 degrees C and/or rapid separation. There was no significant difference between samples handled under sterile, non-pyrogenic conditions and those collected using normal blood collection procedures. The blood collection procedures described in this paper did not induce any of the six cytokines in the unspiked blood. Overall, EDTA-treated samples performed most consistently. The addition of trasylol did not significantly affect the results. Most of the cytokines appeared unaffected by up to three freeze thaw cycles. The stability and recovery of the spiked cytokines varied from least stable to most stable spiked cytokine as follows; TNF-alpha less than IL-6 less than IFN-gamma less than IL-1 alpha less than IFN-alpha less than IL-1 beta. The recovery of spiked IFN-gamma from heparinized plasma samples was considerably higher than any other plasma or serum samples. The recovery of spiked TNF-alpha and IL-6 from serum samples was consistently lower than amounts recovered from plasma samples (anticoagulant treated).(ABSTRACT TRUNCATED AT 400 WORDS)

We evaluated an adjuvant, TiterMax, as an alternative to complete Freund's adjuvant (CFA) for producing antisera in animals. TiterMax, consists of a microparticulate stabilized water-in-oil emulsion of a metabolizable oil, squalene, with the adjuvant block copolymer CRL89-41. This paper reports two evaluations of TiterMax versus CFA and other commercially available adjuvants. In the first study, mice were immunized with a hapten, trinitrophenol, conjugated to hen egg albumin (TNP-HEA) in one of several adjuvants: TiterMax, CFA, Adjuvax, Ribi adjuvant system (RAS), Alhydrogel or Lipovant. TiterMax induced higher longer lasting titers with fewer injections than any of the other adjuvants. The magnitude of the response to TNP varied with species and route of immunization. In the second study, CFA, TiterMax, Adjuvax and RAS were compared in rabbits, mice and goats. Animals were immunized with luteinizing hormone-releasing hormone (LHRH) conjugated to BSA in each adjuvant using comparable protocols. TiterMax induced titers against the peptide equivalent to CFA in all three species. The inflammatory responses induced by TiterMax were mild and transient compared with those induced by CFA. These data suggest that TiterMax is an effective alternative to CFA in many situations.

To detect and compare the capacity of antigen presenting cells to present antigen in a T cell proliferation assay, it is necessary to obtain a pure population of antigen-primed T cells that gives low background proliferative responses. Therefore in this paper we present a newly developed isolation method of antigen-primed T lymphocytes from rat spleen or lymph nodes. This method uses a nylon wool column to deplete most of the adherent cells and B cells, followed by an indirect elimination method with magnetic beads to remove contaminating Ia-positive cells. We compared this method with two commonly used isolation methods, namely a 1.5 h adherence step, followed by a nylon wool column and a Sephadex G-10 column and a 1.5 h adherence step followed by a passage through two consecutive columns of Sephadex G-10. The best T cell enrichment (98% OX-19/52-positive cells) was achieved with the newly developed method, in which the contamination of Ia-positive cells, predominantly B cells and dendritic cells (DC), was diminished to less than 2%. The background response of this population was low and differed significantly with the common methods. Antigen-specific T cell responses induced by splenic DC, expressed as stimulation index, gave very specific responses and showed a steep rise with increasing DC concentrations compared to the common methods. Therefore we conclude that we developed an improved, rapid and reproducible method for the isolation of rat spleen or lymph node T lymphocytes suitable for T cell proliferation assays.

The purification of type II collagen, for the detection of anti-type II collagen antibodies by ELISA procedures, involves removal of proteoglycans by guanidine-HCl, followed by pepsin solubilisation and salt fractionation. However, type II collagen purified in this way may contain contaminants, despite the apparent purity on SDS-polyacrylamide gels. In this paper we demonstrate how additional purification by DEAE chromatography reduces the degree of background binding in the type II collagen ELISA, leading to an increase in disease specificity. The contaminants included proteoglycan and bound serum IgG from both rheumatoid arthritis (RA) patients and healthy controls in ELISA. Furthermore, positive correlations were observed in the sera (n = 24) between degree of reactivity to the contaminants and to (1) purified proteoglycan (r = 0.50, P = 0.01) and (2) pepsin (r = 0.65, P = 0.001). Thus, inadequate purification of type II collagen produces false positive reactions in the collagen ELISA and gives rise to a high background. A lack of specificity has been frequently associated with this assay.

The paper by Herzyk et al. which appears in this issue of the Journal of Immunological Methods is an important and long overdue contribution. The study clearly and convincingly demonstrates that two widely used commercial ELISA kits fail to detect over 90% of the 31 kDa human IL-1 beta precursor (pro-IL-1 beta) in various cells. This is due to the specificity of the monoclonal antibodies raised to the mature 17 kDa IL-1 beta and used in the commercial kits (or in 'home made' ELISAs). The significant amount of pro-IL-1 beta which is not measured by the kits casts doubt on the accuracy of total IL-1 beta synthesis reported in hundreds of published studies. Investigators evaluating cellular synthesis of IL-1 in the development of new drugs or IL-1 levels in health and disease increasingly rely on commercial detection kits. In this commentary, the importance and methods for determining total IL-1 beta synthesis are reviewed. In view of the preliminary positive results reported on the success of the IL-1 receptor antagonist (IL-1ra) in clinical trials, attention should be focused on the amount of IL-1 produced in disease and on the balance of IL-1 versus IL-1ra synthesis. Thus, continued use of these ELISA kits which do not detect pro-IL-1 beta will confound the interpretation of the IL-1 beta determinations.

Colloidal metal particles, in particular gold, have found many biological applications often as probes in light and electron microscopy, and more recently since the 1980s in membrane-based rapid immunoaffinity tests. The surface plasmon resonance absorbance properties in the visible spectroscopy region of gold colloids make them useful tools in medical devices, as the colloids are directly visible to the naked eye. Despite the relative ease with which gold-protein conjugates can be prepared a major issue is the manufacture of poor-quality and poorly characterised bioconjugates that can result in the under performance of subsequent diagnostic tests. This paper describes the preparation of good-quality conjugates for use in immunoassays by optimising the adsorption of antibodies onto the surface of gold colloids, followed by their subsequent characterisation. The conjugates were characterized for size, aggregation and quality using a range of techniques: UV–visible (UV/Vis) absorption spectroscopy, transmission electron microscopy (TEM) and dynamic light scattering (DLS). The biological activities of the conjugated products were also assessed using an immunoassay format and electrochemical measurements. By utilising a number of measurement techniques we aimed to gain a better understanding of the extent of particle aggregation, and the resulting stability and activity of the biological molecule on the surfaces of nanoparticles. The tools developed will enable researchers and companies to ensure the sensitivity, quality and reproducibility of batches of nanoparticle bio-conjugates.

The answer to many fundamental questions in Immunology requires the quantitative characterization of the T-cell repertoire, namely T cell receptor (TCR) diversity and clonal size distribution. An increasing number of repertoire studies are based on sequencing of the TCR variable regions in T-cell samples from which one tries to estimate the diversity of the original T-cell populations. Hitherto, estimation of TCR diversity was tackled either by a "standard" method that assumes a homogeneous clonal size distribution, or by non-parametric methods, such as the abundance-coverage and incidence-coverage estimators. However, both methods show caveats. On the one hand, the samples exhibit clonal size distributions with heavy right tails, a feature that is incompatible with the assumption of an equal frequency of every TCR sequence in the repertoire. Thus, this "standard" method produces inaccurate estimates. On the other hand, non-parametric estimators are robust in a wide range of situations, but per se provide no information about the clonal size distribution. This paper redeploys Poisson abundance models from Ecology to overcome the limitations of the above inferential procedures. These models assume that each TCR variant is sampled according to a Poisson distribution with a specific sampling rate, itself varying according to some Exponential, Gamma, or Lognormal distribution, or still an appropriate mixture of Exponential distributions. With these models, one can estimate the clonal size distribution in addition to TCR diversity of the repertoire. A procedure is suggested to evaluate robustness of diversity estimates with respect to the most abundant sampled TCR sequences. For illustrative purposes, previously published data on mice with limited TCR diversity are analyzed. Two of the presented models are more consistent with the data and give the most robust TCR diversity estimates. They suggest that clonal sizes follow either a Lognormal or an appropriate mixture of Exponential distributions. According to the ecological interpretation of these models, the T-cell repertoire would be divided in several T-cell niches, themselves created in a series of steps. Definitive conclusions, however, would require larger samples. It is shown here that samples 100-fold larger than hitherto available ones would be sufficient to discriminate candidate models. These large sample sizes are currently affordable using massively parallel sequencing technology. Foreseeing this we provide the package PAM for the R software that will facilitate T-cell repertoire data analysis based on Poisson abundance models.

This paper describes the DNA immobilization using carbon multi-walled nanotubes (MWCNTs) for direct and label-free detection of influenza virus (type A). The DNA probe was attached on the sensor surface by means of covalent bonding between the amine and phosphate groups of the DNA sequence. The interaction between the DNA probe and the MWCNTs were characterized by Fourier Transform Infrared (FTIR) spectrometry, Raman spectra. The hybridization of the DNA probe and the target DNA were detected by changes in the conductance on the surface of sensors leading to the change in the output signal of the system. The results show that the DNA sensor can detect as low as 0.5 nM of the target DNA samples; the response time of DNA sensor is approximately 4 min.

Antigen presenting molecules play an important role in both innate and adoptive immune responses by priming and activating T cells. Among them, CD1 molecules have been identified to present both exogenous and endogenous lipid antigens to CD1-restricted T cells. The involvement of CD1-restricted T cells in autoimmune diseases and in defense against infectious diseases, however, remains largely unknown. Identifying novel antigenic lipids that bind to CD1 molecules and understanding the role of CD1-restricted T cells should lead to the successful development of vaccines, because the lipids can be used as antigens and also as adjuvants. In this paper, we have constructed functional recombinant human CD1 dimeric proteins and established a competitive ELISA assay to measure the lipid binding to CD1 molecules using the CD1 dimers. By using the competitive ELISA assay, we were able to show that the lipid extracts from murine malaria parasites can actually be loaded onto CD1 molecules. In addition, we have demonstrated that artificial antigen-presenting cells, which consist of magnetic beads coated with CD1d dimer and anti-CD28 antibody, stimulated and expanded human invariant NKT cells as efficiently as autologous immature DCs. A set of the tools presented in the current study should be valuable for screening various CD1 molecule-binding lipid antigens and for isolating CD1-restricted T cells.

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-β-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to β-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.

In vitro differentiation of human monocytes (Mo) provides large amounts of mature and functionally competent macrophages (M phi) which may be used as potentially powerful anticancer agents for adoptive immunotherapy. Granulocyte macrophage-colony stimulating factor (GM-CSF) was evaluated for its ability to influence long term cultures of Mo-derived M phi. Large quantities of Mo isolated by leukapheresis and elutriation were cultured in non-adherent cell culture bags or in plastic flasks with or without GM-CSF. At various stages of differentiation, GM-CSF treated M phi were recovered and assayed for survival, morphology, surface antigens, functional properties and proliferation in comparison with control M phi. In the present paper, we demonstrate that GM-CSF at a concentration of 50 U/ml (5 ng/ml) promotes better cell survival and the differentiation of Mo into M phi displaying certain morphological differences as compared to control M phi such as an increased expression of Max-1 antigen, CR3 and Fc gamma II receptors, higher phagocytic properties and increased capacities of cytotoxicity and TNF secretion when the cells are further activated by IFN-gamma. Furthermore, GM-CSF treated cells exhibit a low-grade proliferation although the nature of the proliferating cells has not been entirely elucidated. We conclude that the GM-CSF treated M phi would be particularly suitable for adoptive immunotherapy.

Immunomagnetic cell separation uses binding of an antibody to its epitope to identify the target cell, which is then removed by attachment to an anti-immunoglobulin-coated paramagnetic bead, and passage through a magnetic field. This method has previously been shown to be less sensitive to the effects of low target antigen density than are other cell elimination methods, such as complement-mediated lysis. In this paper we demonstrate that, with certain antibody/target cell combinations, the efficiency of immunomagnetic depletion can be adversely affected by high expression of the target antigen. This can occur by two non-mutually exclusive mechanisms. These are (i) steric hindrance of bead binding due to crowding of monoclonal antibodies on the cell surface; and (ii) binding of the monoclonal antibody molecule in a configuration that is poorly-accessible to the anti-immunoglobulin immobilized on the microspheres. The predominant effect operating in any system can be determined by analysis of the cells remaining after the separation procedure. In both cases pre-attachment of the monoclonal to the beads results in improved separation efficiency. These results emphasize the necessity of optimizing experimental conditions in each system that is investigated.

Anti-idiotypic antibodies are powerful reagents for the study of immunoregulation, and have potential interest as vaccines against tumors and infectious diseases. Three immunization strategies for the production of rat monoclonal anti-idiotope antibodies have been compared in this paper. Male Wistar rats were immunized i.p. and at multiple subcutaneous sites with 750 micrograms of purified monoclonal antibody against Plasmodium falciparum for three times and subsequently boosted by (1) intraperitoneal injection with 750 micrograms of the immunogen, (2) intravenous inoculation with 400 micrograms of the IgG, and (3) intrasplenic immunization with 200 micrograms of the idiotype. With the intraperitoneal boost method, the frequency of hybrids with anti-idiotope activity was 0.3-0.9% with 62.8-85.2% of the seeded wells containing hybrids. In the intravenous boost group, the percentage of hybrids demonstrating anti-idiotope activity increased to 11.0-13.3% with 80.2-97.9% of the hybrid efficiency. When immunized by the intrasplenic boost route, the frequency of anti-idiotope hybrids generated rose to 12.9-16.4% with 82.3-96.6% of the hybrid efficiency. There was no obvious effect of the boost immunizing methods on the generation of rat monoclonal anti-mouse IgG antibodies. These results indicated that the multiple-site immunization followed by intravenous or intrasplenic boost injection was an appropriate immunizing method for the production of monoclonal anti-idiotope antibodies.

Several protocols for conjugating peptides in situ to a protein carrier on paper, nitrocellulose, or nylon membranes were explored for their usefulness in dot-ELISA detection of the peptides. The most sensitive method in which peptide diluted in bovine serum albumin is applied to nitrocellulose, then fixed with glutaraldehyde, can detect several peptides, ranging from 4 to 38 amino acids in length, at the level of 2–10 fmol. Both immunohistochemical grade antisera and monoclonal antibodies have been used successfully. The method may be a useful alternative to radioimmunoassay since there is no requirement for radiolabelled peptide, or (for quantitation) for known quantities of unlabelled peptide. The method has been used to monitor, semiquantitatively, the fractionation of FMRFamide-like or CCK-like peptides from the nematode Ascaris, and to detect peptide-like immunoreactivities in tissue extracts.

In a previous publication (Bobrow et al., J. Immunol. Methods (1989) 279-285), we described a novel signal amplification method, catalyzed reporter deposition (CARD), and its application to microplate immunoassays. The method utilizes the analyte-dependent reporter enzyme (ADRE) to catalyze the deposition of additional reporter onto the surface of a solid-phase immunoassay system. In this paper, we describe the utilization of CARD amplification for nonradiometric membrane assays where detection is facilitated by the formation of an insoluble chromogenic product. In the examples described, deposition of reporter is accomplished in two steps: (i) a horseradish peroxidase (HRP) ADRE catalyzes the deposition of either a biotin or fluorescein labeled phenol, and (ii) incubation with either enzyme labeled streptavidin or anti-fluorescein, respectively, results in the deposition of additional enzyme. Using this method, we have improved detection limits from 8- to greater than 200-fold depending on the amplification format and the chromogen used.

Apoptosis, i.e., programmed cell death, may be the mechanism by which both autoreactive and unselected immature CD4+8+ thymocytes are eliminated in the thymus. In the present paper we describe a simple and rapid flow cytometric method which permits one to study the induction and kinetics of apoptosis of CD4+8+ thymocytes using in vivo and in vitro suspension cultures. Analysing the level of surface expression of CD4 and CD8 molecules, forward light scatter and side (90 degrees) scatter as well as staining with ethidium bromide, three distinct stages of apoptosis of CD4+8+ thymocytes were defined. By counting cells passing through different stages of apoptosis one can attempt to quantitate this process. This method should be useful for in vitro studies on the mechanisms of negative and positive selection of CD4+8+ thymocytes, i.e., induction and inhibition of apoptosis respectively.

This report describes a modified dot immunobinding assay (DIA) in microplates using a crude mixture of non-purified antigen. Nitrocellulose filter paper discs exposed to the antigen mixture were inserted into the wells and kept in place by a specially constructed device. To test the efficiency of the modification a set of monoclonal antibodies from a mouse immunized with 58 kDa trpE-Bmyc fusion protein were screened. The advantage of this modified method over conventional ELISA is that it permits the use of non-purified antigen for screening large numbers of monoclonal antibodies.

Feasibility, amount of sample aliquots, processing time and cost are critical considerations for optimizing and conducting assays for large-population based studies. Well designed statistical approaches that quickly identify optimal conditions for a given assay could assist efficient completion of the laboratory assays for such studies. For example, assessment of the profile of secreted cytokines is important in understanding the immune response after vaccination. To characterize the cytokine immune response following smallpox vaccination, PBMC obtained from recently vaccinated subjects were stimulated with varying doses of live or UV-inactivated vaccinia virus and cultured for up to 8 days. In this paper, we describe a novel statistical method to identify optimal operating conditions for length in culture and virus MOI in order to measure a panel of secreted Th1, Th2, and inflammatory cytokines. This statistical method is comprised of two components. It first identifies a subset of the possible time in culture by virus MOI combinations to be studied. It then utilizes response surface analysis techniques to predict the optimal operating conditions for the measurement of each secreted cytokine. This method was applied, and the predicted optimal combinations of length in culture and virus MOI for maximum vaccinia-specific cytokine secretion were identified. The use of the response surface methodology can be applied to the optimization of other laboratory assays; especially when the number of PBMC available limits the testing of all possible combinations of parameters.

This paper reports a method for improving the effectiveness of aggregate removal from monoclonal antibodies by the use of hydroxyapatite (HA) in the presence of polyethylene glycol (PEG). PEG preferentially enhances aggregate retention, thereby increasing the degree of separation between aggregated and non-aggregated antibodies. PEG has a similar effect on ion exchangers but only half the enhancement observed with HA. The HA method is suitable for preparation of aggregate-free IgG and IgM monoclonal antibodies for research, diagnostic, or therapeutic applications.

Quantitative determination of staphylococcal protein A in plasma is often hampered by the interaction between protein A and immunoglobulins. In human plasma, these interactions may not only involve the non-immune binding to the Fc or Fab regions of Ig but also antigen/antibody interaction by specific antibodies directed against protein A. In this paper we describe a method which can be used to quantitate nanogram amounts of protein A in the presence of human plasma. The ELISA used for the quantification of protein A is based on a double antibody solid-phase assay utilizing chicken anti-protein A as both capture and detector antibody. Protein A may be measured down to 5 ng/ml in plasma and 0.5 ng/ml in buffer. The plasma samples were heat-inactivated before analysis and this eliminated interference in the assay caused by interaction of protein A with an excess of immunoglobulins. This assay provides a reliable and convenient method for the detection and quantitation of soluble protein A in human plasma.

Natural killer cells can phenotypically be identified as CD16 positive witha specific monoclonal antibody (B73.1 = Leu-11c) by either immunofluorescence microscopy or by flow cytometry. The standard procedure in flow cytometry is to set a window or gate around the so called lymphocytic population, based on scatter characteristics. In this paper we demonstrate that a substantial part of the NK cell population is situated outside this gate in the total mononuclear cell population. We therefore recommend that the number of CD16+ cells is determined in the total mononuclear cell population. However, in the total mononuclear cell population, a group of dimly CD16 positive cells, probably monocytes, interferes with a clear separation of cells with a positive and negative fluorescence. We describe two methods to overcome this problem.

This paper deals with a new, functional assay of cobra venom factor (CVF) preparations with or without C5-activating property. Existing methods lack sensitivity and use diluted human complement as target of inactivation. An adapted assay using diluted mouse serum as complement source was hampered by underestimation of C3 depletion by bystander lysis and an overvaluation of C5 consumption resulting from C3 inactivation in the reagent used. These disadvantages prompted us to develop the new assay which is based on the incubation of CVF preparations with undiluted mouse serum. After incubation, residual total C activity, as well as functional C3 and C5 are estimated by titration. The procedure permits the assessment of CVF activities with minimal interference from undesired processes. The conditions in the new assay approach the in vivo situation in mice by the use of undiluted serum from the same animal species.

Recruitment of circulating lymphomyeloid cells in the liver during infection often plays a critical role, mediating control or exacerbation of the pathogen growth. This paper describes a simple and rapid technique to recover these lymphomyeloid cells from a normal or an infected liver. After portal perfusion with saline buffer, the liver is gently dissociated on steel screens and the resulting cell population spun in 35% Percoll in 100 IU/ml Calciparine to remove all nuclei and cell debris: the recovery of a pure liver lymphomyeloid cell population is usually achieved in 40–60 min. Phenotypic and functional analysis could then be easily carried out on this cell population. This methodology was applied to normal mouse liver: flow cytometric analysis of the purified free lymphomyeloid cells showed the presence of T lymphocytes (46%±3 with a CD 4 CD 8 ration of 208), B lymphocytes (20%±2 IgG and 30% IgM positive) and myelomonocytic cells (14%±2 complement receptor type III positive).

A modified solid-phase fluorescence immunoassay was developed using bacterial cells as the solid phase to screen antibodies produced against surface antigens from a clinical isolate of Escherichia coli, strain 1–149. The bacterial solid phase was used to analyze both polyclonal and monoclonal antibodies. The bacterial concentration fluorescence immunoassay (BCFIA) showed up to 50-fold greater sensitivity in bacterial cell detection as compared to ELISA (enzyme-linked immunosorbent assay). Moreover, BCFIA was considerably faster than ELISA with uniform reproducibility. This paper demonstrates the utility of using bacteria and their surface antigens as solid-phase matrices for antibody characterization in a FIA.

Although techniques that permit analysis of the clonal composition of T cell populations have been used extensively to provide a better understanding of the mechanisms that influence efficacy of T cell responses in humans and mice, such methods are lacking for other animal species. In this paper we report the establishment and validation of a panel of Vbeta subfamily-specific semi-nested PCR assays, and a CDR3beta heteroduplex technique for analysing the clonal diversity of bovine alphabeta T cell responses. Development of these methods was based on available sequence data for 48 functional Vbeta genes classified within 17 subfamilies. These techniques were used to determine the clonal composition of parasite-reactive CD8(+) T cells obtained from two animals immunised with the protozoan parasite Theileria parva. Analyses of uncloned T cell lines as well as large panels of cloned T cells derived from each of these lines confirmed the specificity and sensitivity of the assays. Specific PCR products were obtained from 96% of the T cell clones examined, indicating that the currently identified Vbeta genes represent most of the functional Vbeta subfamilies in cattle. Heteroduplex analyses, coupled with sequencing of PCR products, identified over 20 clonal expansions within each of the T cell lines, distributed over a large number of Vbeta subfamilies, although a limited number of clonotypes numerically dominated the response in both animals. The development and validation of these methods provides for the first time a generic set of molecular tools that can be used to perform detailed analysis of the TCR diversity and clonal composition of bovine T cell responses.

This paper reports a simple, sensitive, and reusable piezoelectric immunosensor using magnetic hydroxyapatite (HAP)/gamma-Fe(2)O(3)/Au nanocomposite. Use of porous HAP nanocrystals embedded with gamma-Fe(2)O(3) and colloidal gold nanoparticles resulted in a multifunctional HAP/gamma-Fe(2)O(3)/Au nanocomposite. Under optimized conditions, the biocompatible nanocomposites were exploited for direct adsorption of large quantities of rabbit anti-human immunoglobulin G antibodies (anti-hIgG) with well-preserved immunoactivity. In a homogeneous bulk solution, the hIgG analytes were captured by the anti-hIgG-derivatized immunocomposites followed by magnetic separation/enrichment onto a bovine serum albumin (BSA)-sealed QCM probe before measuring. This QCM immunosensor can quantitatively determine concentrations of hIgG ranging from approximately 20 to 800 ng/ml, with a detection limit of approximately 15 ng/ml. Moreover, regeneration of the immunosensor can be simply realized by canceling the controllable magnetic field. With the possibility of performing the analysis automatically and considering its ease of use, high sensitivity, and good reusability, this magnetic separation-assisted QCM immunosensor may have great potential to be further tailored as a general and promising alternative for a broad range of practical applications.

The paper describes a method to use filamentous phage to display specific regions of proteins for immunization in order to direct the immune response towards a pre-defined region of the protein. The method called site-specific immunization (SSI) was evaluated using the E7 protein of oncogenic (high-risk) human papillomavirus (HPV) type 16 as a model system. This protein consists of sequence blocks also present in other viral and cellular proteins and in the corresponding protein of low-risk HPVs. A fragment of the HPV16 E7 oncoprotein specific for a group of high-risk viruses was identified by sequence comparison and displayed on filamentous phages in fusion with the major phage coat protein VIII. The recombinant phages triggered an immune response in mice against the full-length HPV16 E7 protein. Fusion of B-lymphocytes from the immunized animals with myeloma cells resulted in three hybridomas producing monoclonal antibodies (MAbs) with reactivity against the endogenous E7 protein. The specificity of the MAbs for the HPV16 E7 protein in cancer cell lines was confirmed by Western blot analyses and immunocytochemistry. The epitope of each MAb was roughly mapped by determining the reactivity against overlapping E7 fragments displayed on phage particles. The mimotopes of the MAbs were further determined by biopanning against a randomized peptide library displayed on phage and found to be unique for a sub-set of high-risk HPV E7 proteins. The combination of different phage display techniques for immunization and epitope mapping was efficient for generation and characterization of highly specific MAbs.

Although determination of circulating endothelial progenitor cell (EPC) in peripheral blood by flow cytometry is an emerging marker for cardiovascular medicine, a common standardized protocol is still not available, due to the low numbers achieved in peripheral blood. In the present paper we describe a novel technique for EPC quantification as CD34+/CD144+/CD3− cells within the lymphocyte gate, which increases the percentages of EPC positivity described before and also offers high intra-assay reproducibility. These improvements are based on a gating strategy for big-sized lymphocytes, smooth fixation and cytometric clearance of CD3+ lymphocytes (T-cells). This last procedure is able to increase intra-assay Pearson's correlation from 0.8517 to 0.8908. Therefore, the technical setting described here offers a high-performance and clinically oriented EPC determination strategy in human peripheral blood.

Genomic DNA extraction for genotyping analysis is performed from blood samples and is time consuming. We describe a more rapid DNA extraction method, “DBS-miniMAG”, that combines filter paper dried blood spots (DBS) with the NucliSens® miniMAG™ semi-automated instrument (bioMerieux). To assess the performance of this method, a post-PCR HLA-DR shared epitope (SE) oligotyping assay was used as a read-out in a cohort of 72 arthritis patients. This new method was compared to the standard manual DBS extraction protocol using FTA® reagents (Whatmann Bio-Science), and to a reference phenol-chloroform-based method using EDTA whole blood samples. Higher yield of PCR amplicons was observed with DNA extracts obtained using “DBS-miniMAG” method. The intra- and inter-assay variability of the “DBS-miniMAG” method was similar to that obtained with “DBS-FTA” washing process. Concerning the HLA-DR SE genotyping, “DBS-miniMAG” and “DBS-FTA” methods gave 100% concordance compared to the reference phenol-chloroform method. More importantly, the hands-on time and the turnaround time for “DBS-miniMAG” were both two-times shorter than for “DBS-FTA” protocol. Therefore, the “DBS-miniMAG” combination could facilitate polymorphism analysis in routine clinical practice and the creation of large DNA banks using very small amounts of blood.

Mapping of epitopes is a crucial step for the study of immune pathways, the engineering of vaccines and the development of immunoassays. In this work, the Bacillus licheniformis beta-lactamase BlaP has been engineered to display heterologous polypeptides in a permissive and solvent-exposed loop. When combined with phage display, this modified enzyme can be used for epitope mapping by cloning random gene fragments. The procedure presented in this paper allows the selection of large infectious phage libraries with high diversity and efficient beta-lactamase activities. A useful aspect of the proposed technique results from the possibility of using the beta-lactamase activity carried by phages to evaluate the proportion of immobilised phages during the successive enrichment steps of the library or competition experiments with the selected phages. Another advantage of the technique derives from the fact that the epitope is selected as a bifunctional hybrid protein, which can be overproduced and purified. The resulting recombinant protein associates an epitope with a specific and efficient enzymatic activity. This constitutes an original tool for immunoassay development. A virus influenza hemagglutinin (HA1)-gene fragment library has been generated with this system and used to identify a linear epitope.

This paper describes the evaluation and optimisation of boric acid antigen retrieval (AR) in rat joint tissue immunohistochemistry (IHC), with reference to two sample IHC targets, CD31 (PECAM-1) and Proliferating Cell Nuclear Antigen (PCNA). Sections of buffered formalin-fixed arthritic tibial/talus joints, decalcified with EDTA, EDTA/formalin or Surgipath(R) Decalcifier I(R), were subjected to one of a number of pre-treatments (none, 0.1% trypsin, 0.2 M acetic acid pH 7.0 or 0.2 M boric acid pH 7.0) and then immunostained for CD31 or PCNA. Of the pre-treatment AR regimens, boric acid gave the most consistent and specific immunostaining of both antigens in joints from the three different decalcification protocols. Satisfactory CD31 and PCNA staining was also achieved in EDTA decalcified joints with no pre-treatment. Likewise, PCNA could be demonstrated in Surgipath(R) decalcified tissue without pre-treatment, albeit at slightly lower staining intensity than achieved following boric acid. The remaining decalcification/pre-treatment conditions were unsatisfactory for IHC of the two antigens investigated because of lack of staining, non-specific staining or consistent loss of sections from the slides. Boric acid pre-treatment provides a valuable alternative low temperature AR method where conventional heat-mediated AR methods are normally required but cannot be used due to tissue type.

Autoimmune (Hashimoto's) thyroiditis is a chronic inflammatory disease which affects >3% of the population and shows an increasing prevalence with increasing age. Anti-thyroid autoantibodies, particularly against thyroperoxidase (also known as thyroid peroxidase or TPO), occur commonly in humans with autoimmune thyroid disease, and assays for anti-TPO autoantibodies are used in clinical diagnosis. In contrast anti-TPO autoantibodies have not been observed in classical mouse models of autoimmune thyroiditis, except in cases where mice were deliberately immunized with TPO. In the past, detection of anti-TPO autoantibodies in mice has relied on an indirect immunofluorescence assay (iIFA) which screens for thyroid follicle membrane staining in frozen sections of mouse thyroid glands. Since recent transgenic mouse models of autoimmune thyroiditis spontaneously develop anti-TPO autoantibodies, an assay other than serial dilution and iIFA would be useful to detect and quantify these autoantibodies. In this paper we describe such an assay, based on the capacity of autoimmune mouse sera to bind to the extracellular domain of mouse TPO which was produced in a radioactively labeled form using a coupled in vitro transcription/translation system. The same approach, using human TPO, could provide a highly sensitive method to detect anti-TPO autoantibodies in humans.

We describe a rapid, inexpensive and scalable hybrid bioseparation technique for one-step purification of humanized monoclonal antibodies from mammalian cell culture supernatant. It involves the selective and reversible retention of the monoclonal antibody within a membrane module utilizing a combination of microfiltration of precipitated antibody and simultaneous hydrophobic interaction-based membrane chromatography of soluble antibody on the same microfiltration membrane. The retained monoclonal antibody is subsequently recovered from the membrane module in a highly pure form by changing the solution condition to that which favours simultaneous antibody dissolution and elution from the membrane. This combination of separation principles results in high-capacity, high-resolution separation. The monoclonal antibody purities and recoveries obtained in the experiments described in this paper were very high.

The recent expansion of studies on hypermutation may benefit from a fast and uncomplicated way to measure mutation rates. In this paper we compare different retroviral vector designs for monitoring hypermutation in vivo. Retroviral vectors combine a high transduction rate with integration at random sites within the host cell genome, thus equalizing positional effects on the reporter gene. The vectors contain a reporter gene with a premature TAG termination codon; upon reversion, a full-length fluorescent protein is expressed. Any single point mutation at the amber codon activates the reporter--except the transition from G to A, which only creates the stop codon TAA. In the construct, the reporter gene is followed by an internal ribosome entry site and a second marker that allows selection of stably transduced cells. As a reporter gene, we tested the green and yellow fluorescence proteins (GFP and YFP); and various proteins with red fluorescence (dsRed). The second marker was either a drug resistance gene, or a second fluorescent protein. We also introduced various cis-acting enhancer elements into the reporter construct, to study the simultaneous activity of enhancers on transcription and hypermutation. We found that GFP as a reporter, combined with a drug selection marker, gave the most consistent and convenient mutation rate measurements. DsRed is a good alternative to GFP, but variants with greater fluorescence intensity are needed when combined with green fluorescence measurements. We also confirm that no immunoglobulin specific sequence is needed to target hypermutation. Depending on their position in these ectopically expressed constructs, enhancers can have positive or negative effects on hypermutation.

Time-resolved fluorescence has been developed for immunoassay to obtain higher sensitivity than usual immunoassays. In this paper, a simple, sensitive and specific method was developed for immunoassay of serum carcinoembryonic antigen (CEA) by combining time-resolved fluoroimmunoassay (TRFIA) and flow injection analysis. Based on a sandwich immunoassay format, a monoclonal antibody immobilized immunoaffinity column inserted in a flow system was used for immunoreactions. The cleaved solution was collected after the reaction between the immunocomplex in the immunoaffinity column and the enhancement solution that was used to cleave the Eu-labels from the immunocomplex, and then detected by time-resolved fluorescence. Serum CEA could be detected in the linear range from 2.5 to 100 ng/ml with a correlation coefficient of 0.997 and the detection limit of 1.0 ng/ml. Twenty human serum samples detected by this method were in good agreement with the results obtained by the electrochemiluminescence immunoassay. This method could be further developed for fast practical clinical detection of serum CEA levels.