The haemolytic plaque reduction technique to measure cytotoxicity with hybridoma cells as a target

The use of hybridoma cells as targets to measure cell-mediated cytotoxicity has been established. The ability of target hybridoma cells to form haemolytic plaques has been used as an indicator of target viability and therefore, cytotoxicity has been detected by plaque reduction (PR). Allogeneic responses, spontaneous cytotoxic responses and anti-hapten responses have been measured by the PR assay. Compared with the standard [51Cr]chromate release assay, small numbers of target cells (400 or less) can be used in the PR assay and this results in a greatly enhanced sensitivity in detecting small amounts of cytotoxicity. The ability to construct a range of hybridoma targets with the appropriate cell surface determinants presents a new approach, of general applicability, to the sensitive detection of cytotoxic lymphocytes.     

A method for testing the specificity of influenza A virus-reactive memory cytotoxic T lymphocyte (CTL) clones in limiting dilution cultures

This paper describes a system for determining the frequency and fine specificity of influenza A virus-immune memory cytotoxic T cell (CTL) clones from limiting dilution (LD) microcultures. We found that such experiments can only be performed (1) by analyzing the clonal response of CTL from wells of replica plates containing fractions of one original plate, (2) when it has been ascertained that splitting is possible at the clonal level, and that each fraction of a microculture well gives an identical response on target cells infected with the stimulating virus. With these requirements fulfilled we found that short term CTL clones from LD microcultures from influenza A virus (A/X-31)-immune mice (C57BL/6) occur at a frequency of f = 1:546 to f = 1:6303. The effector cells carry the Lyt-2.2 marker and are specific for target cells infected with the immunizing virus (influenza virus A/X-31). They do not lyse NDV (Newcastle disease virus) or HSV (herpes simplex virus)-infected or NK (YAC) target cells.     

Limiting dilution analysis of the allo-MHC anti-paternal cytotoxic T cell response II: recurrent spontaneous abortion and the effect of immunotherapy

Using limiting dilution analysis (LDA) we determined anti-paternal cytotoxic T lymphocyte precursor (CTLp) frequencies in the peripheral blood of 10 women with unexplained recurrent spontaneous abortion (RSA) before and after immunization with paternal lymphocytes. The women and their partners were HLA tissue-typed and none of the women had anti-paternal cytotoxic antibodies (APCA) before immunization. All other known causes of RSA were excluded. All 10 women were found to have high frequencies of specific anti-paternal cytotoxic T cells before immunization (range I 1/1030 to 1/9574). Splitwell analysis showed that these cytotoxic cells were specific to paternal MHC antigens. These frequencies rose significantly following immunization (range 1/683 to 1/4652). The cytotoxic T lymphocyte frequencies against an HLA-mismatched third party varied from woman to woman, but were not affected by the immunization. The LDA data conformed lo single-hit kinetics, indicating that only cytotoxic T ceils were limiting in the assay. Our data are in sharp contrast to the previously held view that women with RSA may be hyporesponsive to paternal MHC antigens. Immunizing such women with paternal leucocytes further sensitizes them. These findings cannot be reconciled with a favourable outcome in the treatment of RSA with immunotherapy. We would argue that this treatment is al best of unproven value, and may even be harmful. Thai these women may sometimes have successful pregnancies following immunotherapy testifies to the effectiveness of the classical MHC antigen-deficient trophoblast as an immunological barrier between mother and fetus.     

Longitudinal study of the frequency of cytotoxic T cell precursors in kidney allograft recipients

Clonal deletion or inactivation of donor-specific alloreactive cells are important mechanisms that are believed to account for acquired immune tolerance in allograft recipients. Serial assessment of precursor cytotoxic T lymphocyte frequencies (CTLpf) by limiting dilution analysis (LDA) provides information at the clonal level on changes in the alloimmune response of graft recipients. We performed a longitudinal study of 15 cadaveric kidney recipients before and every 3 months throughout the first year after transplantation (Tx). Pre-Tx values of donor CTLpf showed high interindividual variability without a predictive value for the clinical outcome. All patients with well functioning kidneys had decreased CTLpf at 3 months post-Tx in comparison with pre-Tx values. This decrease was donor-specific in four patients and was permanent in two cases throughout the study. Most patients presented decreased anti-donor CTLpf values from 6 to 9 months, whereas a partial recovery of donor CTLpf was observed in three patients. Reversible acute rejection was diagnosed in three patients, and it was associated with a marked increase in anti-donor CTLpf, returning to pre-Tx values by 9 months post-Tx. In addition, one patient with chronic rejection displayed a transient increase in CTLpf 6 months after Tx. The results of this sequential study indicate the establishment of a state of either hyporesponsiveness or functional clonal inactivation, transient or permanent, which could facilitate allograft acceptance.     

Demonstration of immunoglobulin-secreting cells in a sensitive reversed hemolytic plaque assay

A sensitive modification of a reversed hemolytic plaque assay demonstrating individual immunoglobulin-secreting cells (ISC) is presented. The test is performed by adding test cells to a monolayer of indicator sheep erythrocytes coated with an IgG fraction of rabbit antiserum against the F(ab')2 portion of human immunoglobulins (Ig). After having added a developing antiserum and complement, hemolytic plaques with a central ISC could be counted in an inverted microscope. In addition, when ISC were killed and fixed in situ in the monolayer, the cells could be further studied by immunofluorescence of their cytoplasmic Ig.     

Optimal conditions for titration of SV40 by the plaque assay method

The parameters of the Simian Virus 40 (SV40) plaque assay on African green monkey kidney cells were optimized for reproducibility and maximum plaquing efficiency. Plaques were visible as early as 8 days postinfection; maximum titers were obtained with a 10- to 11-day incubation period. Titers read 12-16 days postinfection were not significantly higher than those observed after 10-11 days. Adsorption volumes greater than 0.1 ml/60 mm Petri dish decreased plaque forming units (PFUs) detected. Times greater than 60 min for adsorption of virus to the cell monolayer did not significantly increase the titer; adsorption times less than 60 min resulted in decreased titers. Under standard conditions, 3 ml of overlay medium containing 0.8% agar was applied following virus adsorption and again on days 5 and 10. Concentrations of fetal calf serum (FCS) in the overlay medium of 2.5 to 7.5% gave equal plaque formation. FCS concentrations of 1 and 10% resulted in slightly decreased and increased plaquing efficiencies respectively. Of the reagents tested, agar or agarose containing overlay media produced plaques of maximum number and size. An overlay of methyl cellulose resulted in the same number of plaques, but their size was reduced by approximately 70% relative to those observed in agar; thus longer incubation times were required. Gum tragacanth overlay medium was actually inhibitory to plaque development. DEAE-dextran, dextran sulfate, or DMSO added to agar overlay medium did not enhance plaque number or size, nor did they shorten the incubation period required for their detection.     

A rapid limiting dilution assay for measuring frequencies of alloreactive, interleukin-2-producing T cells in humans.

A limiting dilution analysis (LDA) has been established which measures the total numbers of alloreactive interleukin-2 (IL-2)-secreting T cells in human peripheral blood mononuclear cells (PBMC). A significant advantage over most previous LDA is that the assay may be completed in approximately 48 h since an IL-2-dependent 'indicator' cell line is used to reduce assay time. Results are reproducible and correlate with the degree of HLA class II antigenic disparity between responder and stimulator cells. Use of both PBMC and Epstein-Barr virus-transformed B lymphoblastoid cell lines (B-LCL) as stimulator cells permits estimation of the frequency of Epstein-Barr virus-specific T cells in different responder individuals. A modification of the assay may also be used to measure the frequencies of 'primed' alloreactive cells, i.e., those alloreactive cells which have previously encountered their specific stimulating alloantigen. Use of the assay in the clinical context of bone marrow and renal transplantation is discussed.     

The preparation of stable phosphorylcholine-erythrocyte conjugates for use in plaque assays

A method for the preparation of stable phosphorylcholine-conjugated erythrocytes is described. This method employs the formation of active esters of phosphorylcholine hydroxyphenylacetic acid (PC-HPA) and results in coupled red cells which are completely stable for 2-3 weeks. Using this procedure up to 80% of the activity was demonstrable after 6 weeks. Cells coupled with PC-HPA are suitable for the demonstration of anti-idiotype effects in a plaquing assay.     

The use of phagocytic peritoneal exudate cells as targets for the estimation of cytotoxic T cell activity

A new method for measuring cytotoxic T lymphocyte (CTL) activity has been developed with peritoneal exudate cells (PECs) as target. A monolayer of target PECs was labelled with Indian ink and exposed to CTL. After incubation, detached or damaged PECs were rinsed out of the target monolayer, and the remaining phagocytes were fragmented and dissolved by the addition of 2 N NaOH. The number of undamaged target cells was estimated by colorimetric reading of the amount of Indian ink in the phagocytes surviving CTL attack. In studies with several strains of mice, only PECs from the same strain as the stimulator cell donors used for CTL induction were damaged by CTL effectors. When CTLs generated against TNP-modified syngeneic stimulators were used as effectors TNP syngeneic PECs, but neither unmodified syngeneic PECs nor allogeneic TNP-PECs, were damaged. These results demonstrate the antigen specificity of immunolysis of PECs. Macrophage target cells were stable and showed no tendency to spontaneous cell damage during 24 h incubation without effector cells.     

In vitro frequency analysis of spleen colony-forming and marrow-repopulating hemopoietic stem cells in the mouse

Summary An assay is described for Day-12 spleen colony-forming cells (CFU-S-12) and hemopoietic stem cells with marrow-repopulating ability (MRA) in the mouse using a miniaturized stroma-dependent bone marrow culture assay in vitro. Bone marrow cells are grown in liquid culture in microtiter wells, and the resulting adherent stromal layers are depleted of all hemopoietic activity by 20 Gy gamma irradiation. Subsequently, single cell suspensions containing stem cells are overlaid in a range of concentrations, and the presence of one or more emerging phase nonrefractive cell clones (cobblestone areas) in a single well scored as positive. The frequencies of cobblestone area-forming cells (CAFC) are then calculated by employing Poisson statistics. It is shown that the CAFC Day-10 and CAFC Day-28 frequencies closely correlate with those of CFU-S-12 and MRA cells, respectively.     

A filter immuno-plaque assay for the detection of antibody-secreting cells in vitro

A plaque assay has been developed that is based on enzyme immunoassay principles and capable of screening several hundred samples in one day. Single cell suspensions of in vivo or in vitro immunized mouse splenocytes are incubated on antigen-coated nitrocellulose membranes in microfilter plates or in petri dishes. The antibody production of individual cells is detected using a horse radish peroxidase-labeled second antibody, and the insoluble products of the enzymatic reaction are visualized as blue plaques on the membranes. The nitrocellulose membrane of the microfilter plates, which readily absorb a variety of antigens, and the filtration unit used for the washing steps greatly facilitates the plaque assay. Furthermore, this procedure only needs small amounts of antigen for the enumeration of isotype-specific antibody-secreting cells in a defined medium containing low protein levels or in a completely serum-free medium. The plaque assay may be used to evaluate the optimal conditions required for in vitro immunizations.     

Comparison of the plaque assay and 50% tissue culture infectious dose assay as methods for measuring filovirus infectivity

Two common methods of quantifying filovirus infectivity, a plaque assay and 50% cell culture infectious dose (TCID₅₀) endpoint dilution assay, were compared. The two assays were performed side by side using the same virus stock sample to determine the correlation between the results of the two assays. The TCID₅₀ assay appeared to be more sensitive but slightly more variable, and there was a tenfold difference in the numerical results of these methods of enumeration. The advantages and disadvantages of both assays are discussed. Both methods are useful and practicable in filovirus research, and this comparison will be hugely beneficial to the filovirus research community as it seeks to become more united. Further work in this area should be performed to ensure consistency in filovirus research.     

A flow cytometric method to estimate the precursor frequencies of cells proliferating in response to specific antigens

Fluorescent dyes that stain cell membranes or cytoplasm and then partition between daughter cells at division have been used in conjunction with flow cytometry to measure the proliferation of cells. In this paper, using peripheral blood mononuclear cells responding to tetanus toxoid, we describe an extension of this dye methodology to calculate the precursor frequency of antigen-specific T-cells. With mathematical deconvolution of the fluorescence histograms providing information about the proportion of cells in each of the daughter generations, information can be derived about the precursor frequency of cells in the original population that responded to the specific stimulus. Data from a model system with different proportions of fixed and viable cells indicate that the flow method returns accurate values for precursor frequency. Based on the characteristics of flow cytometric data acquisition, it is estimated that the flow method could detect proliferation of cells that represented, before addition of the stimulus, approximately 1/10⁵ of the population. When comparing results to those from the limiting dilution technique, the flow cytometric method returns values that indicate higher precursor frequencies. Possible reasons for this discrepancy are discussed. The flow cytometric method offers the advantage of simplicity as well as the additional ability to phenotype the responding cells and determine their rate of proliferation. The flow method may find use as a simple, routine assay in the fields of allergy, transplant rejection, and autoimmunity and for quantitating responses to vaccination and cancer immunotherapy.     

Simplified plaque reduction neutralization assay for dengue viruses by semimicro methods in BHK-21 cells: comparison of the BHK suspension test with standard plaque reduction neutralization.

A newly modified semimicro plaque reduction neutralization test (PRNT) in BHK cells was compared with a standard PRNT in bottles with LLC-MK2 monolayers and with an LLC-MK2 PRNT adapted to semimicro methods. The BHK semimicro PRNT compared favorably in terms of sensitivity in detecting dengue antibody (96%), specificity at a screening dilution (95%), and ability to detect seroconversion to dengue viruses of three serotypes (93%). Disagreements between the BHK test and the LLC-MK2 tests were attributed to greater sensitivity of the BHK test in detecting dengue type 2 (DEN-2) antibody in acute-phase sera and to apparent low-level DEN-1/DEN-3 cross-reactions in some sera in all three tests. The BHK PRNT was easier, faster, and more economical than either of the LLC-MK2 tests. Many of the benefits of the BHK PRNT derive from the fact that cells are infected while still in suspension, at the time of cell splitting, hence the term "BHK suspension test."     

Enzyme-linked immunosorbent assay for the detection and identification of coxsackie B antigen in tissue cultures and clinical specimens

An enzyme-linked immunosorbent assay (ELISA) has been developed for the identification of Coxsackie B antigens. This assay was capable of identifying and distinguishing all six Coxsackie B serotypes at concentrations one hundredfold to ten thousandfold less than could be detected by complement fixation (CF) systems. In addition, the Coxsackie B ELISA correctly identified the presence of Coxsackie B antigen in 19 of 21 tissue culture fluids and five of nine rectal swab specimens. Two additional rectal swab specimens reacted with Coxsackie B antisera but could not be conclusively serotyped. Tissue culture fluids and rectal swab specimens containing other viruses such as ECHO virus, Coxsackie virus A, rhinovirus, rotavirus and Norwalk virus were consistently negative in the assay. The Coxsackie B ELISA offers potential for the rapid identification of Coxsackie B antigens in clinical specimens and tissue culture systems.     

用ELISA方法检测埃可病毒感染的特异性IgM抗体

目的 研究埃可病毒感染的无菌性脑膜炎的诊断方法。方法 采用埃可病毒混合抗原包被酶标板，用抗人γ链处理人脑脊液标本，通过酶标二抗及底物显色，建立了抗埃可病毒IgM的间接ELISA检测方法；并用特异性试验、对照和重复性试验证实方法的可靠性和实用性。结果 在临床诊断为无菌性脑膜炎的78例患者脑脊液中有14例阳性（17.9%)，而细菌性脑膜炎的36例患者脑脊液中仅有1例阳性（2.8%)，28例脑外伤患者脑脊液均为阴性，ELISA阳性的5份脑脊液中和试验4例阳性，而ELISA阴性的5份脑脊液中和试验均为阴性；该方法与脊髓灰质炎病毒、柯萨奇A组病毒7型和柯萨奇B组病毒1-6型无交叉反应；ELISA阳性的6份标本经特异性IgM破坏和阻断试验均全部转为阴性。结论 本方法快速，简便、可靠、适合于临床早期特异性诊断。     

A rapid colorimetric assay for the determination of IL-2-producing helper T cell frequencies

Interleukin 2 (IL-2) activity is tested in conditioned media by assessing its ability to support proliferation of selected IL-2 dependent T cell lines, conventionally measured by [3H]thymidine incorporation. Here, we compare this [3H]thymidine uptake test for measuring IL-2 activity with a rapid and sensitive colorimetric method which is based on the ability of viable cells to cleave 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT). The sensitivity of the colorimetric method was dependent on the indicator cell line used, being greatest with the cytotoxic T cell line 16 (CTLL-16). The colorimetric method is at least as sensitive as [3H]thymidine uptake tests, does not rely on radioactivity, and is ideally suited to screen large numbers of individual samples for IL-2 activity. The latter point was demonstrated by calculating IL-2-producing helper T cell frequencies in heterogeneous murine lymphocyte populations: in this assay, splenic T cells were clonally expanded under limiting dilution conditions and supernatants conditioned by these in vitro growing T cell clones were tested for IL-2 activity with the colorimetric method. This allowed us to obtain reliable estimates of the frequency of progenitor cells of IL-2-producing T cell clones in various populations.     

Development of a specific monoclonal antibody assay and a rapid testing strip for the detection of apramycin residues in food samples

A specific monoclonal antibody (MAb) against apramycin (AP) was produced and used to develop an indirect competitive enzyme-linked immunosorbent assay (idcELISA) and a rapid testing strip for the detection of AP residues in foods. MAb exhibited negligible cross-reactivity with other aminoglycosides. Under optimized conditions in 0.01 M PBS, the half maximum inhibitory concentration (IC₅₀) of MAb was 0.41?ng/ml with a limit of detection (LOD) of 0.15?ng/ml. The ELISA results were obtained within 90?min. The mean recoveries from all the spiked food samples were within the range of 79.02–105.49%, with coefficients of variation in the range of 2.21–11.4%. The strip test results obtained within 5?min had visual LODs in the range 2.5–5?µg/kg (ng/ml) for all food samples tested. Therefore, the developed strip test represents a fast and convenient detection method of AP residues in foods.     

The fluorolysis assay,a highly sensitive method for measuring the cytolytic activity of T cells at very low numbers

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard 51Cr-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few (> or =6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers (> or =25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies.     

应用免疫荧光搭桥法检测小鼠肠粘膜内特异性抗体形成细胞

肠粘膜固有膜内特异性抗体形成细胞(ACC)的检测是研究肠道粘膜免疫应答机理的重要指标之一,组织免疫荧光技术是检测粘膜固有膜内特异性ACC的主要方法。目前,在粘膜免疫应答机理的研究中,主要