An improved method to create nitrocellulose particles suitable for the immobilization of antigen and antibody

A method is described for producing 1-3 microns sized particles of nitrocellulose (NC) which are able to absorb protein. Protein is absorbed onto preformed particles made by first dissolving a sheet of nitrocellulose paper in DMSO, and then precipitating it with sodium carbonate/bicarbonate buffer. The efficiency of binding is the same as that of an equivalent sheet of non-processed NC filter paper. Antibodies absorbed onto preformed particles are not exposed to DMSO and carbonate buffer and therefore retain a high antigen binding capacity. Antigen and antibody-absorbed NC preformed particles were used to capture antibody and antigen, respectively. Using lysis buffer, the captured antibodies and antigens were readily released from the NC particles. This makes the latter an appropriate matrix for immunoprecipitation assays either for an antigen or for specific antibody. Antigen-coated NC particles were specifically aggregated ('agglutinated') by specific antibodies and thus can be used in semi-quantitative tests.     

Measurement of antigen concentration by an ultrasound-enhanced latex immunoagglutination assay

A novel ultrasonic technique that increases the rate and sensitivity of latex agglutination tests (LATs) has recently been described. The technique is based on the fact that suspended latex particles become concentrated in an ultrasonic standing wavefield, thereby increasing the rate of particle-particle collisions compared to the standard LAT procedure. The present work extends earlier qualitative assessments of agglutination and seeks to establish whether quantitative measurement of agglutinate size may be used as an indicator of antigen concentration. The agglutination of latex microparticles coated with antibody to C-reactive protein (CRP) is studied here as a model system to determine the dependence of agglutinate size on analyte (CRP) concentration. Agglutinate size is characterised by image analysis techniques. The results show that agglutinate size decreases with decreasing CRP concentration. A near linear relationship is shown between analyte concentration and the size of agglutinate formed over a 100-fold dilution range. The threshold concentration of 230 pg/mL for detection of CRP in the ultrasonic test is 2560 times lower than that required for a conventional test-card CRP latex agglutination assay.     

Circadian rhythmic plaque-forming cell response of spleens from mice immunized with SRBC

Studies under controlled conditions of lighting and temperature revealed clear evidence of circadian periodicity with respect to the number of PFC present in the spleens of BALB/c mice 3 or 4 days after immunization with SRBC. Striking differences in proliferative responses of spleen lymphocytes to PHA or Con A were also observed at two different circadian times. Large proliferative responses occurred at the time when injection of antigen and/or sampling for PFC yielded a low PFC formation (early in the daily dark span) and small proliferative responses occurred at the time when antigen injection and sampling yielded high formation of PFC (early in the daily light span). These findings indicate that care should be taken to control the circadian timing of stimulation and sampling in studies of immune responses, and that rhythmically varying aspects constitute a new dimension of immunologic processes awaiting further analysis in both the circadian and weekly spectral regions.     

Influence of 6-metoxy-cumaranon-2-acetic acid and its derivatives on the process of humoral and cellular immunogenesis

The influence of 6-metoxy-cumaranon-2-acetic acid and its four derivatives on the humoral and cellular immunogenesis was determined. The study of immunosuppressive properties included: the control of the level of peripheral blood lymphocytes, the estimation of transplantation immunity, GvH reaction, PFC production for SRBC and LPS and the determination of the number of cells possessing receptors for antigen, Fc fragment and complement. Furthermore, blast transformation of lymphocytes stimulated with PHA was estimated as well as the cytotoxic effect of sensitized lymphocytes. The results indicate immunosuppressive activity of acetamides HKCMF and HKCHF. They lowered the level of circulating lymphocytes, the number of cells possessing receptors for complement and they hindered PFC production for SRBC and LPS. Moreover, acetamide HKCHF weakened the cytotoxic activity of lymphocytes sensitized to alloantigens.     

Modelling the kinetics of antigen-antibody reactions at particle enhanced optical immunoassays

Functionalized latexes coated by antibodies are used in diagnostic tests for the detection of antigens in biological fluids. A simple kinetic model is presented which is related to the optical monitoring of the formation of specific complexes between antigen and antibody amplified by latex beads. The antibodies are chemically coupled onto chloromethylstyrene (CMST) particles. The kinetic model is able to describe the immunoprecipitin curves of immunolatex beads. The number of fitting parameters is relatively reduced (only three), and the meaning of these parameters can be interpreted in terms of the chemical equilibrium constant, the percentage of active IgG on the latex beads, and optical response. The model explains very well the optical response of immunolatex prepared by covalent coupling of antibodies on polymer carriers.     

Sensitive enzyme immunoassay of antibodies to HIV-1 p17 antigen using indirectly immobilized recombinant p17 for diagnosis of HIV-1 infection

Recombinant p17 (rp17) antigen of HIV-1 and maltose binding protein-rp17 fusion protein (MBP-rp17) were immobilized onto polystyrene beads in different ways: rp17 and MBP-rp17 were immobilized directly onto polystyrene beads by physical adsorption; biotinyl-rp17 and biotinyl-MBP-rp17 were immobilized indirectly onto streptavidin-coated polystyrene beads; and 2,4-dinitrophenyl (DNP)-MBP-rp17 was immobilized indirectly onto (anti-DNP) IgG-coated polystyrene beads. These directly and indirectly immobilized antigens were incubated with urine samples containing antibody IgG to p17 antigen and subsequently with rp17-beta-D-galactosidase conjugate or (anti-human IgG gamma-chain) Fab'-beta-D-galactosidase conjugate. Beta-D-galactosidase activity bound to the polystyrene beads was assayed by fluorometry. When rp17-beta-D-galactosidase conjugate was used, signals (fluorescence intensities for bound beta-D-galactosidase activity) were much higher with the indirectly immobilized antigens than those with the directly immobilized antigens. By experiments using (anti-human IgG gamma-chain)Fab'-beta-D-galactosidase conjugate, the binding of rp17-beta-D-galactosidase conjugate to antibodies against p17 antigen bound to directly immobilized rp17 antigen was shown to be seriously limited as compared with that to antibodies against p17 antigen bound to indirectly immobilized DNP-MBP-rp17. When rp17-beta-D-galactosidase conjugate and serum samples were used, serum interference was much less with indirectly immobilized DNP-MBP-rp17 than with directly immobilized rp17 antigen, and the sensitivity of enzyme immunoassay for antibody IgG to p17 antigen using indirectly immobilized DNP-MBP-rp17 was 1,000- to 3,000-fold higher than that of enzyme immunoassay using directly immobilized rp17 antigen and Western blotting for p17 band. This sensitive enzyme immunoassay indicated positivity in HIV-1 seroconversion serum panels as early as conventional methods for antibodies to HIV-1 and earlier than Western blotting for p17 band.     

Kinetics of the reactive cell clones after immunosuppression and induction of tolerance: (1) inhibition of 19 S and 7 S plaque-forming cells in the primary and secondary immune response to sheep red blood cells by cyclophosphamide and 036.5122 (Asta)

The kinetics of the reactive cell clones after primary and secondary immunization with SRBC1) modified by cyclophosphamide and a newly synthesized cyclophosphamide analogue 036.5122 (Asta), have been studied. After primary immunization, both substances caused a severe and dose dependent depletion of 19 S PFC2). The 7 S PFC in the late primary response were only slightly inhibited by cyclophosphamide in low dose ranges, indicating, that sensitization could not be prevented by this substance. In contrast, 0.36.5122 was fully able to suppress 7 S PFC. Thus, treatment with 0.36.5122 after primary immunization can fully prevent the expression of the specific response. Experiments dealing with inhibition of a secondary immune response revealed that both test substances were able to strongly suppress the 19 S as well as the 7 S PFC. In general, 036.5122 demonstrates a higher suppressive potency, and the timing of its application for optimal suppression is less delicate than that of cyclophosphamide. 036.5122 was equally well inhibitory, whether given directly before or after antigenic challenge. The hypothesis is discussed, whether the immunosuppressive effect of 036.5122 given before antigenic challenge in the secondary immune response is due to cytotoxic damage of antigen reactive clones stimulated by persisting antigen.     

Detection of irradiated fruits by gas-chromatographic methods

The purpose of this study was to investigate the effects of prolactin, both in vitro and in vivo, upon heterophil phagocytic activity of the ring dove, Streptopelia risoria. In vitro incubation of heterophils with either 0.1 or 100 micrograms/mL of ovine prolactin for 2 h-significantly increased both latex bead phagocytosis and Nitroblue tetrazolium (NBT) reduction, an index of phagocytic metabolic activity. Ring doves given antigen demonstrated an increase in latex bead phagocytosis and NBT reduction. The greatest increase in both of these parameters was observed in those birds which possessed elevated plasma prolactin concentrations, either through exogenous administration or naturally through being in the later stages of incubation. These results suggest that during the incubation period of the avian breeding cycle there is an increase in phagocytic activity which is a direct consequence of the elevation shown by these birds in the concentration of plasma prolactin.     

Effects of lysozyme dimer on humoral response to sheep erythrocytes in non-treated and cyclophosphamide-immunosuppressed mice

The effects of lysozyme dimer on humoral response to sheep erythrocytes (SRBC) and restoration of the response impaired by a single cyclophosphamide dose (200 mg/kg) were tested on mice. The effect of lysozyme dimer on the humoral response to SRBC in non-treated with cyclophosphamide mice was determined in relation to doses (0.2, 2, 20 or 200 micrograms/kg) and the time of the drug administration with respect to the antigen before or after SRBC immunization. Moreover, the effect of lysozyme dimer on the humoral response in cyclophosphamide-treated mice was studied depending on the dose applied and time of exposure to the drug in relation to SRBC. It has been found that lysozyme dimer potentiates the humoral response to SRBC in mice, resulting in an increased number of splenocytes producing haemolytic antibodies (PFC) and the total and 2-mercaptoethanol resistant level of anti-SRBC antibodies. A single exposure to lysozyme dimer gave the strongest stimulating action on SRBC when the doses of 2 or 20 micrograms/kg were administered 2 h prior to the antigen. The potentiating effect of the drug was reduced when it was administered 24 h before the antigen and also when single doses were as high as 200 micrograms/kg and as low as 2 micrograms/kg. Exposure to four doses of lysozyme dimer at 24 h intervals was more activating than a single injection. A strong potentiating effect on the specific response to SRBC was noted after four injections of lysozyme dimer at doses from 0.2 to 20 micrograms/kg. The effect of the drug did not depend on the time of exposure to the antigen. It has also been found that lysozyme dimer significantly reduces the suppressive effect of a high cyclophosphamide dose (200 mg/kg) on the humoral response of SRBC-immunized mice. The protective action of lysozyme dimer was dose- and time-dependent. The strongest protection was observed after three doses of 20 micrograms/kg administered prior to pharmacological immunosuppression. Reduction in the dose to 2 micrograms/kg and shorter treatment resulted in reduced protective effects. We have also found that the protective action of three doses of lysozyme dimer (2 or 20 micrograms/kg each) administered between cyclophosphamide injection and the antigen, or after antigen administration is weaker than such a treatment prior to cyclophosphamide immunosuppression.     

Competitive enzyme-liked immunoassay with use of soluble enzyme/antibody immune complexes for labeling. I. Measurement of human choriogonadotropin

We described the principle of a new enzyme-immunoassay, competitive enzyme-liked immunoassay (CELIA), for quantitative measurement of soluble antigens and haptens. In the assay, binding of antibody to antigen-immunosorbent is competitively inhibited by the free antigen to be measured. The amount of first antibody bound to the immunosorbent is measured by an enzymatic technique in which a heterologous bridging antibody and a soluble antibody/enzyme immune complex are applied in sequence. The soluble complex we used was rabbit antiperoxidase/horseradish peroxidase. Peroxidase activity is inversely proportional to the concentration in the original sample of the substance to be assayed. The enzyme-linked reagents are potentially widely applicable to any substance to be measured. To demonstrate the feasibility of CELIA, we report a preliminary study of its application to the measurement of human chloriogonadotropin in serum and urine. The assay described for this hormone has a working range of 1 to 50 int. units per milliliter of sample. The technique obviates the disadvantages associated with measurement and handling of radioisotopes in radioimmunoassays and the only major instrumentation required is a centrifuge and a conventional spectrophotometer.     

Complement consuming antibodies against a modified human serum protein in pigeon breeders' disease

The sera of patients with pigeon breeders' disease contain precipitating and human complement consuming antibodies against pigeon dropping antigens. Cessation of antigen exposure results in a decrease of precipitins below the level of detection in immunodiffusion. Complement consuming antibodies remain present, however, despite antigen avoidance. A close correlation is observed between human complement consumption tests with pigeon dropping antigens PDF1-A or pigeon cropmilk IgA and a modified human serum gamma-globulin. Isolation of this protein is readily achieved by its non-specific adsorption onto activated Sepharose 4B and subsequent elution with 1 M acetic acid. This modified protein may act as an autoantigen in pigeon breeders' disease, maintaining human complement consuming antibodies for years in subjects with no further bird antigen contact.     

Latex immunoassays: comparative studies on covalent and physical immobilization of antibodies. I. F(ab')2 fragments

Colloidal particles coated with antibodies are currently used in diagnostic test systems for the detection of antigens in biological fluids. Immobilization is usually carried out by physical adsorption. Covalent coupling of antibodies to particles, however, offers certain advantages. The present research deals with the study of these possible advantages. A sulphonated polystyrene latex has been used to prepare an immunolatex with physically adsorbed antibodies, while a functionalized latex with chloromethyl groups on the surface has been used for the partly covalent coupling of the antibody (F(ab')2 fragments). The immunoreactivity was studied by measuring the variations in scattered light intensity after mixing a solution of CRP antigen and the sensitized latex. The influence on the immunoresponse of the scattering angle (5, 10, and 20 deg), protein coverage and storage time have been studied for both systems.     

Simultaneous identification of antibodies to Brucella abortus and Staphylococcus aureus in milk samples by flow cytometry

Two flow cytometric assays are described herein. The single cytometric test (SCT) detects antibodies to either Brucella abortus or Staphylococcus aureus in the serum or milk of a cow or water buffalo. The double cytometric test (DCT) detects both anti-B. abortus and anti-S. aureus antibodies concurrently. In the SCT, the sample to be tested is incubated in succession with the antigen (either B. abortus or S. aureus) and the proper secondary antiserum (fluorescein isothiocyanate-labelled rabbit anti-cow immunoglobulin antiserum or rabbit anti-water buffalo immunoglobulin antiserum). In the DCT, the sample to be tested is incubated first with B. abortus and S. aureus antigens and then with the secondary antiserum. The B. abortus antigen used in the DCT is covalently bound to 3-microm-diameter latex particles. The difference in size between B. abortus and S. aureus permits the establishment of whether the antibodies are directed against one, the other, or both antigens. When compared to the complement fixation test, the SCT and DCT each show a specificity and a sensitivity of 100%. The SCT has been used previously to detect anti-S. aureus antibodies. Here its use is extended to the detection of anti-B. abortus antibodies. The DCT is described here for the first time. The DCT appears to be useful for large-scale brucellosis eradication programs. It offers the possibility of using one test to identify animals that are serologically positive for both B. abortus and S. aureus.     

Mechanisms of increased sensitivity to noise and light in migraine headache

The neuroimmunomodulation (NIM) function of substance P (SP) in some brain areas of rats was investigated by the hemolytic plaque-forming cell (PFC) technique for detecting humoral immune function, and by radioimmunoassay for assessing SP content in some brain areas. The results showed that the contents of SP in the hypothalamus, hippocampus and pons were significantly decreased, while SP contents in the hypophysis, midbrain and medulla were not significantly changed at the peak of the immune response to sheep red blood cells (SRBC). Intracerebroventricular injection of capsaicin and SP antiserum had no effect on the immune response to SRBC. These results suggest that the immune response could affect the metabolism of SP in some brain areas, and provide further evidence that the nervous system (NS) can both regulate immune function and be affected by it.     

Measurement of 'blocking antibodies' to pollen antigens by radiolabelled protein A from Staphylococcus aureus

Radiolabelled protein A from Staphylococcus aureus (125I-SpA) was utilized to measure the relative concentrations of the so-called 'blocking antibodies' (BA) in sera from patients subjected to two different forms of desensitization therapy. In addition, sera from rabbits immunized actively with pollen antigens were assayed. Antigens coupled to CNBr-activated papear discs were used as solid phase, and BA bound to the solid-phase antigens were detected by 125I-SpA. The results obtained with this technique correlated excellently with those of a double-antibody technique for IgG, indicating the 125I-SpA assay to be suitable for the measurement of the antigen-specific IgG. The BA concentration in sera from patients subjected to conventional injection therapy increased significantly during the treatment period, whereas no increase could be detected in sera from patients subjected to sublingual treatment. The increases in BA concentrations of rabbit antisera to pollen antigens could be readily traced by this technique.     

Influence of subchronic exposure to lindane on humoral immunity in mice

Lindane suppressed both primary and secondary antibody responses to sheep red blood cell (SRBC) in albino mice, the effects being more pronounced on the secondary than the primary response. However, a longer duration of pesticide exposure induced similar degrees of immunosuppression on both responses. The sequential study of plaque forming cells (PFC) kinetics revealed that suppression of plaque formation not only occurred at peak days but also on pre and post peak days, and there was no delay in peak antibody formation. Moreover, reduction in the primary PFC was not associated with decrease in the antibody response to SRBC. The results indicate that lindane suppresses both primary and secondary humoral immune responses in a time and dose dependent manner, and suggest a threshold susceptibility to exposure.     

The nature of antigen in the eye has a profound effect on the cytokine milieu and resultant immune response

The eye is endowed with a number of mechanisms that protect it from immune-mediated injury. One such mechanism, termed anterior chamber-associated immune deviation (ACAID), evokes the antigen-specific, systemic down-regulation of Th1 responses to antigen inoculated into the anterior chamber of the eye. ACAID has been correlated with the selective production of IL-10 by the antigen-presenting cells (APC) and the development of a cross-regulatory Th2-like response. A small subset of antigens do not induce ACAID, but instead provoke IL-12 and normal Th1 immunity. Remarkably, all soluble antigens tested are capable of inducing ACAID; only cell-associated antigens do not induce ACAID. We hypothesized that the nature of antigen plays a decisive role in the resultant immune response. This hypothesis was tested with two well-characterized antigens, ovalbumin (OVA) and SV40 large T antigen (SV40 Lg T Ag). The soluble forms of OVA and SV40 Lg T Ag induced ACAID in both in vivo and in vitro models of the eye. In contrast, the particulate forms of these antigens, i.e. OVA passively absorbed onto inert latex beads (OVA-latex) and SV40 Lg T Ag expressed in two different cell lines, 99E1 and SV-T2, did not induce ACAID in either in vivo or in vitro models of the eye. In addition, the cytokine profiles of ocular APC pulsed with OVA or OVA-latex showed that soluble OVA induced the production of IL-10, whereas OVA-latex induced the production of IL-12. These data suggest that the nature of the antigen in the eye, whether soluble or particulate, is a crucial determinant in the resultant immune response. Moreover, they suggest a mechanism in which soluble antigens preferentially induce the release of ACAID-inducing IL-10 whereas particulate antigens preferentially induce the release of Th1-inducing IL-12 by responding APC.     

Adjuvant effect of Bordetella pertussis vaccine to sheep erythrocytes in mice: enhancement of cell-mediated immunity by subcutaneous administration of adjuvant and antigen

The subcutaneous route (s.c.) was used to study the adjuvant effect of Bordetella pertussis vaccine (PV) on cell-mediated immunity to sheep erythorcytes (SRBC). The immune response was measured by a sensitive assay procedure in which the antigen is injected intracutaneously into the mouse ear and the inflammatory swelling is measured with calipers. PV significantly enhanced cell-mediated immunity to SRBC, and the enhancement persisted for at least 3 weeks. PV administered up to 6 days before SRBC also significantly enhanced the response; PV injected 1 or more days after SRBC was not effective. In addition, it was found that PV per se released into the suspension medium a cell-free component(s) (pertussis supernatant) that contributed significantly to adjuvanticity. The adjuvanticity of both PV and PS was completely eliminated by heat.     

Methodology for Determining Laboratory and In Situ Hydraulic Conductivity of Asphalt Permeable Friction Course

The permeable friction course (PFC) is a layer of porous asphalt pavement overlain on conventional impervious hot-mix asphalt or portland cement concrete. The drainage properties of PFC are typically considered to be governed primarily by two hydraulic properties: hydraulic conductivity and porosity. Both of these hydraulic properties change over the life cycle of the PFC layer due to clogging of the pore space by sediment. Therefore, determination of the hydraulic conductivity and porosity of PFC can be problematic. Laboratory and particularly field tests are necessary for accurately determining the hydraulic conductivity of the PFC layer. Taking multiple measurements over the life of the pavement shows how these hydraulic characteristics change with time and the varying roadway conditions at which they are evaluated. Constant head laboratory testing has shown that PFC experiences a nonlinear flow relationship as described by the Forchheimer equation. In addition to the laboratory analysis of the hydraulic characteristics, a falling head field test is recommended to determine the in situ hydraulic conductivity. This incorporates the modeling techniques used in the laboratory testing and applies them to the falling head conditions used in the field. The result is a nondestructive test procedure for determining the in situ hydraulic conductivity which is necessary for estimating the extent to which the benefits associated with the drainage characteristics of the PFC layer will persist.     

Interleukin-2-dependent augmentation of the anti-TNP antibody production by sodium butyrate in cultured murine splenic B cells

Previously we found that sodium butyrate (NaBu) markedly enhanced production of the antibody specific for a T-cell-dependent antigen, sheep red blood cells (SRBC) in murine splenocytes (Kishiro, Y., Ueda, K., Fujiwara, M. and Yamamoto, I., Jpn J. Phamacol., 1994 66, 369-376. To gain a better understanding of the target cells for NaBu's action on antibody responses, we have utilized the T-cell-independent antigen, trinitrophenyl-lypopolysaccharide (TNP-LPS) as a stimulant and have examined an effect of NaBu on the anti-TNP antibody production in vitro. NaBu markedly increased the anti-TNP plaque-forming cell (PFC) responses in murine whole splenocytes, but not in murine splenic B cells. Addition of T-cells or the concanavalin A supernatant (CAS) from murine splenocytes to the B cell cultures completely restored the enhancing effect of NaBu. This effect of CAS was totally blocked by an anti-interleukin (IL)-2 antibody and partially by an anti-IL-1 beta or anti-IL-4 antibody. The full enhancing effect of NaBu was also detected when IL-2 was added to the B cell cultures, while IL-2 alone had no stimulatory effect on the control PFC response. IL-1 beta alone significantly stimulated the antibody production and adding NaBu to this IL-1 beta-supplemented culture caused a further increase. Neither IL-4 alone nor NaBu plus IL-4 had any effect on the PFC response. NaBu did not affect the expression of the IL-2 receptor alpha- and beta-chains in B cells stimulated with TNP-LPS. These results suggest that NaBu is an agent that promotes B cell differentiation in vitro in an IL-2-dependent manner.