Nonspecific Antigen Research Articles

Development of a novel FRET immunosensor technique

We report on a novel technique to develop an optical immunosensor based on fluorescence resonance energy transfer (FRET). IgG antibodies were labeled with acceptor fluorophores while one of three carrier molecules (protein A, protein G, or F(ab′) 2 fragment) was labeled with donor fluorophores. The carrier molecule was incubated with the antibody to allow specific binding to the Fc portion. The labeled antibody–protein complex was then exposed to specific and nonspecific antigens, and experiments were designed to determine the ‘in solution’ response. The paper reports the results of three different donor–acceptor FRET pairs, fluorescein isothiocyanate/tetramethylrhodamine isothiocyanate, Texas Red/Cy5, and Alexa Fluor 546/Alexa Fluor 594. The effects of the fluorophore to protein conjugation ratio (F/P ratio) and acceptor to donor fluorophore ratios between the antibody and protein (A/D ratio) were examined. In the presence of specific antigens, the antibodies underwent a conformational change, resulting in an energy transfer from the donor to the acceptor fluorophore as measured by a change in fluorescence. The non-specific antigens elicited little or no changes. The Alexa Fluor FRET pair demonstrated the largest change in fluorescence, resulting in a 35% change. The F/P and A/D ratio will affect the efficiency of energy transfer, but there exists a suitable range of A/D and F/P ratios for the FRET pairs. The feasibility of the FRET immunosensor technique was established; however, it will be necessary to immobilize the complexes onto optical substrates so that consistent trends can be obtained that would allow calibration plots.

Potential of Laser Immunoassay for Detection of HIV in Human Blood Serum and Urine

The potential of Light Scattering Immunoassay (LIA) for detection of HIV in human blood serum has been explored by monitoring the agglutination of antigen coated polystyrene particles by dynamic light scattering. Elisa tested human sera having HIV, TB, Filaria along with normal sera have been analyzed using two specific synthetic peptide antigen (SP1, SP2) and one nonspecific peptide antigen (NSP). Few paired human sera and urine samples and nonspecific (of nonHIV diseases) urine samples have also been tested using the same antigens to check the possibility of replacement of sera by urine.

Species-specific field testing of Entamoeba spp. in an area of high endemicity

Entamoeba histolytica has been separated in recent years into 2 morphologically identical species: the apathogenic E. dispar and the pathogenic E. histolytica, only the latter being pathogenic. Although various laboratory techniques allow discrimination between the 2 species there is a lack of field data about the suitability of available diagnostic tests for use in epidemiological studies and few epidemiological studies using species-specific diagnosis have been performed at community level in endemic areas, especially in sub-Saharan Africa. We conducted a repeated cross-sectional study of 967 schoolchildren in central Côte d'Ivoire to compare and evaluate light microscopy, 2 different antigen detection assays, and one polymerase chain reaction (PCR) assay. Microscopy and a non-specific antigen capture Entamoeba enzyme-linked immunosorbent assay (ELISA) were used for the primary screening of all children (time t 0). The prevalence of the E. histolytica/E. dispar species complex at t 0 was 18 · 8% by single microscopical examination and 31 · 4% using the non-specific ELISA. Approximately 2 months after the initial screening, fresh stool specimens were collected on 2 consecutive days ( t 1, and t 2) from (i) all the children who were positive by microscopy at t 0 ( n = 182) and (ii) 155 randomly selected children who were negative at the primary screening. These samples were tested with a second antigen detection ELISA specific for E. histolytica ( n = 238) and with a species-specific PCR assay ( n = 193). The second and third examinations ( t 1, and t 2) revealed an additional 43 infections with the species complex E. histolytica/E. dispar, so that the cumulative microscopical prevalence for t 1 and t 2 was 27 · 7%. The overall prevalence of E. histolytica by species-specific ELISA antigen detection was low (0 · 83%), while the prevalence of E. dispar was 15%. When analysing only microscopically positive samples by PCR ( n = 129), the ratio E. histolytica: E. dispar was very low (1:46), suggesting that the vast majority of Entamoeba infections in this area were apathogenic. Both species-specific tests performed well but the ELISA was easier to use for large-scale field screening.

Effect of Pneumovax ® II vaccination in high-risk individuals on specific antibody and opsonic capacity against specific and non-specific antigen

Pneumovax ® II vaccine has been used in many countries including the UK for adults in high-risk groups to prevent pneumococcal infections. However, its effectiveness has been a controversial issue. In this study, the specific antibody concentration and opsonic capacity of serum against specific and non-specific antigens were measured in 16 high-risk adults vaccinated with Pneumovax ® II. The results indicated that 38% of these individuals could make an antibody response to specific antigens post-vaccination; vaccination had a clear effect in increasing the opsonic capacity of serum against specific pneumococcal antigen (S14 serotype) in 31% of patients, but did not have any effect against non-specific antigen. The specific antibody concentration did not significantly positively correlate with the opsonic capacity of serum. Therefore, it is concluded that the protective effect of Pneumovax ® II vaccination against a specific serotype in high-risk individuals will only be around 30%.

Biochemical markers of type 1 diabetes: clinical use

In type 1 diabetes, a number of specific and non-specific antigens have been identified. The major autoantigens involved in the destructive process of cells leading to the development of type 1 diabetes are proinsulin/insulin, glutamic acid decarboxylase (GAD) and the transmembrane protein tyrosine phosphatase (IA-2). These are the only autoantigens that partially satisfy the criteria by which an autoantigen or cross-reactive nonself antigen could be evaluated for a pathogenic role in autoimmune diseases. Antigens by definition induce antibody production and in type 1 diabetes, such (auto) antibodies are accepted as biochemical markers for the disease. Here we describe the main features and usefulness of these markers for disease prediction.

Characterization of the impaired antipneumococcal polysacharide antibody production in immunosuppressed pediatric patients following cardiac transplantation.

We previously have demonstrated impaired pneumococcal polysaccharide IgG antibody responses in children immunosuppressed following cardiac transplantation in early childhood. We have further characterized the antibody defect. To further investigate the production of antibody, antipneumococcal polysaccharide (PPS) specific IgM, IgG, IgG subclasses, and IgA were measured in postvaccination sera by enzyme-linked immunosorbent assay. Two groups were studied: posttransplant children who made pneumococcal antibody in vivo following natural exposure or PPS immunization (R) and those with an impaired response (NR). There was no difference in IgM or IgA levels between R and NR. IgG and IgG2 levels were higher in R than NR (P = 0.002), even after adsorption of nonspecific common cell wall antigen antibody. Differences in anti-pneumococcal antibody levels suggest that immunoglobulin isotype switching from IgM to IgG and particularly IgG2 is impaired in patients immunosuppressed at a young age. These findings confirm data regarding the effect of immunosuppressive agents derived from animal models in humans.

TH2 Lymphocytes Secrete Functional VIP upon Antigen Stimulation

In addition to the peptidergic innervation, immune cells may also represent a source for VIP in the lymphoid organs. Previous studies reported increased VIP mRNA and protein expression in mitogen-stimulated B and T lymphocytes. To determine whether specific T cell subsets are responsible for VIP production, we derived TH1 and TH2 effector cell lines from T-cell receptor transgenic mice. TH1 and TH2 cells were stimulated with the specific (pigeon cytochrome C peptide) or nonspecific (ovalbumin) antigen presented by MHC class II compatible antigen-presenting cells. Upon stimulation with the specific antigen, TH2, but not TH1 cells express VIP mRNA and intracelllular VIP protein, as determined by Northern blots and FACS analysis. Supernatants harvested from antigen-stimulated TH2 cells contain secreted VIP, as determined by Elisa, and induce cAMP in HEK293 cells transfected with the specific VIP/PACAP receptor VPAC1. These results confirm that TH2, but not TH1 cells, express and secrete functional VIP following specific antigen stimulation. The release of VIP within the lymphoid microenvironment following antigenic stimulation provides a physiological basis for the immunoregulatory effects of VIP on neighboring immune cells, such as downregulation of macrophage activation, effects on lymphocyte migration, on antigen-induced T cell apoptosis, and on T cell differentiation.

Incomplete Freund's adjuvant reduces diabetes in the non-obese diabetic mouse.

As the study of type 1 diabetes moves towards preventive therapy, the role of adjuvants needs to be addressed. Incomplete Freund's adjuvant (IFA) is thought of as "immunologically inert" as, unlike complete FA (CFA), it has no components designed to provoke an immune response. We investigated the effect of IFA as an immunomodulator on the disease process leading to type 1 diabetes in the non-obese diabetic (NOD) mouse. 24 NOD mice were injected intradermally (i.d.) at 8 and 12 weeks of age with a 1:1 mixture of IFA and saline; 24 controls received saline alone. Splenocytes were tested against antigens thought to be involved in the disease process, namely insulin, a GAD peptide, a beta-casein peptide, a Glut-2 peptide and concanavalin A (ConA) as a non-specific antigen. In the IFA experiment diabetes incidence was 13% compared to 38% in the controls (p < 0.05). In vitro, splenocytes from IFA treated animals showed non-specific immunosuppression with ConA (p < 0.01), whereas the response to 1-casein and Glut-2 was raised in IFA treated animals with respect to controls. ELISA using supernatants from IFA treated animals, showed a typical Th2 cytokine pattern, whereas controls showed a Th1 pattern. In conclusion, IFA alone can reduce diabetes incidence in the NOD mouse apparently by modulating the immune response towards beta-cell related specific antigens. As IFA has been adopted as an adjuvant in preventive trials in the NOD mouse, this might have implications for the interpretation of previous and future results.

Site-Specific Immunosuppression

The cellular mechanisms by which topical cyclosporine A (tCsA) induces site-specific immunosuppression were investigated. Experiments were designed to elucidate how cyclosporine A (CsA) suppresses activated immunocytes in animals that are undergoing local alloactivation and concomitant tCsA immune suppression. Lewis rats received dual Lewis x Brown Norway rat skin allografts; the rats were treated with systemic CsA (sCsA) at 8 mg/kg/day for 10 days after grafting and then tCsA and vehicle thereafter. CsA added to mixed lymphocyte reactions 24 hours after culture initiation modeled the local effects of CsA on alloactivated immunocytes, and tCsA in conjunction with limited sCsA prolonged local skin allograft survival. CsA inhibited both antigen-specific and nonspecific activated alloresponses of immunocytes from animals that had received allografts and that underwent limited sCsA treatment only in a dose-dependent manner. When tCsA had been applied, immunocyte responses to a nonspecific antigen were extremely CsA-resistant as compared with those induced by antigen-specific suppression. However, this nonspecific alloresponse was fully suppressible with the use of elevated CsA doses (66 microg/mL); thus alloresponding immunocytes were significantly more sensitive to CsA if they were challenged with the donor antigen and preexposed to limited sCsA followed by tCsA in vivo.

Immunoglobulin E suppression and cytokine modulation in mice orally tolerized to beta-lactoglobulin.

This study was designed to confirm the tolerogenic properties of beta-lactoglobulin in a mouse model and to assess specific oral tolerance induction in humoral and cellular compartments. BALB/c mice were fed beta-lactoglobulin (BLG) or whey proteins at different ages and subsequently intraperitoneally challenged 5 days later with both BLG and a non-specific antigen, ovalbumin (OVA). Three weeks later, oral tolerance induction was analysed in CMP-fed, versus saline-fed mice, by measuring specific seric and intestinal antibody responses, delayed-type hypersensitivity (DTH), specific splenocyte proliferation, and cytokine secretion patterns. Three-week-old mice fed high doses of either whey proteins or BLG (respectively 3 mg/g or 5 mg/g of body weight) were found to achieve oral tolerization. At humoral and mucosal levels, anti-BLG immunoglobulin E (IgE) were suppressed in these groups when compared with saline fed mice. With respect to cellular responses, systemic DTH and lymphocyte proliferation to BLG were also inhibited in CMP-fed mice. Weaning time was determined to be the best period for oral tolerance induction. Kinetic analyses showed however, that a minimum of 2 weeks was required for oral tolerance detection. Finally, cytokine profiles indicated a reciprocal decrease of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) versus an increase of IL-10 and transforming growth factor-beta (TGF-beta) secretions in tolerized mice. Taken together, these results clearly showed that oral administration of high doses of cows' milk proteins can induce significant hyposensitization in mice, in a specific inhibition of T helper 1 (Th1) lymphocytes with the participation of suppressor cytokines.

Effect of Bacterial Extracts on the Immunologic Profile in Chronic Relapsing Brucellosis Patients

Brucellosis is an intracellular bacterial disease of common incidence in Greece. Existing therapy is inadequate and a considerable proportion of patients become chronically ill and are immunocompromised. Defects of the monocyte-macrophage system and T-lymphocytes have been described in chronic brucellosis and can be restored after immunopotentiation therapy. Bacterial (Klebsiella pneumoniae) extracts exert immunostimulating effects on the monocyte-macrophage system and have already been used successfully in the prevention of common infections of the respiratory track. So we decided to investigate: 1) Leukocyte Migration Index (LMI), 2) Monocyte-macrophage random and directed migration against both nonspecific leukoattractant (casein) and disease specific antigens (Brucella melitensis, Brucella abortus), 3) Monocyte-macrophage phagocytosis index, 4) Delayed-type hypersensitivity (skin tests) against seven antigens, before (TO), during (T2), and after (T3) oral administration of bacterial (Klebsiella pneumoniae) extracts at conventional doses plus antibiotics or not. Our results show that: 1) Concerning the LMI, 4 out of 19 remained anergic at time T3 of the study, 2) Random migration was not affected during treatment, 3) Directed migration increased significantly without reaching control group values, 4) Phagocytosis index increased significantly and reached normal values at T3, 5) Delayed type hypersensitivity reactions (skin tests) increased significantly at the end of the study period. Reaction against Tuberculin and Candida antigens showed the most pronounced increase in skin reactivity. In conclusion, bacterial (Klebsiella pneumoniae) extracts improve peripheral monocyte locomotion and restore phagocytosis index, thus enhancing cellular immunity parameters in immunocompromised chronic brucellosis patients.

Immunological Phenotype Analysis of Patients with Fanconi’s Anaemia and Their Family Members

In contrast to patients affected with Fanconi’s anaemia (FA), who are homozygotes, parental heterozygotes are generally considered normal. In this preliminary work the main immunological phenotypes of circulating mononucleated cells were studied, both in homozygous and heterozygous subjects. The statistical analysis of results showed that two sets of variables may be distinguished: (a) the first set, including CD20, CD4, CD8 cell markers and CD20/CD3 and CD4/CD8 ratios, that was able to differentiate between FA patients and the other subject groups; (b) the second set, including CD25, HLA-DR, HLA-DP, HLA-DQ cell markers, that was able to differentiate healthy subjects from the other groups. Therefore, in contrast with the literature data, immunological abnormalities may already be present in parental heterozygotes of FA patients. These subjects displayed a reduced number of cells expressing both specific (CD25) and non-specific (HLA) antigens. This defect was more severe in FA homozygous patients, who showed, in addition, a reduced total lymphocyte count, reduced levels of T helper (CD4+) and B lymphocytes (CD20+), and a reduced CD4+/CD8+ cell ratio. In conclusion, our results suggest the presence of a grading of immunological defects in FA patients and family members. Our suggestion needs to be confirmed by functional studies.

The Detection of Pseudomonas aeruginosa Using the Quartz Crystal Microbalance

The development of a piezoelectric immunosensor for the detection of Pseudomonas aeruginosa in milk and dairy samples was undertaken here. This was achieved primarily by optimising the system using ELISA, investigating capture, competitive and displacement assays. Results from ELISA supplied information on detection limits and linear ranges obtained with each assay. A displacement assay was chosen to be transferred to the piezoelectric system and the reduction in mass on the surface of the crystal due to antigen displacement was measured by recording the frequency changes of the quartz crystal microbalance. The linear range obtained was from 2x106 cell/ml to 1x108 cell/ml and the limit of detection was 100,000 cells. The system was also tested for cross reactivity with a non-specific antigen, Pseudomonas fluorescens.

Immune Mechanisms of Platelet Refractoriness

Multiple platelet transfusions may prove ineffective in approximately 40% of patients treated for bone marrow aplasia. This condition is known as platelet refractoriness and is diagnosed by evaluating the corrected count increment following platelet transfusion. Immune factors still represent an important cause of platelet refractoriness and, among these, HLA alloimmunization plays the most relevant pathogenetic role. Less important are other specific and non-specific antigens, that can be detected on platelet surface. Several assays have been developed to reveal anti-platelet antibodies, which include the lymphocyte cytotoxicity test, the platelet immunofluorescence assay and the mixed passive hemagglutination assay. It is now clear that leukocytes contaminating the platelet concentrates represent the main cause of HLA alloimmunization which can be prevented by leukocyte depletion to less than 5 × 106 cells per unit. Transfusion of HLA-matched platelets in alloimmunized platelets may be quite effective, but it can also be fairly expensive considering the large number of donors to be typed for HLA in order to find HLA compatible platelets. A more practical approach would be to select the platelet concentrates on the basis of the negative crossmatch.

Immunological enhancement of breast cancer.

Breast cancer is a complex disease. Its aetiology is multifactorial, its period of development can span decades, and its clinical course is highly variable. Evaluation of the role of the immune response in either the development or control of breast cancer is also complex. Nevertheless, there is substantial information that in this disease, the immune response is not a host defence reaction and may even serve to facilitate cancer development. This evidence comes from a variety of sources including clinical-pathological investigations in women that show a correlation between the intensity of lymphocytic infiltration into the tumour mass with poor prognosis, studies in breast cancer patients that demonstrate a similar correlation between delayed hypersensitivity reactivity or in vitro assays of immune reactivity to tumour cell membranes or non-specific antigens and poor prognosis, and analyses of cancer incidence in chronically immunosuppressed, kidney transplant recipients who develop an unexpectedly low incidence of breast cancer. The overall conclusions from these human studies are corroborated by observations in mouse mammary tumour models that also demonstrate immune enhancement of breast cell proliferation in vitro and of breast cancer development in vivo. Potential mechanisms for these effects include production, by inflammatory cell infiltrates, of direct or indirect modulators of breast cell growth, e.g. cytokines, peptide or steroid hormones, enzymes involved in steroid metabolism, as well as of antibodies to growth factors or their receptors. These immune facilitatory mechanisms must be overcome if immune-based therapies are to be applied successfully in breast cancer.

The study of the antigen-antibody reaction by fluorescence method in LB films for immunosensor

The antigen-antibody reaction for immunosensor was investigated in LB lipid monolayers by fluorescence method. It was obtained the difference in mechanisms of interaction between the antibodies in LB monolayer (on modified quartz plate) and the specific, non-specific antigens (in solution). The sensitive element for immunosensor with fluorescent registration was fabricated.

Lymphocyte reactivity to hepatitis C virus (HCV) antigens shows evidence for exposure to HCV in HCV-seronegative spouses of HCV-infected patients.

Lymphocyte reactivity against hepatitis C virus (HCV) antigens was studied in 20 couples in which 1 member had chronic hepatitis C. This was done to investigate the possibility of HCV transmission between spouses that was not followed by seroconversion. Twenty healthy subjects without any risk factors for HCV transmission served as negative controls. All the patients' spouses and the healthy controls were negative for HCV RNA and for anti-HCV antibody. Lymphocytes were cultured with recombinant HCV core and nonstructural antigens (c22, c33, c100, c200, and NS5) and with control antigens (sperm whale myoglobin, chicken lysozyme, and superoxide dismutase). Lymphocytes from 10 patients and 4 seronegative spouses proliferated in the presence of at least one HCV antigen. No proliferation was shown with nonspecific antigens or in the control group. This study gives evidence for possible in vivo priming with HCV antigens that did not lead to seroconversion in spouses of HCV-positive patients.

Summary of serological test results on the monoclonal antibodies to non-specific erythrocytes antigens.

Monoclonal antibodies from the 2D3 Section (Other Non-specific Erythrocyte Antigens) of the Third International Workshop & Symposium on Monoclonal Antibodies Against Human Red Blood Cell and Related Antigens were tested using standard serological tube methods. Each antibody was tested to determine the presence of prozoning. A selected panel of red blood cells was tested against each antibody at different phases. Anti-CD44 specificities were weaker or non-reactive with Lu(a-b-) InLu cells and 2 sources (2D3-1 and 2D3-2) were slightly decreased with Ko cells. Anti-CD47 and antibodies to Rh related molecules showed decreased or no reactivity with Rh null cells. Anti-CD99 showed weak or no reactivity with Gy-, LW:-3 and cord blood cells.

Flow cytometric investigation of non-specific erythrocyte antigens.

Thirteen monoclonal antibodies submitted to the Third Workshop on Erythrocyte Antigens from the panel "non-specific erythrocyte antigens" were tested for their reactivity with different types of cells. Most of them were defined as specific for adhesion antigens. The CD 44 antibodies 2D3-1, 2D3-2, 2D3-3 and 2D3-4 reacted as expected for CD 44 except their negative reactivity with the myeloid cell line HL 60 and B-cell line Raji. The CD 47 antibodies 2D3-5 and 2D3-6 reacted specific. Only with Raji and T-cell line MOLT 4 the CD 58 antibodies 2D3-7 and 2D3-8 showed reactivity as expected which indicates that they are "CD 58 related". The CD 99 antibody 2D3-9 shows similar results as expected for a CD 99 specific antibody except its high reactivity against Raji. From the RBC-related antibodies 2D3-11 and 2D3-12 the latter becomes completely negative with trypsin treated erythrocytes. The antibody is negative on normal peripheral blood lymphocytes but reacts with transformed cell lines like Raji and MOLT 4. With a view to their reactivity to the cells tested at least 2D3-13 of the Rh-related antibodies seems to be similar to CD 47 antibodies.

Surface Marker Analysis in Acute Leukemias

We studied the expression of the cell surface antigen associated with myeloid and lymphoid leukemias on bone marrow or peripheral blood blast cells from 153 leukemic patients including 61 cases of acute myelogenous leukemias(AML), 46 of acute lymphocytic leukemias(ALL) and 12 of acute leukemias. They were analyzed by direct or indirect immunofluorescence method for reactivity with the monoclonal antibodies to B cells(CD10, CD19, SmIg), T cells(CD2, CD5, CD7, CD3, CD4, CD8), myeloid antigen(CD13, CD14, CD33, CD61) and a nonspecific antigen, HLA-DR. Lymphoid associated markers detected on AML is CD7 32.8%, CD10 14.8%, CD5 13.1%, CD2 6.6% and CD19 1.6%. TdT was positive in 4.9% of AMLs. Hybrid leukemias were 8 cases out 61 AML cases and were mainly composed of monocytic lineage, M4 and M5a. Myeloid markers detected in ALL were CD13 2.2% and CD33 2.2%. In this study, immunologically classified ALLs were composed of 65.2% of CALLA (+) B precursor type, 10.9% of CALLA (-) B precursor pattern, 8.7% of T cell type, 2.2% of B cell type, 4.5% of mixed lymphoid lineage(B&T), 2.2% of undifferentiated leukemia, and 6.5% of hybrid leukemia. Twelve cases of acute leukemias ware finally diagnosed to be 5 cases of hybrid leukemia, 3 cases of B lineage, 3 case of T lineage and 1 case of mixed lymphoid(B&T) leukemia. In summary, we think the best method for typing acute leukemias is by using a combination of FAB classification and immunophenotying.