Low Molecular Weight Antigens Research Articles

Purification and Characterization of IgM-like Immunoglobulin from Turbot (Scophthalmus maximus L.)

A total of 40 turbot (Scophthalmus maximus) were immunized 3 times during a 3 months period using DNP-HSA whereafter serum samples were collected and pooled. Specific immunoglobulins (Ig) were affinity purified on an agarose column with immobilized DNP-BSA and further purified by gel filtration whereafter monospecific rabbit anti Ig serum was generated. Size exclusion chromatography and non-reduced SDS-PAGE indicated a MW of 8-900 kDa of the dominant antigen binding proteins from turbot serum. Reduced SDS-PAGE showed this fraction to be composed of disulphide linked heavy and light chains with MWs of 79 and 27-29 kDa, respectively, indicating a tetrameric structure. Isoelectric focusing of the 800-900 kDa Ig showed several bands between pH 5.5 and pH 5.8. Mean Ig concentration in serum of 10 turbot was measured to 6.48 mg/ml (SD 5.4) using rocket immunoelectrophoresis. Low molecular weight antigen binding molecules were copurified with the dominating immunoglobulins with an estimated MW of 500 kDa. Reducing SDS-PAGE of this fraction revealed molecules with MWs of 97, 79, 57, 29, and 27 kDa.

Immunolocalization and characterization of the low molecular weight antigen (4-5 kDa) of Toxoplasma gondii that elicits an early IgM response upon primary infection.

A striking feature of toxoplasmic seroconversion is the prominent and early IgM response to a low molecular weight antigen of 4-5 kDa. Two different monoclonal antibodies directed against the 4-5 kDa antigen have been generated and used to characterize this molecule. Using these monoclonal antibodies, we could demonstrate the surface localization of the low M(r) antigen by immunofluorescence and immuno-electron microscopy assays. By immunoblotting, we observed that one of the monoclonal antibodies was unable to recognize the 4-5 kDa antigen in tachyzoites propagated in cell culture, indicating an epitope variability between Toxoplasma gondii tachyzoites grown in vivo and in vitro. We discuss the implications of this latter finding in the design of diagnostic reagents.

Serological distinction between syphilis and Lyme borreliosis

The serological distinction of an immune response to the agent of syphilis, Treponema (T.) pallidum, and Lyme disease borreliae is difficult due to the existence of cross-reacting antibodies. In this study, the immunoblot technique is used to compare the immune response of sera from patients with syphilis or Lyme borreliosis to either T. pallidum or Borrelia (B.) burgdorferi. Patients with syphilis showed an IgG response to the 17 and 15 kDa antigens of T. pallidum, whereas sera from patients with Lyme borreliosis-especially in late stages of the disease-reacted with the 95 and 19-17 kDa antigens of B. burgdorferi. Thus, IgG antibodies against low molecular weight antigens of T. pallidum (17, 15 kDa) or B. burgdorferi (19-17 kDa) may represent useful markers for the serological diagnosis of syphilis or Lyme borreliosis.

An immunodominant 30-kDa antigen of a candidate anti-leprosy vaccine,Mycobacterium w, shares T and B cell determinants withM. leprae andM. tuberculosis

Earlier we reported that vaccination of leprosy patients with Mycobacterium w induces an immune response directed predominantly against low molecular weight antigens. One of these antigens, with a molecular mass of 30-kDa, was recognized by a majority of the vaccinated subjects as well as the tuberculoid leprosy patients and healthy contacts. In the present communication we report further characterization of this antigen. Immunofluorescence and Western blot studies with antibodies raised against this antigen demonstrate that it is associated with the cell surface and has homologues present in M. leprae and M. tuberculosis. Delayed-type hypersensitivity studies carried out in guinea pigs immunized with the 30-kDa antigen show that in addition to sharing B cell determinants, this immunodominant antigen of M. w also shares T cell determinants with M. leprae and M. tuberculosis.

Anisakis simplex: antigen recognition and antibody production in experimentally infected mice

The kinetics of antibody response to intraperitoneal infection of mice with third stage larvae of Anisakis simplex was investigated by ELISA. Maximum antibody response to excretion-secretion (ES) antigens was reached before maximum response to somatic (SA) antigens. Total immunoglobulin (Ig) production (consisting mainly of IgM and IgG1 isotypes) was very similar in both cases. Immunoblotting was used to characterize the antigens recognized by the host in the presence or absence of the metabolic products released by the parasite in vivo. Sera from mice infected with live larvae (anti-L3 L serum) and immunized with dead larvae (anti-L3 D serum) recognized a similar pattern of bands in immunoblots of ES and SA antigen preparations. In the latter, however, three bands at 14, 17 and 18 kD were only recognized by the anti-L3 L serum. A possible explanation is that these low molecular weight antigens are ES products released only in vivo. Finally, the immune response in mouse was compared using ELISA and immunoblotting with the response of a human anisakiasis reference serum, and was found to display considerable similarities. This suggests that the mouse may be a useful model for studying the immunobiology of A. simplex in man.

Identification and initial characterization of Pneumocystis carinii soluble antigens in rabbit serum and lung lavage

Lung lavage and serum samples from Azathioprin-treated (acute-phase infection) and untreated (non acute-phase infection) rabbits were used in the immunoblotting technique to look for Pneumocystis carinii (Pc) soluble antigens, using rabbit polyclonal antibodies raised against rabbit-derived Pc antigens and labeled with peroxidase. Analysis of the supernatant of lavage fluid after centrifugation to sediment intact organisms revealed components of approximately 80, 60-65, 55, 39 and 27 kDa in acute-phase samples. The components in the regions of 80, 60-65, 55 kDa and to a lesser extent 39 kDa were also present in non acute-phase lung lavage samples. In acute-phase serum samples, a major component of 80 kDa and minor components of about 65 and 39 kDa are detectable. The 80 and 65 kDa components are also detectable in some of the serum samples from the untreated rabbits. Immunofluorescent staining with FITC-conjugated affinity-purified antibodies to the 80, 60-65, 55, 39 or 27 kDa-components showed that they shared epitopes with both Pc cysts and trophozoites. The affinity-purified antibodies also cross-reacted in immunoblotting with several antigens in the Pc whole preparations. The putative Pc soluble antigens in serum and lung lavage were then isolated by affinity chromatography with polyclonal antibodies to Pc. Preliminary characterization of the column-extracted antigens revealed complete inactivation by trypsin whereas only the 55 and 80 kDa antigens bind to Concanavalin A. In conclusion, the results of this study suggest that Pc soluble antigens are present even in non acute-phase samples and only the low-molecular weight antigens (39 and 27 kDa) seem specific for the acute-phase. These findings are consistent with previous investigations reported by others that development of Pc could occur in nonimmu-nosuppressed rabbits.

Purification and Characterization of Alt a-29 from <i>Alternaria alternat</i><i>a</i>

A major protein component reactive with pooled human atopic sera was isolated from a lyophilized broth extract of Alternaria alternata 34-016. By successive chromatography on Whatman DE-52, Sephadex G-100 and Mono Q HR5/5, a low molecular weight antigen was obtained. Comparison with standard proteins on Sephadex G-100 indicated its molecular weight was 31 kD. Non-reduced samples run on SDS-PAGE showed a band at 29.2 kD which reacted strongly with human IgE. After reduction, it produced a doublet pattern on SDS-PAGE with MW 14.5 and 16.0 kD. The doublet pattern was confirmed by Western blotting with pooled human atopic sera. IEF of the protein showed a major component with a PI of 4.15 and two minor components at 4.25 and 4.40. Immunoblots of the IEF bands showed all three were reactive with human IgE. Ion exchange chromatography of the protein on Mono Q HR5/5 resulted in three resolved components, all of which are immunoreactive. Together with the IEF data, this suggests that there are several conformational or structural isoforms of this protein.

Localization of a Low Molecular Weight Antigen of Eimeria tenella by Use of Hybridoma Antibodies

Three monoclonal antibodies (Mabs), found by western blot analysis to recognize 10-kDa bands of Eimeria tenella sporozoite preparations, were used with immunoelectron (IE) microscopy, immunogold-silver staining (IGSS), and indirect immunofluorescent antibody (IFA) light microscopy to determine the location and distribution of the antigens in or on extra- and intracellular parasites. All 3 of the Mabs (designated C3, E5, and 1231) were found by IE microscopy to label amylopectin granules of extracellular sporozoites. Additionally, these Mabs extensively gold-labeled the sporocyst wall. In cultured primary chicken kidney cells inoculated with sporozoites of E. tenella, IGSS showed surface labeling of the parasite and intense labeling of the infected host cells by 6 hr postinoculation (PI). At 24 hr PI, host cell vacuoles in infected and uninfected cells were labeled by the 3 Mabs by IFA. The E5 and C3 Mabs also were seen to label the host cell membrane of newly infected cells. The C3 and 1231 Mabs showed little label of the host cells by 48 hr PI, but the parasites still were labeled up to 96 hr PI. The E5 Mab had intense IFA labeling of infected host cells at 48 hr PI. The results of this study indicate that parasites apparently release antigenic material during the early stages of parasite development and that this material is found internally and/or on the surface of the infected host cells.

The intestinal and serum humoral immune response of mice to orally administered antigens in liposomes: II. The response to liposome-entrapped bacterial proteins

Effective oral adjuvants are needed to improve the intestinal immune responses to oral vaccines that are based on relatively low molecular weight antigens refined from veterinary pathogens. Liposomes prepared by different methods and composed of phospholipids of varying transition temperature were used to entrap cholera toxin (CT) and fed to mice. No significant increase in the intestinal antibody nor the serum IgA antibody response was detected but levels of serum IgG anti-CT antibody were significantly elevated in the group fed CT in phosphatidylcholine-based liposomes. Levels of antibody were significantly reduced in the groups fed CT in dipalmitoylphosphatidylcholine liposomes. Escherichia coli wall extract (ECWE) entrapped in certain liposome types and fed to mice elicited significantly increased serum anti-ECWE antibody responses but intestinal antibody responses were insignificantly different from the controls. These results suggest that orally administered liposomes fail to act as potent intestinal adjuvants for the entrapped antigens of bacterial origin used in this study.

T‐Cell Responses to Fractionated Antigens of Mycobacterium w, a Candidate Anti‐Leprosy Vaccine, in Leprosy Patients

Mycobacterium w, an atypical cultivable mycobacterium, is undergoing phase III clinical trials as a vaccine against leprosy in India. It has brought about lepromin conversion and histopathological upgradation in a significant number of patients studied so far. It is important to identify antigens of M. w that trigger T-cell responses in leprosy patients vaccinated with this organism. In the present study the peripheral T-cell repertoire of 12 M. w-vaccinated leprosy patients, 10 unimmunized leprosy patients, 8 tuberculoid and 5 healthy contacts was analysed with fractionated antigens of M. w. The lepromatous leprosy patients who are in general anergic to antigens of M. leprae did not respond to antigens of M. w. However, peripheral blood mononuclear cells obtained from leprosy patients who had been vaccinated with M. w responded to many antigens. These responses were frequently directed against low molecular weight entities of 14-45 kDa. T cells from tuberculoid leprosy patients and healthy contacts also responded predominantly to a number of low molecular weight antigens of M. w. The study also identified an immunodominant 28-31 kDa antigenic fraction carrying T- as well as B-cell activating determinants.

Development of a serologic assay for cysticercosis, using an antigen isolated from Taenia spp cyst fluid

SUMMARY An ammonium sulfate-soluble fraction of Taenia hydatigena cyst fluid (Thfas) was further evaluated for use in the immunodiagnosis of cysticercosis. Analysis of Thfas by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein immunoblot analysis confirmed earlier reports of a highly specific, low molecular weight antigen in this preparation; in contrast, other components of Thfas were shown to react nonspecifically. Antibodies against the < 12-kD diagnostic antigen were detected in sera from 10 cattle and 4 swine inoculated with metacestodes of T saginata and T solium, respectively, but not in animals inoculated with Fasciola hepatica, Trichinella spiralis, Brucella abortus, or Toxoplasma gondii, or in noninoculated controls. Isolation and immobilization of the < 12-kD antigen on a hydrophobic transfer membrane resulted in development of an unambiguous dipstick assay capable of correctly identifying fully developed (10-week) experimentally induced infections in cattle and swine. In addition, the dipstick assay was highly specific for diagnosis of the disease in human beings, and offers the potential of distinguishing between human clinical cases of cysticercosis and taeniasis. A similar reactive antigen of diagnostic potential was also identified and isolated from T crassiceps and T taeniaeformis cyst fluids.

Delayed-Type Hypersensitivity Antigens Detected in Culture Supernatants of <i>Salmonella</i><i>typhimurium</i>

Protein antigens eliciting delayed type hypersensitivity (DTH) were analyzed and purified from the supernatants of protein-free cultures in which Salmonella typhimurium TV148 organisms had grown. DTH activity was measured by the footpad swelling test in mice immunized with living organisms of S. typhimurium TV148 or Escherichia coli K-12. DTH activity in the culture supernatant was specific to TV148-immunized mice. This activity was destroyed by pronase. DTH activity was unable to pass through an ultrafilter with an exclusion limit of 10 kD. After condensation of the supernatant and following centrifugation (100,000 g for 1 h), the DTH activities of the sediment and the supernatant were examined, and both showed DTH activity. Further analyses of DTH antigens in the supernatant by HPLC gel filtration separated the activity into three portions. The most active portion was further fractionated by hydroxyapatite HPLC, revealing the presence of two DTH antigens, with molecular weights of 65 and less than 10 kD. These results indicate that the culture supernatant of S. typhimurium TV148 organisms contains a variety of macromolecular protein DTH-eliciting antigens, and one of the antigens is 65 kD, which is dissociated partly by organic solvents into a low molecular weight (less than 10 kD) antigen.

Isolation and structural characteristics of a monoclonal antibody-defined cross-reactive phospholipid antigen from Mycobacterium tuberculosis and Mycobacterium leprae.

A low molecular weight antigen of Mycobacterium leprae and other mycobacteria was previously defined in our laboratory by means of IgG2a monoclonal antibody termed L4. The antigen had an apparent molecular mass of 4.5-6 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was assumed to be a glycoprotein on the basis of its staining with periodic acid Schiff and sensitivity to periodate treatment. In the present work, the cross-reactive and phospholipidic nature of the antigen, present in Mycobacterium tuberculosis as well as in M. leprae sonicates, was demonstrated and this enabled us to undertake its purification from crude M. tuberculosis phospholipidic extracts. The L4-reactive antigen from M. tuberculosis called L4-PIM, was purified by means of silicic acid high pressure liquid chromatography. Its characterization by gas chromatography and FAB-MS showed the antigen to be the common mycobacterial dimannosylated phosphatidylinositol (PIM2), the structure of which had been previously established by others (Lee, Y. C., and Ballou, C. E. (1964) J. Biol. Chem. 239, 1316-1327; Lee, Y. C., and Ballou, C. E. (1965) J. Biochem. (Tokyo) 4, 1395-1404). Delineation of the L4 epitope on M. tuberculosis L4-PIM revealed the involvement of the axial 2-hydroxyl of the alpha-D-mannosyl residues, without any detectable contribution from the myo-inositol. Consequently, L4 was shown to react with PIM5, the structure of which contains twice the number of epitopes as does PIM2. By using both immunostained thin layer chromatography and indirect enzyme-linked immunosorbent assay, similar L4-PIM epitopes were demonstrated in M. leprae sonicate, thereby explaining the cross-reactive nature of the L4-monoclonal antibody. Antibodies of IgG class directed against M. tuberculosis L4-PIM were detectable in sera from patients with leprosy, but no evidence of T cell reactivity to L4-PIM was obtained. The demonstration of a correlation of anti-L4-PIM IgG and anti-disacharide-conjugated bovine serum albumin IgM antibody titers in the sera of leprosy patients indicates that measurement of antibodies directed against L4-PIM may have the potential to be used as a complementary assay to the disaccharide-conjugated bovine serum albumin test for diagnosis and monitoring of patients undergoing leprosy therapy.

A continuous flow immunoassay for rapid and sensitive detection of small molecules

An immunosensor operating in continuous flow and capable of detecting low molecular weight antigens is described. The approach differs from previously described continuous flow assays by not requiring incubation steps or the introduction of additional reagents following the loading of the sample into the system. Detection of the antigen is rapid, occurring within 3 min in the system described. The assay is based on the binding of labeled antigen to an immobilized antibody, with subsequent displacement of the labeled antigen when antigen is present in the buffer flow. Signal detection occurs downstream of the antigen recognition event. In this study, the hapten 2,4-dinitrophenol DNP as (DNP)-lysine was used as model antigen. To generate a labeled antigen, DNP was coupled to the terminal amino group of insulin A chain (tetra S-sulfonate form) which provides two tyrosine residues for the introduction of an 125I-label (DNP-Ins- 125I) or three carboxyl groups for the attachment of three fluorescein residues (DNP-Ins-Fl). The radiolabeled antigen was used to establish assay conditions. Subsequently, fluorescein was substituted for the radioisotope label in order to develop an assay independent of the restrictions associated with isotopes. Using this flow immunoassay, we were able to detect DNP-lysine down to a detection limit of 143 nM (29 pmol/200 μl) using DNP-Ins- 125I or DNP-Ins-Fl as labeled antigen. The density of immobilized antibody and the flow rate were identified to be critical parameters for the sensitivity of the assay.

Protection against Leishmania major in BALB/c mice by adoptive transfer of a T cell clone recognizing a low molecular weight antigen released by promastigotes.

We have shown previously that BALB/c mice can be protected against a fatal infection with Leishmania major by adoptive transfer of a T cell line recognizing a protective soluble fraction (fraction 9) of promastigotes. We now describe the isolation and characterization of a T cell clone (9.1-2) that also transfers protective immunity against Leishmania. After Ag or mitogen stimulation, this clone secrets IL-2 and IFN-gamma, but not IL-4 or IL-5. The clone preferentially recognizes L. major fraction 9, and in addition, soluble Ag from Leishmania donovani, Leishmania amazonensis, and Leishmania braziliensis, but not from the related flagellates, Trypanosoma cruzi or Crithidia fasciculata. Besides being contained in fraction 9, the stimulatory Ag is also released from the parasite, because concentrated promastigote culture supernatants induced IFN-gamma production by 9.1-2. By means of T cell immunoblotting, we determined that 9.1-2 recognized a protein with a relative m.w. between 8,000 and 12,000, and within this region is a predominant protein contained in fraction 9 of approximately 10,000 m.w. These data identify a new candidate Ag for immunization against protozoa belonging to the genus Leishmania.

Guidelines for preparation and characterization of chemical-protein conjugate antigens: Report of the Subcommittee on Preparation and Characterization of Low Molecular Weight Antigens

Occupational asthma is a significant cause of morbidity in the workplace for which immunologic and nonimmunologic causative agents have been identified.’ For nonimmunologic forms of occupational asthma, a diagnosis may rely on a careful occupational history accompanied by documentation of work-related changes in pulmonary function associated with exposure to the causative agent. In some cases in which immunologic markers are not identifiable, diagnostic confirmation of occupational asthma may require specific bronchoprovocation challenge testing to the suspected causative substance. However, occupational immunologic lung disease (OILD) can be diagnosed with the assistance of either in vivo or in vitro immunologic tests. This report will present recommended guidelines for preparing and characterizing antigens that can be useful for the diagnosis of OILD caused by reactive low molecular weight (LMW) chemicals. These guidelines are based on established methodologies used to prepare antigens with LMW agents that have already been recognized to cause OILD. The acid anhydride, diisocyanate, and precious metallic salt groups of chemicals are prime examples of such substances that have been successfully used to prepare suitable antigens for both in vivo or in vitro testing of workers with OILD.’

Pneumocystis Antigen Appearance during Immunosuppression

ABSTRACTThe sequential appearance of Pneumocystis carinii (Pc) antigens during the progression of immunosuppression in rats was studied using the immunoblotting technique and specific immunologic probes. Putative Pc soluble antigens, with molecular weights of approximately 70 and 90 kd, were detected as early as 2 wk after initiation of immunosuppression in rats using a pool of monoclonal antibodies produced to Pc isolated from lungs using enzymatic digestion. Monoclonal antibodies produced to Pc isolated by massaging the lung tissue using a Stomacher apparatus and infecuon‐derived sera did not detect soluble antigens until at least the 6lh wk of immunosuppression. Analysis of Pc pellets obtained from Stomacher‐ and lavage‐processed lungs revealed that the lower molecular weight antigens ( 40, 45 and possibly 55–60 kd) were recognized early during the immunosuppression process.

Pneumocystis antigen appearance during immunosuppression.

The sequential appearance of Pneumocystis carinii (Pc) antigens during the progression of immunosuppression in rats was studied using the immunoblotting technique and specific immunologic probes. Putative Pc soluble antigens, with molecular weights of approximately 70 and 90 kd, were detected as early as 2 wk after initiation of immunosuppression in rats using a pool of monoclonal antibodies produced to Pc isolated from lungs using enzymatic digestion. Monoclonal antibodies produced to Pc isolated by massaging the lung tissue using a Stomacher apparatus and infection-derived sera did not detect soluble antigens until at least the 6th wk of immunosuppression. Analysis of Pc pellets obtained from Stomacher- and lavage-processed lungs revealed that the lower molecular weight antigens (less than or equal to 40, 45 and possibly 55-60 kd) were recognized early during the immunosuppression process.

Interferon Review

Although IFN proteins were recognized first for their potent antiviral properties, it has now been established that they may profoundly affect other vital cellular functions. The IFNs are divided into three main classes, alpha, beta, and gamma, and are defined by their differences in amino acid sequences, physicochemical properties, and induction by different agents from different cell types. The inducing agents include viruses, bacteria, bacterial products, polymers, low molecular weight compounds, and antigens or mitogens. Studies on the mechanisms of action of IFNs have mainly been focused on their antiviral actions. However, many of the facts revealed by these studies are equally relevant for understanding other actions of IFN. IFNs are extremely potent, they interact with specific receptors, and they induce the expression of specific genes, the products of which mediate their various actions. There is almost a complete lack of knowledge of what happens between the interaction of IFN with its receptor and induction of new RNA synthesis. However, we are beginning to understand how some of the IFN-inducible enzymes impair viral replication. The discovery of the dsRNA-dependent enzymes has implications beyond the IFN system. It is quite possible that they are used for other physiologic regulatory systems as well. The identities and functions of many other IFN-inducible proteins remain to be elucidated. Principally, IFNs alpha and beta are cytokines in that they may be produced by the cellular components of the immune system and have immunoregulatory effects on the cells of the immune system. These effects include enhancement of surface structures such as histocompatibility antigens, pleiotropic hormone-like effects, and stimulation or inhibition of the activities of a number of different effector cells such as B cells, T cells, macrophages, and natural killing cells. IFN levels may be below detection and yet mediate important biologic functions. Perhaps the most interesting IFN subtype regarding immunoregulation is IFN gamma, which is a product of T lymphocytes. Few drugs have stimulated as much research interest or clinical promise as the IFNs. Clinical trials in patients have shown most promise in coryza, herpes virus infections, papilloma virus tumors, hairy cell leukemia, multiple myeloma, and renal cell carcinoma. IFN gamma employed alone and in combination with IFN alpha may dramatically increase IFN's activity. IFN treatment combined with chemotherapy also may give enhanced antitumor activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Isolation of an antigenic fraction for diagnosis of Onchocerciasis

A surface enriched fraction was prepared from adults of Onchocerca volvulus by brief extraction of entire worms with detergent. This was then gel filtered to yield a low molecular weight fraction which functioned specifically in ELISA analysis. An identical result was also obtained when the related cattle parasite, O. gibsoni, was similarly fractionated and tested. The low molecular weight fraction contained at least four antigenic components when examined by coprecipitation and immunoblotting studies. One ml of packed worms yielded sufficient low molecular weight antigen to examine about 2,000 human sera by the ELISA procedure, and the test was sensitive at human serum dilutions down to 1/400. A preliminary study with individual sera from Onchocerciasis endemic and non-endemic areas of Southern Mexico yielded 0/24 false positives, 3/24 false negatives and a significant ELISA value in 21/24 sera from proven cases of Onchocerciasis.