Epstein-Barr viral antigens used in the diagnosis of nasopharyngeal carcinoma

The major antigen complexes of Epstein-Barr virus (EBV) include the latent infectious proteins, early antigens, membrane antigens and viral capsid antigens. The various polypeptides within each antigen complex have been identified and isolated through gene-cloning technology. These polypeptides are exploited to be used as serological markers for the diagnosis of nasopharyngeal carcinoma (NPC) through enzyme-linked immunosorbent assay. This paper reviews the recent studies on the profile of antibodies in patients with NPC towards these EBV polypeptides of each antigen complex. The sensitivity and specificity of each polypeptide when used as serological markers to NPC patients' sera are summarized.     

Investigation of populations heterogeneous according to O-antigen in Vibrio cholerae cultures

The O-antigenic composition of 36 cultures of Vibrio cholerae agglutinating simultaneously with 01 cholera sera and 0 sera to NAG vibrios of the Sakazaki collection was investigated. It has been established experimentally that under the effect of medium and environmental conditions such cultures dissociate to subcultures differing in their affiliation to different serological groups according to 0 antigen. The passage of these cultures in the organism of susceptible animals promotes preservation of 01-group clones whereas the passage in peptone water or prolonged storage under unfavourable conditions result in the predominance of clones of different serological affiliation. The proposition has been put forward that the observed vibrio cultures are genotypically capable of producing, besides the 01 group, a number of 0 antigens. Phenotypical manifestation of the antigenic structure in the respective individuals of the population depends on the conditions of the environment.     

Evaluation of Micro Complement Fixation Tests for Antibodies Against Group A Streptococcal M and M-Associated Antigens in Rabbit and Human Sera

The microslide gel-diffusion and macro-tube agglutination techniques to detect Brucella canis antibodies in dogs were compared. Sera from dogs experimentally infected with B. canis and a random sample of dog sera with unknown histories of exposure to this organism were examined. The results of the gel-diffusion method employing specific rough Brucella saline-extract antigens of B. canis and Brucella ovis were comparable to those obtained by the tube agglutination test. The easily prepared, stable R antigen in the freeze-dried form offers a convenient, simple, and reliable diagnostic method for the serological detection of canine brucellosis by the gel-diffusion test.     

Further environmental factors affecting the antigenicity of Trichophyton rubrum

Biochemical determinations performed on ammonium sulfate soluble and insoluble fractions of crude mycelial extracts ofTrichophyton rubrum indicated that these antigens were either carbohydrates or carbohydrates with peptide side chains. The antigens contained considerable amounts of galactose and mannose.Gel filtration techniques proved to be effective in separating these antigens. One antigen had a molecular weight greater than or equal to 2.0 × 10⁶. A smaller, more reactive antigen was also found; however, the elution time of this antigen varied with the concentration of dextrose in the medium.Quantitative precipitation tests used to differentiate the serological reactivity of crude antigens, recovered from mycelia grown on media containing variable concentrations of dextrose, indicated that the serological reactivity of the crude antigen was inversely proportional to the concentration of the carbohydrate source, with an optimum reactivity occurring with antigens prepared from mycelia grown in low dextrose concentrations.Nitrogen and carbohydrate concentrations performed on whole mycelia and cell free extracts demonstrated that the total nitrogen, soluble nitrogen, total carbohydrate and soluble carbohydrate concentrations were influenced by the concentration of the carbohydrate source. The optimum carbohydrate concentration necessary for the maximum ratio of protein and carbohydrates per gram of mycelium was 15.0 g/l. This is less than the amount used in most Sabouraud's dextrose media formulations. The effect of these environmental factors on the serological reactivity was discussed.Supported in part by NIH Environmental Health Tranining Grant ES00081-02. The help of Mrs. Gary Swecker is gratefully acknowledged for preparing the graphs.     

Toxoplasma gondii-positive human sera recognise intracellular tachyzoites and bradyzoites with diverse patterns of immunoreactivity

Antibody detection assays have long been the first line test to confirm infection with the zoonotic parasite Toxoplasma gondii. However, challenges exist with serological diagnosis, especially distinguishing between acute, latent and reactivation disease states. The sensitivity and specificity of serological tests might be improved by testing for antibodies against parasite antigens other than those typically found on the parasite surface during the acute stage. To this end, we analysed the reactivity profile of human sera, identified as positive for anti-Toxoplasma gondii IgG in traditional assays, by indirect immunofluorescence reactivity to acute stage intracellular tachyzoites and in vitro-induced latent stage bradyzoites. The majority of anti-Toxoplasma gondii IgG positive sera recognised both intracellularly replicating tachyzoites and in vitro-induced bradyzoites with varying patterns of immune-reactivity. Furthermore, anti-bradyzoite antibodies were not detected in sera that were IgM-positive/IgG-negative. These results demonstrate that anti-Toxoplasma gondii-positive sera may contain antibodies to a variety of antigens in addition to those traditionally used in serological tests, and suggest the need for further investigations into the utility of anti-bradyzoite-specific antibodies to aid in diagnosis of Toxoplasma gondii infection.     

Autoantibody biomarkers in the detection of cancer

By definition, tumor biomarkers are selective molecules that can distinguish between patients with cancer and controls. Serum tumor markers have been the most widely used approach for cancer detection. However, the limitations of these markers, which are based on the measurement of tumor antigens, preclude their general use in cancer screening and diagnosis. Here we give an overview of recent cancer biomarker developments based on the detection of autoantibodies produced against tumor antigens in patients' sera. This new detection method can measure the autoantibodies for a spectrum of tumor antigens in a single assay, with sensitivity and specificity exceeding those obtained using the conventional antigen determination method. Autoantibodies against serum cancer biomarkers offer a novel technology for cancer detection.     

Detection of polysaccharide, teichoic acid, and protein antigens in bacterial colonies on an agar surface

An improved method of using fluorescein-labeled antibody for the detection of polysaccharide, protein, and teichoic acid antigens synthesized by streptococcal colonies on an agar surface is described. The bacteria were grown on the surface of an agar medium contained in the shallow well of an immunodiffusion slide. An agar overlay containing the fluorescein antiserum was dispensed over the colonies, excess antiserum was washed out of the overlay agar, and the fluorescent colonies were observed under an ultraviolet microscope. The shallow well in the immunodiffusion slide prevented the agar from floating loose during washing, and the agar overlay prevented the fragmentation and loss of colonies. The thin layer of agar facilitated microscopic examination and the counting of fluorescent and nonfluorescent colonies. Colonies producing an antigen against which the antiserum was directed could readily be distinguished from colonies not producing the antigen. The specificity of the method was shown by using mixtures of streptococci representing six serological groups and five types. Those not known to possess cross-reacting antigens were specific in their reaction to the fluorescein antibody. Cross-reactions between the group antigens of A, C, and G, as reported previously by fluorescent staining of streptococcal suspensions, were also seen. Group A colonies reacted weakly with fluorescent E antibody and vice versa. The extraction of this antigen with cold trichloroacetic acid indicates it was related to the teichoic acids. Colonies possessing polysaccharide, protein, and teichoic acid antigens gave equally strong fluorescent reactions. This procedure permits detection of the synthesis of antigen which could not be observed by the use of a selective medium; it also eliminates the necessity for subculture of each colony and testing by appropriate serological means. Such a technique has value for studies in classification and biochemical genetics, and should be applicable to other genera of bacteria.     

The increased presence of repetitive motifs in the KDDR-plus recombinant protein,a kinesin-derived antigen from Leishmania infantum,improves the diagnostic performance of serological tests for human and canine visceral leishmaniasis

Visceral leishmaniasis (VL) is caused by protozoa belonging to the Leishmania donovani complex and is considered the most serious and fatal form among the different types of leishmaniasis, if not early diagnosed and treated. Among the measures of disease control stand out the management of infected dogs and the early diagnosis and appropriate treatment of human cases. Several antigens have been characterized for use in the VL diagnosis, among them are the recombinant kinesin-derived antigens from L. infantum, as rK39 and rKDDR. The main difference between these antigens is the size of the non-repetitive kinesin region and the number of repetitions of the 39 amino acid degenerate motif (6.5 and 8.5 repeats in rK39 and rKDDR, respectively). This repetitive region has a high antigenicity score. To evaluate the effect of increasing the number of repeats on diagnostic performance, we designed the rKDDR-plus antigen, containing 15.3 repeats of the 39 amino acid degenerate motif, besides the absence of the non-repetitive portion from L. infantum kinesin. Its performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and rapid immunochromatographic test (ICT), and compared with the kinesin-derived antigens (rKDDR and rK39). In ELISA with human sera, all recombinant antigens had a sensitivity of 98%, whereas the specificity for rKDDR-plus, rKDDR and rK39 was 100%, 96% and 71%, respectively. When evaluated canine sera, the ELISA sensitivity was 97% for all antigens, and the specificity for rKDDR-plus, rKDDR and rK39 was 98%, 91% and 83%, respectively. Evaluation of the ICT/rKDDR-plus, using human sera, showed greater diagnostic sensitivity (90%) and specificity (100%), when compared to the IT LEISH (79% and 98%, respectively), which is based on the rK39 antigen. These results suggest that the increased presence of repetitive motifs in the rKDDR-plus protein improves the diagnostic performance of serological tests by increasing the specificity and accuracy of the diagnosis.     

Serum Antibodies to the Catalase Antigen of Aspergillus fumigatus in Cattle

Aspergillus fumigatus is an important agent of mycotic infection in cattle and a potent source of antigens. However, the efficacy of serological diagnosis of aspergillosis in cattle remains controversial. Corbel (1972) and Knudtson et al. (1974) considered a precipitin assay useful as a supplementary test in the diagnosis of mycotic abortion, whereas Wiseman et al. (1984) found the specificity too low to justify its routine use. We have studied 1) the antibody response to the catalase antigen of A. fumigatus in experimentally infected cattle and 2) the prevalence of catalase antibodies and A. fumigatus precipitins in healthy and diseased cattle. The aim was to ascertain how far detection of antibodies to a defined fungal antigen can contribute to the often difficult diagnosis of mycosis.     

Serological differentiation between Echinococcus granulosus and E. multilocularis infections in man

An enzyme-linked immunosorbent assay (ELISA) was adapted for the serological differential diagnosis of cystic or alveolar echinococcosis in man caused by Echinococcus granulosus or E. multilocularis respectively. By affinity chromatography using rabbit anti hydatid fluid IgG coupled covalently to CNBr-Sepharose 4B a protein fraction (Em 1) containing shared antigens of both parasites could be isolated from an extract of E. multilocularis metacestode tissue. From the same source another antigen fraction (Em 2) with a high degree of specificity for E. multilocularis was prepared by immunosorption. Antigen Em 1 was equally sensitive for the detection of antibodies against E. granulosus and E. multilocularis, whereas antigen fraction Em 2 appeared to be more specific for E. multilocularis. A correct serological differential diagnosis was achieved in 95% of 57 confirmed cases of human cystic or alveolar echinococcosis by the simultaneous use of both antigen fractions in the ELISA and by comparison of their reactivities.     

结核分枝杆菌抗原蛋白的研究及其应用新进展

结核病(tuberculosis, TB)是由结核分枝杆菌(Mycobacterium tuberculosis, MTB)感染引起的慢性传染病,是仅次于正在暴发的新型冠状病毒肺炎(COVID-19)的第二大单一感染致死病因。COVID-19的大流行对TB的诊断及治疗造成了破坏性的影响,全球实现终结TB目标的进展偏离了轨道。因此,早诊断、早治疗依然是防控TB蔓延的关键。TB精准诊断一直受MTB抗原特异性、检测技术特异性和灵敏度的影响,因此亟需挖掘高特异性新抗原、开发新检测技术。随着蛋白质基因组学(proteogenomics)和质谱技术的快速发展,从临床体液、组织样本中高效、精准靶向检测MTB特异性已知、甚至新抗原的表达,以及监测治疗过程中的抗原表达量的动态变化,是TB诊断及治疗的发展趋势。在MTB标准菌株H37Rv的4 008个注释基因中(NC＿000 962.3, NCBI),国内外报道的已注释抗原虽有140多个,但仅有极少的抗原应用于TB的筛查及辅助诊断,离世界卫生组织(World Health Organization, WHO)的诊断标准尚远。本文通过对MTB已报道抗原以及基...     

Luminescent-serologic analysis of the antigenic interrelationship between R. canada and classic representatives of rickettsiae of the typhus group

The results of studying the antigenic relationships of R. canada, a new Rickettsia species, and classical Rickettsia species of the typhus group are presented. The study was carried out by luminescent serological analysis with the use of corpuscular antigens and the live infectious agent cultures. R. canada and Rickettsiae of the typhus group were similar in their antigenic structures; this, however, could be revealed only in the study of the live cultures of the infectious agents. The study of corpuscular antigens revealed unilateral relationship: R. prowazeki antigen could be detected with homologous and heterologous sera, R. canada antigen with homologous serum only. In the CFT and the agglutination test corpuscular R. canada antigen reacted with homologous and heterologous sera. The study of the live cultures of the infectious agents revealed that different R. prowazeki and R. typhi strains vary in the degree of their similarity to R. canada.     

嗜肺巴氏杆菌外膜蛋白和脂多糖抗原在血清学诊断中的意义

目的评价嗜肺巴氏杆菌外膜蛋白(OMP)和脂多糖(LPs)作为血清学诊断抗原的敏感性和特异性.方法用OMP、LPS和全菌(WC)作为Western blot和ELISA的诊断抗原检测自然感染和实验感染嗜肺巴氏杆菌小鼠相应的IgG抗体滴度,同时测定3种抗原与实验动物常见致病菌的交叉反应.结果与嗜肺巴氏杆菌自然感染和实验感染小鼠血清的ELISA反应中,不同时期,LPS作为诊断抗原时血清抗体阳性率最高,WC次之,OMP最低.自然感染小鼠群中,出生4周LPS抗体阳性率即可达80%,而同期的WC和OMP仅为25%和20%,故LPS敏感性最高.与实验动物常见致病菌免疫血清和阴性种鼠血清的ELISA反应中,WC抗原表现出较高的吸光度(A)值,经Western blot证实,其反应为非特异性反应,LPS抗原特异性最强,OMP抗原次之.结论混合多株具有型或种特异性的OMP或LPS作为ELISA的诊断抗原,无论从特异性和敏感性上均高于全菌抗原.     

Detection ofLegionella antibodies by enzyme-linked immunosorbent assay (ELISA) using whole cell and carbohydrate antigens

The systematic study ofLegionella as a human pathogen and a bacterium widely disseminated in the environment requires simplification of present methodology. We describe a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the detection of serum antibodies that can also be used for the detection of antigen.Legionella pneumophila serogroups 1 and 3 (Philadelphia 2 and Bloomington 2),L. bozemanii (WIGA), andL. micdadei (TATLOCK) were grown in diphasic medium consisting of charcoal yeast extract agar (CYE) overlayed with yeast extract medium (YEM) for the production of whole cell antigen and CYE for the extraction of carbohydrate antigen. The whole cells were inactivated with 0.5% formalin. The carbohydrate was obtained from the supernatant of cells resuspended twice in phosphate buffered saline (PBS). The antigen was sterilized and concentrated by filtration and purified by chromatography through a Sepharose 4B column. The highest molecular weight fractions were used for chemical characterization, which confirmed the carbohydrate nature of the antigen, and for micro-ELISA. Titers ranging from 5×10³ to 3×10⁵ (inverse of serum dilutions) were obtained from rabbit sera collected after 1, 2, or 3 injections of whole cells. The titers were somewhat higher and more consistent with the higher of 2 antigen concentrations used (5 or 15g/ml protein or dry weight), and with the carbohydrate rather than the whole cell antigen. The reactions were serogroup and species specific and only low titers were obtained with some of the heterologous antigens. The sensitivity and specificity of the reactions were not diminished when as many as 4 antigens were mixed in the same well. Thus, the micro-ELISA can be used as a test of highly specific antigens as well as a screening test with mixtures of antigens. A preliminary test withLegionella containing water specimen concentrates and high-titer rabbit sera indicated that the micro-ELISA can also be used for the detection of antigen. This investigation appears to have paved the way for the simplification of the serological methodology for the study ofLegionella. On temporary leave from: Department of Microbiology, University of Maryland School of Medicine, 660 West Redwood Street, Baltimore, MD 21201.     

Immunochemistry of Yersinia enterocolitica O3 grown at different temperatures.

Comparative agglutinations of homogeneous stable suspensions prepared with Yersinia enterocolitica growth at 37 degrees C and at 25 degrees C were performed with anti-sera prepared in rabbits with the bacteria grown at both these temperatures. Sera prepared with live Y. enterocolitica grown at 37 degrees C agglutinated both suspensions at a much lower titre than the sera prepared with formaldehyde-treated bacteria is grown at 25 degrees C. All the sera in which strongly precipitating antibodies were induced reacted, in agar-gel, against native and heated proteins. The small amounts of antipolysaccharides induced in all the sera reacted only in the ring test against the bacterial polysaccharides. The absorption of the sera prepared with live Y. enterocolitica grown at 37 degrees C, with antigens synthesized at 25 degrees C did not remove all the homologous antibodies; apparently, some determinants are specific for the bacteria grown at 37 degrees C. Morphological changes of the small rods to elongated bacilli and filamentous forms were observed in most cultures of the Y. enterocolitica grown at 37 degrees C; these changes coincided with a low yield of proteins and point to an inhibitory effect of the 37 degrees C temperature.     

ELISA for the detection of specific IgM and IgG in human leptospirosis

ELISA was used to detect specific IgM and IgG in sera from humans with current or past leptospirosis. A serological pattern of a high IgM titre (greater than or equal to 1280), or moderately increased IgM (160-640) in conjunction with a low IgG titre (less than or equal to 20), with serovar copenhageni antigen was characteristic for approximately two-thirds of the sera from serovar icterohaemorrhagiae patients obtained in the first two months of the disease. The antigen was the supernatant of a heated and centrifuged culture of leptospires. Antigens were prepared from serovars copenhageni, grippotyphosa, hardjo and patoc. Sera from patients with icterohaemorrhagiae, grippotyphosa and hardjo infections showed cross-reactivity when different antigens were used. In past infections the IgG titres were clearly higher with the homologous antigen. ELISA for IgM and IgG allows the rapid diagnosis of acute leptospirosis.     

Cross reaction of recombinant equine infectious anemia virus antigen to heterologous strains and application for serological survey among horses in the field

Cross reactivity of equine infectious anemia virus (EIAV) antigen prepared using a recombinant baculovirus containing the p26 gene of strain P337-V70 was examined by the agar gel immunodiffusion (AGID) test and enzyme-linked immunosorbent assay (ELISA). Serum samples serially collected from 13 horses experimentally infected with six different EIAV strains (two or three horses per strain) were subjected to the test. Positive reactions were observed in the AGID test and ELISA before or soon after the first feverish period and continued persistently in most of the horses. The results with recombinant antigens were essentially the same as those with the virion antigen prepared from horse cell cultures both in the AGID test and ELISA. The reactivities of the antigens were further compared using serum samples collected from horses in 1999 in certain districts of Mongolia where equine infectious anemia has been prevalent, and from horses in Japan in 1973 when EIA had not been eliminated completely from Japanese horses. These results were completely concurrent. Generally, recombinant antigens have high specificity but low cross reactivity to heterologous strains. However, the present study showed that the recombinant EIAV p26 antigen has cross reactivity to the heterologous strain and is useful for diagnosis of EIA in the field.     

Isolation of a New Soluble Antigen from the Yeast Phase of Histoplasma capsulatum

A method is described by which a soluble antigen was prepared from the yeast phase of Histoplasma capsulatum. This soluble preparation had a specificity greater than that of whole-cell yeast-phase antigens. In complement fixation tests with sera from human cases of histoplasmosis, blastomycosis, and coccidioidomycosis, the soluble antigen reacted in 12.1% of 141 tests with heterologous sera, whereas conventional whole-cell yeast antigens reacted in 47.3% of 91 tests with heterologous sera. The reactivities of the two types of antigens with homologous sera were essentially the same.     

Synthetic Peptides Mimic gp75 from Paracoccidioides brasiliensis in the Diagnosis of Paracoccidioidomycosis

Paracoccidioidomycosis (PCM) is a systemic granulomatous disease, endemic in Latin America, caused by the thermal dimorphic fungus Paracoccidioides brasiliensis. Although some fungal antigens have already been characterized and used for serological diagnosis, cross-reactions have been frequently observed. Thus, the examination of fungal forms in clinical specimens or isolation of P. brasiliensis by culture is still the most frequent method for the diagnosis of this mycosis. In this study, a random peptide phage display library was used to select mimotopes of P. brasiliensis, which were employed as antigens in an indirect enzyme-linked immunosorbent assay. The protective monoclonal antibody against experimental PCM (anti-gp75) was used as molecular target to screen a phage display library. That approach led to a synthetic peptide named P2, which was synthesized and tested against PCM patients' sera to check whether it was recognized. There was significant recognition of P2 by sera of untreated PCM patients when compared with normal human sera. Sera from treated PCM group, patients with other mycosis or co-infected with HIV had much lower recognition of P2 than untreated patient group. The test showed a sensitivity of 100 and 94.59% of specificity in relation to human sera control. These data indicate a potential use of P2 as diagnostic tool in PCM. Its application for serological diagnosis of PCM may contribute to the development and standardization of simpler, faster and highly reproducible immunodiagnostic tests at low cost.     

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.