Rapid and simultaneous detection of Ureaplasma parvum and Chlamydia trachomatis antibodies based on visual protein microarray using gold nanoparticles and silver enhancement

Based on gold-labeled silver stain (GLSS) method, we developed the visual protein microarray for simultaneous, sensitive, and specific detection of Ureaplasma parvum and Chlamydia trachomatis using N-terminus multiple-banded antigen (NMBA) of U. parvum and major outer membrane protein of C. trachomatis. The specific antigens were immobilized on glass surface that was treated with 3-glycidoxypropyltrimethoxysilane, and they were used as the capturing probes to recognize the complementary target antibodies binding to the detecting probes of Nano-gold-Staphylococcal protein A (SPA). In the “sandwich” format, Nano-gold-SPA probe was used as an indicator and GLSS was applied to amplify the detection signals and produce black image on array spots, which were visible with naked eyes. In our model arrays, the detection limit of protein microarray was as low as 2 ng/mL, and the lowest titer of detectable antibody was 1:128; thus, this sensitivity was comparable to the fluorescent detection method. The visual simultaneous protein microarrays were used to detect total 186 clinical samples, which had been determined by enzyme-linked immunosorbent assay (ELISA) and fluorescence quantitative real-time polymerase chain reaction; the results were identical and no distinct difference (P > 0.05) existed between them. Our results demonstrate that we have developed the visual protein microarray technique, which is of high sensitivity and high specificity, and it may have potential in clinical applications.     

Parallel detection of autoantibodies with microarrays in rheumatoid diseases

BACKGROUND: Clinical needs often dictate testing for several autoantibodies in a single patient with evidence of autoimmune disease. We developed a microarray containing 15 autoantigens for the detection of autoantibodies in rheumatoid autoimmune diseases. METHODS: We synthesized recombinant centromere protein B, cytokeratin 19, SSA 52-kDa antigen, SSA 60-kDa antigen, SSB antigen, and Jo-1 antigen and prepared anti-nuclear antibody antigens. Cyclic citrullinated peptide, histone, goat IgG for detection of rheumatoid factor, double-stranded DNA, and single-stranded DNA were purchased, as were recombinant small nuclear ribonucleoprotein U1, topoisomerase I, and Smith antigen (Sm). All 15 antigens were of human origin except calf thymus Sm. Proteins were printed on polystyrene. The arrays were incubated with serum samples and then with horseradish peroxidase-conjugated secondary antibodies and chemiluminescent substrates, and light signals were captured by a charge-coupled device camera-based chip reader. Antibodies were quantified by use of calibration curves. Positive samples were confirmed by commercially available methods. RESULTS: The detection limit of the microarray system was 20 pg of IgG printed on the polystyrene support. More than 85% of the confirmed positive sera were detected as positive with the microarray system based on cutoff values established with the microarray system. The imprecision (CV) of the microarrays was <15% for all 15 autoantibody assays, with the exception of single-stranded DNA (18% and 23%) within and between batches. Characteristic autoantibody patterns were seen in patients with clinical diagnoses of rheumatoid arthritis (n = 83), systemic lupus erythematosus (n = 71), systemic sclerosis (n = 36), polymyositis (n = 38), and Sjogren syndrome (n = 20). CONCLUSIONS: This microarray system provides results similar to those by conventional methods. Assessment of the diagnostic accuracy of the system remains to be done.     

Follow-up of HIV-infected individuals by semiquantitative detection of antibodies to p24 in ELISA

The purpose of this study was to develop and evaluate an enzyme-linked immunosorbent assay (ELISA) test for the semiquantitative detection of antibodies to p24 in the serum of human immunodeficiency virus (HIV)-infected individuals and AIDS patients. The assay is based on a recombinant p24 antigen coated onto polystyrene plates, and uses a protein-A/peroxidase conjugate as probe. The titres of antibodies to p24 were cross-sectionally studied in a cohort involving 35% of the total of Cuban HIV-seropositive individuals, including AIDS patients. Of these, 123 individuals were followed up for 3 yr. Our results confirm that anti-p24 low responders are at higher risk for the progression of disease to AIDS than high responders. In 68.4% of the group IV patients antibody titres were under 80, with statistically significant differences between this group and cases in groups II (P<10⁻⁵) and III (P<10⁻⁵). All 22 individuals who died with AIDS-related illnesses during the study had anti-p24 titres lower than 160. The new assay is an alternative tool in the follow up of HIV-seropositive individuals.     

An optical pickup enzyme-linked immunosorbent assay (ELISA) with a microfluidic disk

We evaluated optical pickup ELISA with an original microfluidic disk that contains eight radially arranged channels, which enable semi-automatic sample loading and washing. This disk-shaped chip composed of acrylic plates was fabricated by CO₂ laser machining and capture antibodies were immobilized in the channels. After the immunoreaction with antigens and enzyme-linked secondary antibodies, an enzyme-catalyzed nanoaggregation of o-phenylenediamine was detected by measuring the reflectivity change of a laser beam focused in the channel. The assay of C-reactive protein (CRP) was successfully performed in a short amount of time (approximately 20 min from CRP loading). The limit of detection was determined to be 2 ng mL⁻¹, which is more sensitive as compared with conventional ELISA using microplates.

Optical pickup ELISA with an original microfluidic disk, which enable semi-automatic sample loading and washing, was developed. The rapid and sensitive assay of C-reactive protein (CRP) was successfully performed.     

Study on the antigens and antibodies of Mur and Mia blood groups in southern China

BackgroundThe frequency of Mur and Mi^a blood group antigens in Asian population is much higher than that in Caucasian population. However, due to the scarcity and high price of commercial detection reagents, there are few studies on antigen and antibody detection and comparative analysis in large samples.ObjectiveTo study the occurrence frequency, antigen correlation and antibody properties of Mur and Mi^a antigens and their corresponding antibodies in southern China.MethodsMur and Mi^a antigens and antibodies in local blood donors and patients were detected by routine serological microplate method. Statistical methods were used to calculate the incidence of two antigens and antibodies and analyze their correlation.ResultsAmong blood donors, the positive rates of Mur and Mi^a antigens were 6.4 % and 6.5 % respectively, with no significant difference (P > 0.05). In this region, the incidence of anti-"Mur" and anti-"Mi^a" was 0.65 % and 0.45 % respectively. But significant difference existed between blood donors and patients (P < 0.05). Among the anti-"Mur" and anti-"Mi^a" positive patients, most of the antibodies were IgM or IgM + IgG mixed type and had saline activity.ConclusionMur and Mi^a antigens and their corresponding antibodies have a high frequency in the population of southern China. In the routine clinical detection of irregular antibodies, Mur or Mi^a positive (GP.Mur) should be added to screen erythrocytes. Moreover, given the high correlation between Mur and Mi^a antigens expression on red blood cells, using monoclonal antibodies against Mi^a could predict the presence of Mur antigen.     

STUDIES ON THE BINDING BETWEEN STREPTOCOCCAL M PROTEIN AND ANTIBODY

A method for detecting type-specific antibody to type 12 β-hemolytic streptococci is described. The method is based on the utilization of a partially purified I¹³¹ labeled streptococcal acid extract and the solubility of this extract in 40 per cent saturated ammonium sulfate as compared to the insolubility of antibody and antigen-antibody complexes at this salt concentration. The salient features of this technique are: (a) When absorbed sera were used, no non-type-specific reaction with antigen occurred. (b) The sensitivity of the system allowed detection of type-specific antibody in hyperimmune antisera that had been diluted 1200-fold. (c) The method does not rely on secondary manifestations of antigen-antibody interaction for the detection of antibody. (d) As little as 0.03 µg of M₁₂ protein was detected when soluble unlabeled M protein was used to inhibit the reaction between I¹³¹ M₁₂ protein and anti-M₁₂ antibody. (e) The kinetics of the reaction between M protein and anti-M antibody can be studied, thereby making information available as to the quality as well as the quantity of antibody produced in this antigen-antibody system.     

Development of up-converting phosphor technology-based lateral-flow assay for rapidly quantitative detection of hepatitis B surface antibody

An up-converting phosphor technology-based lateral-flow (UPT-LF) assay system was developed for rapid and quantitative detection of hepatitis B surface antibody (HBsAb). To evaluate its performance, we compared it with the Abbott Axsym AUSAB (ABBOTT Diagnostics Division, Wiesbaden, Germany) assay and conventional ELISA (Wantai Biological Pharmacy Enterprise, Beijing, China) using 13 standard positive sera and 306 clinical sera. In both laboratory evaluation and clinical application, UPT-LF assay showed the best sensitivity (99.19%) and detection agreement (97.43% for the adjusted agreement) with true results. The concordance rate between UPT-LF and ELISA, as shown by correlative regression analysis, was the highest (R² = 0.6389), whereas that between UPT-LF and AUSAB was the lowest (R² = 0.5702). In conclusion, UPT-LF assay for quantitative detection of HBsAb is sensitive and rapid, promising this new assay a bright future.     

Utility of a combination of RD1 and RD2 antigens as a diagnostic marker for tuberculosis

We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel detection of antigens and antibodies in the serum samples of pulmonary tuberculosis (PTB) patients resulted in higher sensitivity as compared to either of the single tests in both smear-positive (90%) and smear-negative (60%) PTB patients. In addition, combined detection of antigens and antibodies in the fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the patients with high specificity. These results demonstrate the ability of the combination of antigen and antibody detection assays based on the cocktail of RD antigens to diagnose a substantial number of PTB and EPTB cases with high specificity.     

Effect of centrifugal enhancement of infectivity on the rapid detection of human cytomegalovirus in cell culture by immunofluorescence using a monoclonal antibody to an immediate early nuclear antigen

A murine monoclonal antibody to a human cytomegalovirus (HCMV) immediate early nuclear antigen was used to detect HCMV in human diploid fibroblast cultures inoculated with the laboratory-adapted HCMV strain AD169 or clinical specimens and examined at 24 and 48 h post-infection (p.i.) by indirect immunofluorescence. The effect on the sensitivity of this rapid diagnostic test of centrifugation of the inoculum onto the cell monolayer was evaluated, and the results obtained were compared with conventional virus isolation.Centrifugal enhancement of infectivity significantly increased the sensitivity of HCMV immediate early nuclear antigen detection at 24 h p.i. with both AD169 (P < 0·001) and clinical specimens (P < 0·05). The 48 h test with centrifugation provided additional sensitivity, but was less sensitive than 28-day conventional virus isolation with AD169 (significant, P < 0·001) and clinical specimens. HCMV immediate early nuclear antigen detection was significantly more likely than routine virus isolation to produce a result with clinical specimens (P < 0·002).HCMV immediate early nuclear antigen detection at 24 and 48 h p.i. with centrifugation represented an ideal combination of rapidity and sensitivity for the early diagnosis of HCMV infection.     

Development of a gold-nanorod-based lateral flow immunoassay for a fast and dual-modal detection of C-reactive protein in clinical plasma samples

Fast and simple detection of C-reactive protein (CRP) is highly significant for the diagnosis and prognosis of inflammatory or infectious diseases. Lateral flow immunoassay has the advantages of rapid detection, simple operation and low cost, but it is usually limited by the quantitative ability and speed of data extraction. Herein, a gold-nanorod-based lateral flow immunoassay was developed to rapidly detect CRP by simultaneously monitoring the colorimetric and temperature signals. In this method, anti-CRP antibody-modified gold nanorods (GNRs) were designed as colorimetric and photothermal conversion probes. A mouse anti-CRP monoclonal antibody and goat anti-mouse IgG were used as test and control lines, respectively. Then, a lateral flow immunochromatographic strip was constructed by a sandwich-type method for detecting CRP by introducing antibody-modified GNRs, and this procedure needed less than 15 min. Finally, the detection signals can be directly observed by eyes and directly read using a thermal imager. The as-synthesized GNR showed high photothermal conversion efficiency (η = 39%) and strong localized surface plasmon resonance (LSPR) absorption. For CRP detection, the proposed immunochromatographic strip exhibited good specificity, high sensitivity, good linearity within the range of 50–10 000 ng mL⁻¹ and a low limit of detection (LOD, 1.3 ng mL⁻¹). This method was successfully applied for CRP detection in clinical plasma samples, and it correlated very well with the diagnostic kit of immunoturbidimetry (r = 0.96). The results indicated that the developed GNR-based immunochromatographic strip has immense potential for use as a rapid and cost-effective in vitro diagnostic kit.

A gold-nanorod-based lateral flow immunoassay for rapid and quantitative detection of CRP by simultaneously monitoring the colorimetric and temperature signals.     

CHARACTERIZATION OF A HUMAN ACUTE PHASE PROTEIN FOUND IN ASSOCIATION WITH RUBELLA VIRUS INFECTION

A precipitating antigen, rho, was first detected in the blood of persons with rubella and in rubella virus-infected cell culture fluids (1). Partially purified antigens from both sources were examined and shown to have similar properties, although antigen from serum sedimented more heterogeneously, with estimated coefficients from 15 to 21 S, while that from culture fluids sedimented in the 11–14 S region. In each case, antigen was located in the β-1 zone after electrophoresis in agarose, and at a density of 1.305 g/ml after centrifugation in CsCl. Stability characteristics were typical of protein antigens. Immunofluorescent microscopy revealed that rubella virus induced the appearance of rho antigen scattered throughout the cytoplasm of infected cells. When cells containing antigen were exposed for 24 h to 5 µg/ml actinomycin D rho was no longer detectable, indicating the probable cellular origin of the antigen. Also, titers in medium of infected cultures showed a reduction after actinomycin treatment, but levels of the virus-specified antigen, iota, were relatively unaffected. Rho appears to be a protein common to man and many animals. In vitro, it was induced by rubella virus and by adenovirus. In vivo, rho titers were shown to be elevated after rubella virus infection and, to a lesser extent, after infection with certain other viruses. High titers were also demonstrated in women late in pregnancy and in patients with rheumatoid arthritis. In man and the chimpanzee, the appearance and decline of rho in the blood after rubella virus infection were temporally similar to the patterns of CRP, although rho seemed to be a more sensitive indicator of infection. The data presented indicate that rho is a newly recognized acute phase protein inducible by certain virus infections and by other unidentified stimuli present prominently in pregnancy and rheumatoid arthritis.     

High diagnostic accuracy of antigen microarray for sensitive detection of hepatitis C virus infection

BACKGROUND: Hepatitis C virus (HCV) can be transmitted through blood transfusion. Screening ELISA, the most widely used method for HCV diagnosis, sometimes yields false-positive and false-negative results, so a confirmatory test is used. This secondary testing is labor-intensive and expensive, and thus is impractical for massive blood bank screening. Therefore, a new massive screening method with high accuracy is needed for sensitive and specific detection of HCV. METHODS: With sol-gel material, we designed novel antigen microarray in 96-well plates for HCV detection. Each individual well was spotted with 4 different HCV antigens. We used this new system to test 154 patient serum samples previously tested for HCV by ELISA (87 HCV positive and 67 HCV negative) (HCV EIA3.0, ABBOTT). We assessed the detection limit of our microarray system with the use of serial 10-fold dilutions of an HCV-positive sample. RESULTS: Our microarray assay was reproducible and displayed higher diagnostic accuracy (specificity) (98.78%) than did the ELISA (81.71%). Our method yielded significantly fewer false-positive results than did the ELISA. The detection limit of our assay was 1000 times more sensitive than that of the ELISA. In addition, we found this novel assay technology to be compatible with the currently employed automated methods used for ELISA. CONCLUSION: We successfully applied the sol-gel-based protein microarray technology to a screening assay for HCV diagnosis with confirmatory test-level accuracy. This new, inexpensive method will improve the specificity and sensitivity of massive sample diagnosis.     

Strain- and Age-Associated Variation in Viral Persistence and Antibody Response to Mouse Parvovirus 1 in Experimentally Infected Mice

The effect of mouse strain and age at infection on viral replication and concurrent antibody response to mouse parvovirus 1 (isolate MPV1f) was evaluated for 305 d after inoculation in 4 strains of mice. The results confirmed previous reports that mouse strain and age at infection are significant factors in viral persistence and antibody development and detection. Random-bred Arc:Arc(s) mice originally bred from CD1 stock inoculated as juveniles (4 wk) or adults (8 wk) developed persistent viral infection for 152 d after inoculation and an antibody response that persisted for 295 d. Mice of C57BL/6J background inoculated as juveniles had detectable viral DNA in large intestinal content and tissues for 24 d after inoculation and an antibody response that persisted for 288 d. However, viral DNA was not detected in tissues of C57BL/6J mice inoculated as adults, although an antibody was detected for 111 d after inoculation; these results suggest probable viral replication in adult C57BL/6J mice but at levels below the limits of detection. BALB/cArc mice inoculated as juveniles or adults had detectable virus DNA in tissues for 108 to 242 d after inoculation, but no antibody was detected. Similarly, BALB/c-Foxn1^nu/Arc mice had detectable levels of viral DNA in tissues for 98 to 131 d but no measurable antibody. The difficulty of detecting antibody in mice with a BALB/c background indicates they are unsuitable for routine surveillance of MPV1f infection.Abbreviation: MPV1, mouse parvovirus 1, rNS1, recombinant nonstructural protein 1, rVP, recombinant virion (capsid) proteinInfection with mouse parvovirus 1 (MPV1) typically is asymptomatic³ but considered to cause dysfunction of T lymphocytes and alter the pattern of graft and tumor rejection.⁸ The detection of MPV1 in and its eradication from laboratory mouse colonies is therefore crucial and requires an understanding of the kinetics of viral replication in different mouse strains.Mouse-strain–associated variation in the antibody response to MPV1 infection has been reported¹ and an understanding of the extent of this variation is important in routine serologic surveillance. Seroconversion was affected by age at infection and the serological assay used but was detected in all C3H mice but only some ICR, DBA/2, and BALB/c mice 4 wk after experimental infection with MPV1b.¹ MPV immunofluorescent and MPV hemagglutination-inhibition assays found seroconversion only in C57BL/6 mice inoculated with high doses of virus.¹Antibody was detected in C57BL/6J mice when an ELISA based on a recombinant structural protein, virion protein 2 (rVP2),⁷ was used, but mice with a BALB/c background continued to manifest a poor antibody response when rVP2 was used as the ELISA antigen.⁵We have described the detection of a variant MPV1 (MPV1f) in a commercial laboratory mouse colony in Australia.² By using recombinant biotinylated truncated VP1 protein (rVP1) as an antigen for ELISA and Western immunoblots, we found that seroprevalence varied among the various strains in the infected colony. Inbred DBA/2JArc, B6D2F1 hybrid, and random-bred Hsd:NIH and Arc:Arc(s) strains yielded high seroprevalence, but strains with a BALB/c background showed low seroprevalence, and antibody was not detected in strains with a C57BL background other than in an F1 C57BL × DBA/2JArc hybrid.To develop an improved understanding of the pathogenesis of infection and determine whether the inability to detect antibody in some strains was associated with the lack of replication, we examined the kinetics of viral replication of MPV1f and the associated antibody response in inbred C67BL/6JArc mice and BALB/cArc and BALB/c-Foxn1^nu/Arc (nude) mouse strains and random-bred Arc:Arc(s) mice; this population originally was bred from CD1 stock (Charles River Breeding Laboratories, Kingston, NY). Antibody had been detected in naturally infected Arc:Arc(s) mice and in some C57BL/6JArc mice but not BALB/cArc mice and, as expected, not in BALB/c-Foxn1^nu/Arc (nude) mice. To determine the effect of age at infection, we infected mice at 4 wk (juveniles) or 8 wk (adults) of age. All mice were examined for 10 mo after infection.     

变性梅毒重组抗原免疫印迹技术的建立及其在鉴别梅毒血清学假阳性实验的应用

目的 为了评估临床梅毒抗体筛查中出现假阳性实验结果的真实性,建立了变性梅毒重组抗原免疫印迹技术,并应用于对临床非高危人群梅毒抗体筛查阳性血清进行验证确认,分析出现假阳性的可能性。方法 采用梅毒TP47,TP45,TP17和TP15重组抗原,将变性后的抗原与非变性的抗原配对包被在硝酸纤维膜载体上,建立梅毒免疫印迹抗体谱检测方法。收集临床经电化学发光免疫分析仪及配套试剂检测的梅毒螺旋体抗体假阳性的非性病高危人群门诊和住院患者血清样本56例、临床确认梅毒患者血清样本25例和健康体检人员样本30例进行方法学验证。并用该方法对1例特殊的“梅毒患者”进行了鉴别。结果 根据建立的变性与非变性抗原配对梅毒免疫印迹抗体谱检测结果判定模式,56例临床梅毒螺旋体抗体筛查假阳性样本,对四种变性重组抗原均不发生反应,无阳性条带。但是对二种以上非变性抗原发生弱阳性反应,出现阳性条带;25例梅毒患者对照样本,对四种非变性和变性重组抗原均有反应,出现强阳性条带;30例健康体检人员样本,对四种非变性和变性重组抗原均无反应。梅毒螺旋体特异性抗体阳性特殊“梅毒患者”血清样本,对四种变性重组抗原均为阴性反应,但对四种非变性重...     

THE ENHANCING EFFECT OF THE MICROBIAL FLORA ON MACROPHAGE FUNCTION AND THE IMMUNE RESPONSE : A STUDY IN GERMFREE MICE

The immune response to bacteria and to a soluble protein was compared in germfree and conventionalized mice. Sixty germfree and 59 conventionalized mice received a suspension of killed Serratia marcescens into one front foot-pad and sterile horse ferritin into the other and were sacrificed in groups from 2 hr to 14 days after inoculation. All mice had no pre-existing antibody to either antigen and the flora of the conventionalized mice never contained Serratia. Lymphatic tissue changes and the fate of the antigens were followed in axillary lymph nodes and the spleens by histologic, fluorescent antibody, and autoradiographic techniques after tritiated thymidine injection. Individual serum antibody titers for both antigens were determined at each time period. The cellular and serologic responses were slightly delayed in the germfree mice but later equaled and sometimes exceeded those of the conventional animals. In all animals, lymph nodes draining the site of Serratia injection showed a more vigorous response than those on the ferritin-injected side but the reaction was qualitatively the same for both antigens. All lymph nodes contained the antigens by 2 hr after foot-pad injection. With time, both antigens lost their particulate nature sooner in conventionalized than in germfree macrophages. In the latter, both antigens persisted throughout the study while no longer demonstrable with fluoresceinated antiserum in conventional macrophages after the first week. While phagocytosis is equal in germfree and conventional mice, a greater digestive capacity of macrophages for antigens seems to result from the continuous exposure of conventional animals to the immunologic effects of the microbial flora. Conversely, the lack of substantial antigenic stimulation of lymphatic tissue in germfree animals fails to develop these macrophage functions beyond their basic ability to degrade foreign substances. Although the onset of the immune response is delayed in germfree mice, the relatively prolonged antigen digestion and the presumably slower release of immunogenic antigen fragments result in a more sustained and sometimes greater response than in conventional animals. This modifying effect of the microflora on the function of macrophages during the immune response is independent of previous experience with, or the nature of, the antigen.     

Evaluation of five Legionella urinary antigen detection kits including new Ribotest Legionella for simultaneous detection of ribosomal protein L7/L12

Urinary antigen tests are a widely used rapid diagnostic method for Legionella pneumonia. However, conventional urinary antigen tests are unable to detect anything other than Legionella pneumophila serogroup 1. The Ribotest Legionella (Ribotest) can detect all serogroups by using antibodies recognizing L. pneumophila ribosomal protein L7/L12 in addition to the conventional L. pneumophila serogroup 1 lipopolysaccharide. The aim of this study was to evaluate the performance of Ribotest against conventional urinary antigen tests, including the detection of Legionellaceae other than L. pneumophila. We investigated the detection sensitivity of various kits using in-vitro culture-soluble antigen extracts of ATCC strains and 22 clinical isolates collected from multiple medical facilities in the Kinki region of Japan. For L. pneumophila serogroup 1, four kits, including Ribotest, had a detection sensitivity of 10⁵ CFU/mL, with only Check Legionella having a sensitivity of 10⁶ CFU/mL. L. pneumophila non-serogroup 1 and Legionellaceae of other species were undetectable by the four conventional kits, whereas Ribotest could detect them with a sensitivity of 10⁵–10⁸ CFU/mL. The Ribotest was also able to detect other species such as Legionella hackeliae, Legionella feeleii, Legionella anisa, Fluoribacter bozemanae, and Fluoribacter dumoffii, but the detection sensitivity of L. hackeliae and L. feeleii was 10⁸ CFU/mL, which was much lower than that of the other strains. The Ribotest has high potential to be applied as a rapid diagnostic method for pneumonia caused by other species of Legionella and Fluoribacter.     

Antigen detection assay with parasite specific monoclonal antibodies for diagnosis of lymphatic filariasis

BackgroundLymphatic filariasis is a painful and profoundly disfiguring disease. Infection is usually acquired in childhood but its visible manifestations occur later in life, causing temporary or permanent disability. The importance of developing effective assays to diagnose, monitor and evaluate human lymphatic filariasis has been emphasized by the WHO.MethodsHigh-affinity monoclonal antibodies (mAbs) specific for recombinant filarial antigen WbSXP-1 were developed. An ELISA based capture assay using monoclonal and polyclonal antibodies for WbSXP-1 was used for detection of circulating filarial antigen.ResultsHigh-affinity monoclonal antibodies (mAbs) were developed that specifically binds both W. bancrofti and B. malayi mf antigens. Two mAbs (1F6H3 and 2E12E3) of subclass IgG2a and IgM showed high affinity, avidity and reactivity to recombinant and mf native antigen. Both the mAbs were used in combination as capture antibodies and polyclonal as detection antibody to develop the assay. The assay showed very high sensitivity towards W. bancrofti mf positive samples compared to endemic normal samples (P < 0.0001).ConclusionA capture assay using high-affinity monoclonal antibodies for WbSXP-1 was developed for the detection of filarial circulating antigen in clinical samples from bancroftian infection. Besides, this would also help in epidemiological studies in endemic areas of filarial infections.     

STRUCTURE OF TYPE 5 ADENOVIRUS : I. ANTIGENIC RELATIONSHIP OF VIRUS-STRUCTURAL PROTEINS TO VIRUS-SPECIFIC SOLUBLE ANTIGENS FROM INFECTED CELLS

Type 5 adenovirus was purified by fluorocarbon (freon 113) treatment followed by banding in a CsCl equilibrium density gradient. This method permitted separation of virus from normal host cell materials and virus-specific soluble antigens. Virus banded in CsCl with a mean bouyant density of 1.3349 gm/cm³. The three virus-specific soluble antigens (group- and type-specific antigens and toxin) banded together with a mean bouyant density of 1.2832 gm/cm³. The group-specific antigen was the predominant antigen of the purified virus particle, whereas the group- and type-specific antigens were present in equal titers in the antigen band. Infectious virus particles were inactivated by prolonged dialysis at pH 10.5. Centrifugation of inactivated virus preparations in a CsCl equilibrium density gradient resulted in separation of virus DNA from specific antigen: the antigens banded with a mean bouyant density of 1.2832 gm/cm³ and the DNA sedimented to the bottom of the tube. The predominant antigen derived from purified virus particles was the group-specific antigen and it was in the same relative proportion to the type-specific antigen as measured in intact particles. The antigens derived from disrupted virus were immunologically identical with the soluble virus antigens present in infected cells.     

Molecular genotyping of Indian blood group antigens amongst regular voluntary blood donors of Surat city,Gujarat, India

BackgroundThere is paucity of data related to the prevalence of the rare blood group antigens amongst South Gujarat blood donor population due to unavailability and high cost of antisera. Therefore it is difficult to screen donors for such rare antigens by gold standard haemagglutination assay. The single nucleotide polymorphism (SNPs) of In^a and In^b antigens is the base of the PCR based detection methods that help to detect these alleles in regular voluntary blood donors.Materials & methodsBlood samples of 200 unrelated regular voluntary blood donors wee collected. DNA was extracted using phenol-chloroform method and genotyped for Indian (In^a/IN*01, In^b/IN*02) blood group alleles by Sequence Specific PCR. In^a antigen positivity was confirmed by serology test.ResultsFour donors were found heterozygous for In^a antigen i.e. In (a + b+) by SS-PCR and their In^a positivity were confirmed by in-house polyclonal Anti-In^a reagent. SS-PCR was standardized using known heterozygous sample of a blood donor. The frequency of In^a antigen (2.0 %) was higher than Caucasians, lower than Iranians and Arabs while comparable to those reported among Indians of Mumbai city.ConclusionIn absence or unavailability of antisera particularly for low frequency alleles like In^a, such PCR based method would be extremely helpful to prepare rare donor registry by screening blood donors’ at large scale. Red cells of In^a positive donors can be used as in-house reagent red cells for screening and identification of corresponding antibody.     

Antiviral Protection and Germinal Center Formation, But Impaired B Cell Memory in the Absence of CD19

Coligation of CD19, a molecule expressed during all stages of B cell development except plasmacytes, lowers the threshold for B cell activation with anti-IgM by a factor of 100. The cytoplasmic tail of CD19 contains nine tyrosine residues as possible phosphorylation sites and is postulated to function as the signal transducing element for complement receptor (CR)2. Generation and analysis of CD19 gene–targeted mice revealed that T cell–dependent (TD) antibody responses to proteinaceous antigens were impaired, whereas those to T cell–independent (TI) type 2 antigens were normal or even augmented. These results are compatible with earlier complement depletion studies and the postulated function of CD19. To analyze the role of CD19 in antiviral antibody responses, we immunized CD19^−/− mice with viral antigens of TI-1, TI-2, and TD type. The effect of CD19 on TI responses was more dependent on antigen dose and replicative capacity than on antigen type. CR blocking experiments confirmed the role of CD19 as B cell signal transducer for complement. In contrast to immunization with protein antigens, infection of CD19^−/− mice with replicating virus led to generation of specific germinal centers, which persisted for >100 d, whereas maintenance of memory antibody titers as well as circulating memory B cells was fully dependent on CD19. Thus, our study confirms a costimulatory role of CD19 on B cells under limiting antigen conditions and indicates an important role for B cell memory.