Determination of immunoreactivity of<Emphasis Type="Italic">Plasmodium falciparum</Emphasis> antigens,serum dilutions and biomaterials

Immunoreactivity properties of serum dilutions andPlasmodium falciparum malaria antigens were measured and compared by ELISA technique using different ELISA plates to evaluate the role of antigens and serum dilutions for optimum binding. Also effort has been made to see the effect of reaction surface and material i.e. ELISA plates for binding capacity. Serological properties were estimated by ELISA methods for detection of malaria and determination of immunological characteristics. Three Pf antigens (PfAg) i.e. ring infected erythrocyte surface antigen: AR-1 (RESA), histidine-rich protein 2 antigen (HRP-2) and glycophospholipid antigen (grown and developed Pf antigen from PSJ-M strain): GPL1 have been used for serological testing of human blood samples by Enzyme Link Immunosorbant Assay (ELISA). 1∶100, 1∶1000 and 1∶10000 dilutions of Pf positive and negative serum (50 samples in each group) and 1∶1000 dilution of Pf antigens were used to measure immunoreactive properties by ELISA method. Result of PfAg-serum immunoreactivity study showed that GPL1 has the highest degree of immuno binding reactivity compared to other Pf antigens. HRP-2 and RESA antigens showed no significant difference to each other. Study also found that Costar and Fastec ELISA plates have a better Ag−Ab binding capability compared to immulon and Falcon plates at all dilutions of serum. Serum dilution of 1∶100 showed best binding and reactivity with Pf antigens followed by 1∶1000 and 1∶10000 showed lowest reactivity.     

Detection of tubercular antibody and antigen in sera of bone and joint tuberculosis

Trichloroacetic acid (TCA) solubilized and DEAE fractionatedMycobacterium tuberculosis H₃₇Ra excretory-secretory (ES) antigen viz., Mtb EST DE1 and affinity purified goat antibodies to the TCA solubilized ES antigen (Mtb EST) were explored in detecting tubercular antibody and antigen respectively in sera of bone and joint tuberculosis by indirect and sandwich ELISA. Out of total 36 bone & joint tuberculosis cases, tubercular antibody was detected by indirect ELISA in 30 patients (sensitivity 83%), while circulating tubercular antigen was detected by sandwich ELISA in 27 patients (sensitivity 75%). Out of 34 non tubercular disease control cases, 10 patients showed positive reaction for antibody while only 4 patients showed positive reaction for antigen. In another group of 34 healthy subjects who were screened, 4 individuals showed positive reaction for tubercular antibody and 2 cases for antigen. This study shows that antigen detection assay using affinity purified anti Mtb EST antigen antibody is superior with overall specificity of 91% as compared to antibody detection assay with 75% specificity in bone & joint tuberculosis.     

Mycobacterium tuberculosis H37Ra ESAS-7—An excretory-secretory antigen fraction of immunodiagnostic potential in pulmonary tuberculosis

A mycobacterial excretory-secretory protein fraction ESAS-7 purified by 50% ammonium sulphate precipitation followed by SDS-PAGE fractionation was evaluated by penicillinase enzyme linked immuno-sorbent assay (ELISA) for its sensitivity and specificity in the diagnosis of pulmonary tuberculosis. At a “cut off” serum dilution of 600, 38 (90%) of 42 sera from bacteriologically confirmed tuberculosis cases, 15 (100%) of 15 sera from bacteriogically negative but anti tubercular therapy (ATT) responded cases, 3 (7%) of 43 sera from normal healthy subjects and 4 (8%) of 48 sera from non tuberculous disease control cases gave positive reaction for tubercular antibody to ESAS-7 antigen fraction containing predominantly 33-kDa protein with a sensitivity of 90% in bacteriologically confirmed cases and specificity of 92%. Further, this diagnostic assay using the ESAS-7 antigen is more sensitive requiring as little as one nanogram antigen per test compared to use of 100 nanogram EST-6 antigen reported earlier. Thus use of ESAS-7 antigen for antibody detection has good diagnostic potential with improved specificity in pulmonary tuberculosis.     

Elisa based diagnostic test for Aspergillosis

Detection of lgE and lgG antibodies in Aspergillosis is of diagnostic significance. The serological methods, such as agglutination, gel diffusion and counter immuno electrophoresis that are commonly used in the laboratories for diagnosis of Aspergillus induced infections, are less sensitive and high cross reactivity is often encountered. We carried out work on characterization and identification of diagnostically relevant antigens ofA. fumigatus. Well characterized antigens were used to develop an ELISA with 92% sensitivity and 89% specificity for detection of specific lgE and lgG in the sera of patients of allergic bronchopulmonary aspergillosis (ABPA), aspergilloma and invasive aspergillosis. Subsequently, a sample kit having “ready to use type” of dry reagents (powder/tableted buffers and lyophilized antigen, conjugate and reference sera) was formulated. The kit was validated with sera from patients of ABPA, related allergic disorders, tuberculosis, post-Kochs cases and thalassemic children receiving repeated blood transfusions. The performance of the kit was found to be satisfactory with coded sera.     

Diagnostic potential of fractionatedMycobacterium tuberculosis H37Ra excretory-secretory (EST-DE1) antigen in pulmonary tuberculosis

Tuberculosis is emerging as a major public health problem in developing and developed world. Early and precise diagnosis is of prime importance in successful control of infection. Indirect ELISA with penicillinase as marker was developed using purifiedM. tuberculosis excretory-secretory (EST-DE₁) antigen for detecting IgG antibodies in pulmonary tuberculosis. The assay System gave a overall sensitivity of 82% for both smear positive and smear negative pulmonary tuberculosis cases with a specificity of 84%. The positive and negative predictive values were 75% and 88% respectivaly. Further studies with EST-DE₁ antigen revealed that, it contains two of the active antigen fractions of Mtb EST antigen i.e. Mtb EST-4 (56–68 KDa) and Mtb EST-6 (37–45 KDa), as demonstrated by inhibition ELISA. Reactivity with monoclonal antibodies HGT 3a showed the presence of 38 KDa molecule in EST-DE₁ antigen.     

Immunodiagnosis of human lymphatic filariasis: A review

A monoclonal antibody-based antigen detection system was used to detect the levels of circulating antigen in filarial patients before and after treatment with DEC and in normal individuals living in an area endemic forW. bancrofti infection in Chennai, India. The present study was to show the use of this assay as a means of efficient screening for filariásis in an endemic area where blood was absorbed onto the filter paper by finger prick during day time. The results of the antigen levels collected onto filter strips correlated with their corresponding plasma antigen levels (r=0.83). In microfilaraemics, DEC treatment did not alter the levels of circulating antigens upto a period of one month. We conclude that this monoclonal antibody based ELISA using filter strips may be used in daytime and can replace the existing routine night blood survey.     

Assay of tubercular antibody, circulating free and immune complexed antigen in the diagnosis of pulmonary tuberculosis

Analysis of tubercular antibody, circulating free and immune complexed antigen (CIC-Ag) was done in confimed pulmonary tuberculosis sera by ELISA, using ES-31 antigen and affinity purified anti ES-31 antibody. Twenty three of 25 (92%) tuberculosis sera were positive for IgG antibody to ES-31 antigen. Using anti ES-31 antibody, free tubercular antigen could be detected in 20 of 25 (80%) cases whereas circulating immune complexed antigen (CIC-Ag) in 18 of 25 (72%) cases by sandwich ELISA. Of the two sera showing absence of antibody, one showed presence of free and CIC-Ag whereas the other showed the presence of free antigen. Thus antigen assay may be used as an adjunct tool for confirmation of pulmonary tuberculosis.     

Serodiagnosis of tuberculosis using two ELISA systems

Serodiagnosis by ELISA has been widely explored over the years, in the diagnosis of tuberculosis. Two ELISA systems were evaluated for detection of mycobacterial antibodies in pulmonary and extra pulmonary tuberculosis. The two test assays explored were ERBA LISA (TB IgG) test (Anda Biologicals) which uses A60 antigen complex found in the cytosol of typical and atypical mycobacteria, and SEVA TB (IgG) ELISA, which uses a 31 kDa, glycoprotein antigen purified fromM. tb H₃₇Ra culture filtrate. Sera from 98 proven tuberculosis [pulmonary TB (48), tuberculous lymphadenopathy (30), tuberculous meningitis (15) & genitourinary TB (5)] were studied along with 32 healthy controls. The overall positivity obtained using ERBA LISA (TB IgG) test and SEVA TB (IgG) ELISA test was 72.9% and 91.6% in pulmonary tuberculosis, 43.3% and 76.6% in tuberculous lymphadenopathy respectively. The sensitivity of ERBA LISA test in tuberculous meningitis and genito-urinary TB was significantly low (26.6% & 40% respectively) compared to sensitivity obtained using SEVA TB ELISA (86.6% & 60% respectively) with overall specificity of 60% and 87.5%. Thus SEVA TB IgG ELISA test was found to be more sensitive than ERBA LISA in detecting IgG antibodies in tuberculous sera, in particular in extra pulmonary tuberculosis cases.     

Analysis of SEVA TB ES-31 antigen specific immunoglobulins IgM, IgA and IgG in sera of sputum and culture positive pulmonary tuberculosis

Tuberculosis remains major health problem in India and developing countries Immunodiagnosis has important role in screening, diagnosis and management of tuberculosis. SEVA TB ES-31 antigen has shown potential in detecting tuberculous IgG antibody in earlier studies from our laboratory. In the present study we have analysedSEVA TB ES-31 antigen specific immunoglobulinsIgM, IgA and IgG in clinically and bacteriologically confirmed pulmonary tuberculosis cases to determine the usefulness of specific immunoglobulin class in the diagnosis of patients attending the hospital. Of the 30 cases of pulmonary tuberculosis 25 (83.3%) were positive for IgG, 19 (63.3%) for IgM and 16 (53.3%) for IgA. On combining IgG and IgM positivity, sensitivity was increased to 93.3%. While combining IgG and IgA positivity, sensitivity increased to 90%. However specificity was decreased to 66.6% and 70% for both of these combinations respectively. It could be envisaged from this study that IgG antibody detection against ES-31 antigen showed acceptable sensitivity (83.3%) and specificity (86.6%) compared to IgM or IgA alone or in combination. When immune responses were analysed according to degree of sputum positivity, IgG response was observed to be predominant in all grades, compared to IgM or IgA antibody. The addition of IgM or IgA as an adjunct test increases the sensitivity but at the cost of specificity. Hence the detection of IgG alone is more useful compared to IgM or IgA assay, in detecting tuberculosis disease cases coming to the hospital.     

<Emphasis Type="Italic">In vitro</Emphasis> released antigens in diagnosis and immunomonitoring of filaria and tuberculosis

In vitro released antigens by living parasites or bacteria underin vitro maintenance or short term culture showing specific humoral immune response have been explored in development of immunodiagnostics for infectious diseases such as filariasis and tuberculosis in our laboratory. ELISA usingB. malayi mf ES antigen has been explored for detecting IgG antibody by Indirect ELISA and antigen by Inhibition ELISA and in immunomonitoring of carriers as well as clinical filarial cases. A ten year follow up of mf carriers with DEC therapy showed disapperance of antigen and antibody followed by reappearance in few cases in an endemic area. None of the cases followed developed clinical symptoms suggesting the need for long term monitoring and treatment of microfilaraemic carriers. Further immunomonitoring was found to be useful in confirming filaria aetiology in the absence of microfilaremia and determining appropriate period of treatment of acute, early clinical and occult filarial infections for clinical relief and cure.Indirect Stick Penicillinase ELISA system using Mtb EST-6 antigen for detecting tuberculous IgG antibody and a Sandwich Penicillinase ELISA system using affinity purified antibody for detecting circulating antigen were explored in tuberculosis. A combination of both the assay systems with a sensitivity of 70% and specificity of 98% was found to be promising in the precise diagnosis of pulmonary tuberculosis. Further antigen detection was found to be useful in bone and joint tuberculosis.     

Isolation ofMycobacterium Tuberculosis 31 kDa antigen protein of diagnostic interest from culture filtrate using anti-ES-31 antibody by affinity chromatography

Proteins secreted into the culture medium byMycobacterium tuberculosis (M. tb) are shown to be source of antigens of immunodiagnostic interest. Anin vitro released 31 kDa antigen ESAS-7F isolated fromM.tb H₃₇Ra culture filtrate by salt precipitation, SDS-PAGE and cation exchange fast protein liquid chromatography (FPLC) was shown earlier to be a diagnostically important antigen fraction. In this report, we describe the isolation of ESAS-7F antigen using monospecific antibody coupled to sepharose CL-4B column. The percentage recovery of ESAS-7F antigen using affinity chromatography was approximately 8% of the total ES antigen proteins compared to 0.05% obtained by conventional purification steps using salt precipitation, SDS-PAGE and FPLC. Similar seroreactivity was observed by the antigen isolated by both the methods in indirect ELISA. Affinity chromatography helped in an increased recovery of ESAS-7F antigen and obviates the need for time consuming conventional purification steps.     

Diagnostic Significance of CA15-3 with Combination of HER-2/neu Values at 85th Percentiles in Breast Cancer

The human epidermal receptor-2/neu (HER-2/neu) oncogene encodes a transmembrane tyrosine kinase receptor. This molecule could have a diagnostic value since the extracellular domain of c-erbB-2 (HER-2) transmembrane is shed into the blood as a circulating antigen. The diagnostic value of serum HER-2/neu was calculated along with the conventional marker carbohydrate antigen 15-3 (CA15-3) and carcinoembryonic antigen (CEA) at 85th percentiles. Serum levels of breast carcinoma antigens HER-2/neu, CEA and CA15-3 were determined in 175 normal individuals and 268 malignant patients. The soluble form of serum HER-2/neu, CEA and CA15-3 was assayed by enzyme linked immunosorbent assay in control and breast cancer patients prior to treatment. Serum levels of the tested tumor markers HER-2/neu and CA15-3 and CEA were significantly higher in cancer patients compared to controls. At 85th percentile the sensitivity of HER-2/neu was 51.12 %; the specificity was 86.29 % and the overall accuracy was 64.56 %. The sensitivity of CA15-3 was 73.13 %; the specificity was 85.14 % and the overall accuracy was 77.88 %. The sensitivity of the combined testing was 82.84 %; the specificity was 73.71 % and the overall accuracy was 80.01 %. The sensitivity and the overall accuracy of combined testing were higher than those of HER-2/neu and CA15-3 testing single. The combined testing of HER-2/neu and CA15-3 can increase the sensitivity and overall accuracy of breast cancer diagnosis. The results of this study suggest that the use of multiple tumor markers may be employed as combination and at 85th percentiles to assess the prognosis.     

Use of specific IgE to fractions of gynandropsis gynandra pollen inin vitro diagnostic test

The study was aimed at presence of specific IgE antibody levelsinvitro to the identified antigen. Based on positive skin test with Gynandropsis gynandra and elevated levels of total IgE (>325 IU/ml) 104 patients were selected. Healthy, asymptomatic individuals (25) with low total IgE (<325 IU/ml) were included as controls. The mean OD values by ELISA for specific IgE were 0.67±0.21, 0.57±0.18 and 0.56±0.18 with whole pollen antigen, 46-37 kD fraction and 36-32 kD fraction, respectively. The specificity and sensitivity between skin test positivity with whole pollen antigen verses fraction with mol.wt 46-37 kD was 90% and 90% and for fraction with mol.wt 36-32 kD was found to be 81.1% and 89.4%. The clusters with molecular weights 46-37 kD and 36-32 kD may be useful inin vitro diagnostic test. Fractions within these clusters need to be identified for a higher specificity.     

Diagnostic role of the antibody response to the 38kDa, 16kDa proteins and lipoarabinomannan of mycobacterium tuberculosis

The antibody response to the 38kDa, 16kDa and Lipoarabinomannan (LAM) antigens ofMycobacterium tuberculosis was evaluated using three different ELISAs based on these antigens. The study group included tuberculosis patients (n=52), patients with HIV and TB co-infection (n=10), other chest symptomatics (n=5), HIV infected individuals (n=10), leprosy cases (n=7) and healthy controls (n=75). The results indicate that the 38kDa and LAM based ELISA for IgM/IgG has a low specificity (ranging from 69–85%) and sensitivity (ranging from 55–78%). When three ELISAs are carried out on a single patient the probability of detection of tuberculosis was significantly increased to 95.2% indicating that a single ELISA test is of low sensitivity and that a combination of ELISA’s may be needed to be of any value as a diagnostic test for tuberculosis. Additionally, a western blot assay of the serum antibody response to protein fraction ofM. tuberculosis was analysed in 15 tuberculosis patients and five healthy controls. A multiple antibody response to various M.tuberculosis proteins was observed which varied from patient to patient as compared to controls who showed a single 38–39 kDa protein band positivity. These finding suggest that a western blot assay which determines the antibody response to a set of antigenic components ofM. tuberculosis could be a better serological test for the diagnosis of tuberculosis in our population.     

伤寒LPS-PHA快速诊断试剂盒的评价

本文对伤寒LPS-PHA诊断试剂盒进行现场应用评价.来自各医院和不同地区977份不同类型的血清标本作LPS-PHA检测的同时与Widal试验作比较,结果伤寒沙门氏菌血培养阳性46份血清标本前者检出率(86.96％)显著高于后者(67.39％),两者在统计学上有显著差异(X~2=4.999,P<0.025),95份Widal试验阳性血清LPS-PHA阳性率为97.89％,漏检2份系乙型副伤寒抗体阳性者.607份对照血清标本LPS-PHA的假阳性率1.48％,低于Widal试验(4.84％).伤寒LPS-PHA的敏感度和特异度(94.33％和98.52％)高于Widal试验(89. 36％和95.72％).其他5项评价指标(阳性和阴性预示值,粗一致性,调整一致性和约登指数)亦都较Widal试验好.因此,LPS-PHA诊断试剂盒能满足目前临床上早期快速诊断伤寒的要求.但不适用于追溯诊断和血清流行病学调查.     

Antigens of diagnostic and prophylactic importance inEntamoeba histolytica

Summary  Amoebiasis, caused by an enteric protozoanEntamoeba histolytica, is one of the major parasitic diseases of mankind. Current estimate suggests that the parasite infects about 10% of the world population at any given time. There is an urgent need to characterize the antigenic molecules ofE. histolytica, and find out antigens which have both immunodiagnostic and prophylactic potential against amoebiasis. The results of somatic antigen analysis, using sera from immune or infected individuals, indicated that the wholeE. histolytica trophozoites, are highly complex and heterogeneous in nature and both immunodiagnostic and immuno prophylaxis activity remain mainly in a surface associated 29 kDa glycoprotein ofE. histolytica. Future studies at molecular level particularly, genes responsible for expression of this protein, their homology with other proteins and structure analysis will give better understanding about this polypeptide. Studies on excretory secretory antigens, clearly demonstrated thatE. histolytica like many organisms, also liberates certain antigenic moieties into the culture medium during in vitro cultivation and this antigen has similar diagnostic values like the conventional somatic antigens. It is important that the ESA should be prepared from the supernatant after collecting the cell and use for immunodiagnosis of amoebiasis. This is an additional source of antigen which will help in carrying out more tests using the same reagents. Further studies are needed to clarify the location of these molecules on the parasite, along with detailed biochemical and immunological characterization and their relation with the pathogenesis. This was presented at the symposium on “Recent Trends in Tropical Disease Research”, held at Sevagram on 5-6 September, 1997.     

Current status of the lateral flow immunoassay for the detection of SARS-CoV-2 in nasopharyngeal swabs

Early detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and diagnosis of coronavirus disease 2019 (COVID-19) are priorities during the pandemic. Symptomatic and suspected asymptomatic individuals should be tested for COVID-19 to confirm infection and to be excluded from social interactions. As molecular testing capacity is overloaded during the pandemic, rapid antigen tests, such as lateral flow immunoassays (LFIAs), can be a useful tool as they allow greater test availability and obtain results in a very short time. This short review aims to present the analytical properties of LFIAs in the detection of SARS-CoV-2 in nasopharyngeal swabs. Lateral flow immunoassay is a method that combines thin-layer chromatography and indirect immunochemical sandwich method and allows the detection of a specific SARS-CoV-2 antigen in nasopharyngeal swabs. Swab specimens should be adequately collected and tested as soon as possible. Users should pay attention to quality control and possible interferences. Antigen tests for SARS-CoV-2 show high sensitivity and specificity in cases with high viral loads, and should be used up to five days after the onset of the first symptoms of COVID-19. False positive results may be obtained when screening large populations with a low prevalence of COVID-19 infection, while false negative results may happen due to improper specimen collection or insufficient amount of antigen in the specimen. So as to achieve reliable results, a diagnostic accuracy study of a specific rapid antigen test should be performed.     

Screening of HLA antibodies in maternal sera from Hyderabad

Antibodies against human leucocyte antigens (HLA) in sera from uni and multiparous women are the potential source of HLA reagent. The present study was undertaken to screen 169 sera from pregnant women for the presence of HLA antibodies employing 26 panel cells (Peripheral blood lymphocytes) having known HLA phenotypes. 20.7% (35/169) sera were found to be positive for HLA class-1 antibodies. Present study generated one monospecific, (r=0.6 for A32) the duospecific sera (r=0.5 for A2 B35, r=0.47 A1 DR6 and r=0.7 A28 B51), and rest multispecific sera (r=below 0.4). These positive sera will be utilized as HLA reagents in future studies for tissue typing.     

Novel and adaptive contribution of the red channel in pre-processing of colour fundus images

A new pre-processing method for colour fundus images with adaptive contribution of the red channel is proposed. Based on a condition that is developed in this paper, this method utilises the intensity information from both red and green channels instead of using only the green channel as in the usual practice. The histogram matching is used to modify the histogram of the green channel by using the histogram of the red channel (of the same retinal image) to obtain a new processed image having the advantages of both channels. This method can be used to correct non-uniform illumination in colour fundus images or as a pre-processing step in the automatic analysis of retinal images.Results show that the use of histogram matched (HM) image give better performance than using the green channel image when employing the two-dimensional matched filter to detect retinal blood vessels. At specificity of 90%, in case of abnormal images, sensitivity increased from 76% when using the green channel image to 82% when using the HM image compared with 81% when using the piece-wise threshold probing method. In case of normal images, at the same specificity, the sensitivity obtained when using green channel image or HM image was 87% compared with 88% for the piece-wise threshold probing method.