Diagnostic utility of monoclonal antibodies raised against microfilarial excretory-secretory antigens in bancroftian filariasis

Two monoclonal antibodiesWuchereria bancrofti E 33 andWuchereria bancrofli E 34 raised againstWuchereria bancrofti microfilarial excretory-secretory antigens were studied for their diagnostic utility.Wuchereria bancrofti E 34 monoclonal antibody was found to be relatively specific and sensitive in detection of circulating filarial antigen. WhenWuchereria bancrofti E 34 monoclonal antibody was used alongwith immunoglobulin G fraction of human filarial serum immunoglobulins in double antibody sandwich enzyme linked immunosorbent assay. 68% of microfilaraemic sera (26 out of 38). 12% of clinical filarial sera (3 out of 25), 13% endemic normal sera (2 out of 15) and none of the 20 non-endemic normal sera showed the presence of filarial antigen. The filarial antigen detected byWuchereria bancrofti E 34 monoclonal antibody in double antibody sandwich enzyme linked immunosorbent assay is possibly associated with the active stage (microfilaraemia) of infection.     

Detection of circulating antigen in bancroftian filariasis by sandwich ELISA using filarial serum IgG

The utility of the IgG fraction of human filarial serum immunoglobulin in detecting circulating antigen by sandwich enzyme linked immunosorbent assay (ELISA) was studied. 27 of 33 sera from persons with microfilaraemia, 19 of 30 sera from clinical cases of filariasis, 4 of 30 sera from normal persons from a region endemic for filariasis showed the presence of circulating filarial antigen. All the 20 normal sera from the area where filariasis was not endemic gave negative reaction for filarial antigen. Those sera from persons with microfilaraemia that showed the presence of circulating antigen also showed an apparent positive correlation between the microfilarial density and the antigen titre.     

Differential reactivity of filarial antigens with human sera from bancroftian filariasis endemic zone

The reacting pattern of circulating filarial antigen fraction-2 fromWuchereria bancrofti and soluble antigen from adultBrugia malayi with bancroftian filarial sera were analysed by immunoblotting technique and enzyme linked immunosorbent assay. Microfilaraemic sera reacted specifically with proteins of molecular weight 200, 120, 97, 56, 54, 43, 26 and 17 kDa of circulating Filarial antigen fraction-2 and 44, 40, 38, 31, 22 and 18 kDa ofBrugia malayi adult soluble antigen. Clinical filarial sera identified protein molecules of 56, 54 and 42 kDa of circulating filarial antigen fraction-2 and 19, 16 and 14 kDa ofBrugia malayi adult soluble antigen. Some components of both the antigen preparation were also identified by endemic normal serai.e.proteins 120, 97, 62, 43 and 33 kDa of circulating filarial antigen fraction-2 and 170, 120, 43, 31 and 12 kDa ofBrugia malayi adult soluble antigen. One of the sodium dodecyl sulphate-polyacrylamide gel electropherosis fractions of circulating filarial antigen fraction-2 (CFA₂-8) andBrugia malayi adult soluble antigen fraction-6 when used in enzyme linked immunosorbent assay could differentiate microfilaraemic sera from endemic normal and clinical filarial sera. The other antigen fractions (CFA₂-2, 6 and 7 andBmA-2) showed a high geometric mean titre of filarial immunoglobulin G antibodies in endemic normal sera when compared to microfilaraemia and clinical filarial sera. These proteins need to be further studied to assess their involvement in protecting from filarial infection in an endemic area.     

Diagnostic utility of fractionated urinary filarial antigen

Urinary filarial antigen isolated from urine samples of microfilaraemic patients was analysed for its antigenic activity by immunoblotting and enzyme linked immunosorbent assay techniques. SDS-PAGE fractionation of urinary filarial antigen showed 11 protein bands, of which two showed reactivity with immunoglobulin-G fraction of filarial serum immunoglobulin in immunoblotting. Antigenic analysis of SDS-PAGE fractions of urinary filarial antigen by inhibition enzyme linked immunosorbent assay using filarial serum immunoglobulin-G andWuchereria bancrofti microfilarial excretory-secretory antigen revealed 3 fractions, numbers 5, 6 and 9 with significant activity. In indirect enzyme linked immunosorbent assay using fractions 5 and 6, filarial immunoglobulin-G antibody was detected in about 90% of microfilaraemics, 80% clinical filariasis and 20% of endemic normal individuals. Further, there was no phosphorylcholine epitope in these fractions. Fractions 5 and 6 can be a candidate antigens for the immunodiagnosis of filariasis.     

Detection of filarial antigen by inhibition enzyme linked immunosorbent assay using fractionatedBrugia malayi microfilarial excretory secretory antigen

Detection of filarial antigen in blood or urine samples would provide an accurate indication of active infection. The absence of a simple, well established animal model and limitations in getting the required amount of parasite material from human sources have been the main obstacles for the diagnosis ofWuchereria bancrofti infection. An inhibition ELISA has been developed for detection of filarial antigen using a partially purifiedBrugia malayi mf ES antigen (BmE DE1) and its affinity purified antibodies. Filarial antigen was detected in the sera of 88% of microfilaraemic, 60% of chronic filarial, 17% of endemic normal and none of the non- endemic normal subjects. The sensitivity and specificity of the assay were 88% and 89% respectively. Moreover, undiluted urine samples from 82% of microfilaraemic and 17% of endemic normal, but none of the non- endemic normal samples showed the presence of filarial antigen. With the limitations on the availability of sufficient homologous parasite materials, the inhibition ELISA using BmE DE1 and anti BmE DE1 antibodies shows promise for the detection of active infection in bancroftian filariasis in man. Moreover, its detection in urine makes it more possible to test patients in field areas.     

Immunodiagnosis of bancroftian filariasis—Problems and progress

The immunodiagnosis of bancroftian filariasis is a major challenge to the immunoparasitologist. Significant progress is yet to be made in developing convenient laboratory animal model and inin vitro cultivation of filarial parasites making it very difficult to obtain required amount of parasite material for research. Parasitological examination techniques are not useful in low microfilaraemia, occult or chronic.filarial infections. A precise and accurate immunodiagnostic technique is very much needed for successful filaria control programmes. Such a test will also avoid the need for laborious night blood examination in bancroftian filariasis. Due to comparatively easy availability, a good amount of work has been done to explore immunodiagnostic potential of heterologous filarial antigens isolated fromLitomosoide carinii, Dirofilaria immitis, Brugia malayi, Setaria digitata, Setaria cervi and number of other filarial species. However, there has been limited or no significant success due to number of false negative and false positive reactions. Extensive study has also been made with antigens isolated fromWuchereria bancrofti microfilariae. Soluble antigens of microfilariae have been used in different immunological techniques such as skin test, counter immuno electrophoresis, indirect haemagglutination test, indirect fluorescent antibody test and enzyme linked immunosorbent assay. Fractionation of Wuchereria bancrofti microfilarial soluble antigens yielded mfS3e antigen fraction which was found to be highly reactive in microfilaraemia by enzyme linked immunosorbent assay, but the yield of the purified antigen was not sufficient enough to make it a practical proposition for large scale isolation of antigen. Wuchereria bancrofti microfilarial excretory-secretory antigens were found to be specific and highly sensitive requiring as little as 0.35 ng antigen protein per well in penicillinase enzyme linked immunosorbent assay for detection of filarial antibody. One ml of culture fluid was found to be sufficient for 400,000 tests. Field evaluation of this test showed that it can replace laborious night blood examination. Assay systems have been developed for detection of filarial antigen in serum, urine, hydrocele fluid and immune complexes using immunoglobulins from chronic filarial sera and antisera to excretory filarial antigens. Further purification of excretory-secretory antigens by affinity chromatography and production of monoclonal antibodies should hopefully give suitable reagents for use in sensitive assays such as enzyme immuno assay and immuno radiometric assay, providing an ideal assay system for detection of active filarial infection in the not too distant future.     

Evaluation of fractionatedWuchereria bancrofti microfilarial excretory-secretory antigens for diagnosis of bancroftian filariasis by enzyme linked immunosorbent assay

TheWuchereria bancrofti microfilarial excretory-secretory antigens were fractionated into ES1, ES2, ES3 and ES4 by ultra-membrane filtration and evaluated for their diagnostic utility by enzyme linked immunosorbent assay. Three of the four fractions showed antigenic activity (ES2, ES3 and ES4). The antigen fractions ES2 and ES4 were highly active in the detection of filarial IgM antibody in clinical filariasis and microfilaraemia respectively. The chemical characterization of the ES2 and ES4 antigen fractions showed that they were glycoproteins     

Stick enzyme-linked immunosorbent assay using the avidin-biotin system for detection of circulating antigen in bancroftian filariasis

Detection of filarial antigen in different groups of sera was carried out by sandwich as well as inhibition enzyme-linked immunosorbent assays using antibody-coated sticks. Both systems were found to be equally sensitive in detecting antigen in 90% of microfilariae carriers. Incorporation of avidin-biotin in the sandwich assay system increased the sensitivity of antigen detection from 10⁻⁶ to 10⁻¹⁶ pg. A 67% decrease in the number of false negative results was observed when the sensitive avidin-biotin inhibition enzyme-linked immunosorbent assay system was used for analysis of filaria blood samples.     

Immunoradiometric assay for detection of filarial antigens in human serum

The immunoradiometric assay (IRMA) for detection of filarial antigens in the serum of patients infected with Brugia malayi (Bm) or the closely related filarial parasite Wuchereria bancrofti (Wb) was investigated, and its performance and clinical utility were examined. Reference sera prepared by the addition of crude Bm antigen (BmA) to negative control human sera provided a reproducible reference curve. The IRMA displayed acceptable precision and reproducibility. Agreement between dilutions (parallelism) was good in sera without specific antibody, but the presence of even modest levels of antibody resulted in nonparallelism in about one-half of the tested sera from endemic areas. Significant reduction in detectable BmA occurred when low levels of specific antibody (less than 1 microgram/ml) were added to BmA containing sera. Thus, antibody interference limited absolute quantitation of antigen in the IRMA. Results were therefore expressed in a semi-quantitative manner by using the mean + 3 SD of the binding of nonexposed human sera as the positive threshold. The frequency of reliable filarial antigen detection in individuals from the Wb endemic areas of India and the South Pacific was the following: microfilaremia, 15 out of 15; elephantiasis, 2 out of 18; tropical pulmonary eosinophilia, 2 out 8. These findings show clearly that a two-site IRMA can effectively detect circulating antigen (and thus be diagnostic of infection) in a great many patients with filariasis, but to enhance the sensitivity of the assay to the point where all patients can be diagnosed, a number of suggested modifications will be necessary.     

Enhanced chemiluminescence ELISA for Listeria specific antigen in cerebrospinal fluid using an FITC-anti-FITC system

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of a soluble Listeria monocytogenes serogroup 4 antigen in cerebrospinal fluid samples (CSFs). In the ELISA an anti-Listeria monoclonal antibody, immobilized onto assay wells, was used to capture antigen from CSFs. the captured antigen was then reacted with a fluorescein isothiocyanate (FITC) conjugate of the same anti-Listeria antibody, which was detected with a horseradish peroxidase conjugate of a monoclonal antibody to FITC. The presence of antigen was detected by an enhanced chemiluminescence assay using a camera luminometer. Antigen was detected in the CSFs taken from five out of seven patients with culture proven L. monocytogenes serogroup 4 central nervous system infections, and in none of the CSFs taken from 25 other patients.     

Development of simple and stable ELISA for detection of copro antigen in intestinal amoebiasis

A simple, sensitive and stable ELISA (enzyme linked immunosorbent assay) was developed using rabbit antibody to fractionated Entamoeba histolytica antigen for the detection of copro antigen in the faeces of individuals with intestinal amoebiasis. In this test none of the healthy individuals, all trophozoite positive, 40% cyst passers and 6% individuals living in endemic area showed the presence of copro antigen. ELISA using polyclonal rabbit antibody could detect 1-5 trophozoites/well and 20-50 ng protein per well of NIH-200 strain of E. histolytica and the sensitivity of the test was comparable with that using monoclonal antibody. Cross reaction was observed only with E. invadens when faeces having other parasites were screened. The reagents of ELISA were stabilized and found to be stable for more than 6 months when stored at 4 degrees C.     

Epidemiological studies on the background of the endemic occurrence of tsutsugamushi disease in Toyama Prefecture. II. Anti-Rickettsia tsutsugamushi antibody-positive rate in inhabitants of endemic and nonendemic areas

With a view to clarifying the actual state of inapparent infection of tsutsugamushi diseases, inhibitants of endemic and nonendemic areas were screened for anti-Rickettsia tsutsugamushi antibody (anti-Rt antibody) by the indirect immunofluorescence test. The anti-Rt antibody-positive rate in the inhabitants of the endemic area (about 50%) was statistically significantly higher than that in the nonendemic area (14.7%). The antibody titer in the inhabitants of the endemic area was 10-160, and the number of inhibitants showing a high antibody titer was 2-4 times larger than that of the nonendemic area. A total of 257 volunteers in the endemic area were analyzed for the changes in anti-Rt antibody titer over 1.5-2 years on an individual basis. An increase in the antibody titer was found in 20 inhabitants. There was no difference in the anti-Rt antibody-positive rate between male and female in either the endemic or the nonendemic area. The positive rate was also compared as to the distribution by 10 years of age. In the endemic area, there were no significant differences in the positive rate between any pair of 10-year age groups from 30s to 60s, whereas in the nonendemic area, the positive rate in the teen-age group was significantly lower than those in the age groups of 20 years or older. In Yamada district, the numbers of serum samples obtained from each age group were about the same, and the distribution of the positive rates showed a normal distribution. The nurse students having their homes in Toyama Prefecture were plotted on the map as for their anti-Rt antibody and geographical distribution. The results showed that many of them having homes in the endemic area were positive for the antibody, while some antibody-positives were scattered all over Toyama Prefecture.     

Seroreactivity of purified Brugia malayi microfilarial soluble and excretory-secretory antigens in different clinical presentations of bancroftian filariasis

Brugia malayi microfilarial excretory-secretory (mf ES) and phosphate buffer saline soluble (mf S) antigens were fractionated by fast protein liquid chromatography (FPLC) on superdex 200 HR 10/30 gel filtration column. The active antigen fractions were identified and explored in comparison with whole mf ES and mf S antigens to detect filarial IgG antibodies in different groups viz microfilaraemics, acute, chronic and occult filarial cases of Wuchereria bancrofti infection and endemic and non-endemic normals. One of the fractions of mf ES antigen (ESF-6) and two fractions of mf S antigen (SF-2 & 3) were identified to be useful to detect filarial antibodies. A pooled preparation of these antigen fractions gave a sensitivity of 86.6% (for microfilaraemic cases) and a specificity of 95% to detect filarial IgG antibodies by indirect ELISA. The pooled FPLC purified mf antigens also showed 55-88% of cases of different grades of clinical filariasis and 65% of tropical pulmonary eosinophilia cases as positive for filarial antibodies. The pooled FPLC purified B. malayi mf antigens with higher specificity are preferable to whole mf ES and mf S antigens to detect active filarial infection in microfilaraemia and as well in different clinical entities of bancroftian filariasis.     

The ICT Filariasis Test: A rapid-format antigen test for diagnosis of bancroftian filariasis

Antigen testing is now recognized as the method of choice for detection of Wuchereria bancrofti infections. Unlike tests that detect microfilariae, antigen tests can be performed with blood collected during the day or night. However, existing enzyme-linked immunosorbent assay (ELISA) tests for filarial antigenemia are difficult to perform in the field, and this has limited their use in endemic countries. In this article, Gary Weil, Patrick Lammie and Niggi Weiss review their experience with a new rapid-format filarial antigen test. They found that the ICT card test was very easy to perform and that it was comparable with ELISA for the detection of filarial antigen in sera from people with microfilaremia. The introduction now of an antigen test suitable for use in the field is especially timely, in that it may facilitate implementation of new strategies proposed by the World Health Organization for control and elimination of lymphatic filariasis.     

Evaluation of Rapid Blood Sample Collection in the Detection of Circulating Filarial Antigens for Epidemiological Survey by rWbSXP-1 Capture Assay

Background

Lymphatic filariasis is a neglected tropical disease leading to profound disfiguring causing socio economic burden in the tropics. Current diagnosis strategies available during field surveys and epidemics are based on traditional microscopic detections and a few antigen/antibody assays. We have compared different sampling methodologies and standardized the highly sensitive and reliable rWbSXP-1 antigen detection assay to our new sampling methodology.

Methodology

Samples collected as serum, whole blood, whole blood on filter paper and whole blood on microscopic slides from patients belonging to various clinical groups of filariasis [endemic normal(EN), chronic pathology(CP), microfilaraemic(MF) and non-endemic normal(NEN)] were collected and standardized the rWbSXP-1 antigen detection assay using monoclonal antibody raised against rWbSXP-1 protein. The whole blood collected on microscopic slide based sampling method was employed in the field and the presence of circulating filarial antigen (CFA) was assessed using the rWbSXP-1 assay.

Principal Findings

The sampling methods were compared and no significant difference was observed for the detection of CFA (MF, P = 0.304, EN, P = 0.675, CP, P = 0.5698, NEN, P = 0.4494). Further the optimized sampling method was utilized to collect the 1106 samples from Polur, Tiruvannamalai. The rWbSXP-1 assay gave 98 antigen positive results whereas the microscopic method gave only 17.

Conclusions

Four sampling methodologies were analyzed and the new sampling methodology of whole blood collected on microscopic slide was found to be convenient for the detection of CFA using rWbSXP-1 antigen detection assay. The 1106 samples from Polur were collected using the new method. The rWbSXP-1 antigen assay perceived a 7.32% increased result which was read as false negatives on the conventional microscopic staining method. This new sampling methodology coupled with the rWbSXP-1 antigen assay can be used in epidemiological surveys for lymphatic filariasis and the same sampling methodology can be expanded to other antigen based high affinity assays.     

Evaluation of recombinant antigens for diagnosis of melioidosis

Burkholderia pseudomallei, the causative agent of melioidosis, is endemic to Southeast Asia and northern Australia. Clinical manifestations of the disease are diverse, ranging from chronic localized infection to acute septicaemia, with death occurring within 24-48 h after the onset of symptoms. Definitive diagnosis of melioidosis involves bacterial culture and identification, with results obtained within 3-4 days. This delayed diagnosis is a major contributing factor to high mortality rates. Rapid diagnosis is vital for successful management of the disease. This study describes the purification and evaluation of three recombinant antigenic proteins, BPSL0972, BipD and OmpA from B. pseudomallei 08, for their potential in the serodiagnosis of melioidosis using an indirect enzyme-linked immunosorbent assay (ELISA) method. The recombinant proteins were evaluated using 74 serum samples from culture-confirmed melioidosis patients from Malaysia, Thailand and Australia. In addition, 62 nonmelioidosis controls consisting of serum samples from clinically suspected melioidosis patients (n=20) and from healthy blood donors from an endemic region (n=18) and a nonendemic region (n=24) were included. The indirect ELISAs using BipD and BPSL0972 as antigens demonstrated poor to moderate sensitivities (42% and 51%, respectively) but good specificity (both 100%). In contrast, the indirect ELISA using OmpA as an antigen achieved 95% sensitivity and 98% specificity. These results highlight the potential for OmpA to be used in the serodiagnosis of melioidosis in an endemic area.     

Competitive antibody binding to soluble CD16B antigen and CD16B antigen on neutrophils in whole blood by flow cytometry

BACKGROUND: Shed receptors from the surface of white blood cells in whole blood have been quantitated using the long and tedious enzyme-linked immunosorbent assay (ELISA) method. A simple rapid flow cytometric method of analysis for shed antigen in the presence of cell-bound antigen can be advantageous. METHODS: Magnetic bead depletion of neutrophils in whole blood with CD16b antibody-conjugated beads as measured by flow cytometric analysis of the remaining cell suspension was inhibited by the presence of soluble CD16b antigen in the blood plasma of normal donors. We describe a competitive binding assay between labeled and unlabeled CD16b antibody for receptors shed from the surface of formed bodies (cells) into solution. Also presented is a new method of obtaining the amount of soluble antigen in a sample. We determine the total unlabeled and labeled ligand concentration at which the fluorescence intensity of the labeled ligand matches the fluorescence intensity in a control run with only the labeled ligand. RESULTS: Normal blood donors showed serum concentration levels of shed CD16b antigen in the range of 1-50 nM as determined by a flow cytometric competitive binding assay. These figures compared favorably with parallel determinations using magnetic bead depletion of targeted neutrophils for washed and unwashed whole blood samples to evaluate the concentration of shed CD16b antigen. CONCLUSIONS: The competitive antibody binding assay for shed and cell-bound CD16b antigen can be applied to similar GPI-linked antigens, for which purified antibody and fluorescent antibody against the same antigenic receptor are available.     

A monoclonal antibody-based immunoradiometric assay for detection of circulating antigen in Bancroftian filariasis

A monoclonal antibody designated Gib 13 has been used in an immunoradiometric assay (IRMA) to detect circulating antigen in the sera of Wuchereria bancrofti-infected subjects from an endemic area of Papua New Guinea. A clear association between the presence of patent infection and the Gib 13 target epitope in serum was established because 93% of microfilaremic individuals were antigen-positive. Moreover, there was a significant correlation between levels of serum antigen and blood microfilarial counts. Detection of circulating antigen in amicrofilaremic subjects with acute symptoms of lymphatic filariasis, and 53% of asymptomatic amicrofilaremic subjects, but not in nonendemic controls, suggests that the Gib 13 IRMA will also be of value in the diagnosis of occult filariasis. However, as in all IRMA based on detection of potentially immunogenic molecules in man, antibodies can be expected to be the major contributor to reduced sensitivity of the assay.     

Analysis,characterization and diagnostic use of circulating filarial antigen in bancroftian filariasis

Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of circulating filarial antigen fraction-2 isolated from plasma of microfilaraemic patients withWuchereria bancrofti infection has shown 21 bands with molecular weights ranging from 12 to 120 kDa. The gel (12 cm) was sliced at an interval of one cm and the eluates of all the gel slicesviz., CFA2-1 to CFA2-12 showed the presence of filarial antigen by sandwich enzyme-linked immunosorbent assay. The low molecular weight circulating filarial antigen fractions were found to share a common epitope withWuchereria bancrofti microfilariae excretory-secretory antigen and urinary filarial antigen. The 3 antigen fractions CFA2-1, CFA2-9 and CFA2-12 showed higher sensitivity in detecting filarial immunoglobulin M antibodies than immunoglobulin G antibodies. However CFA2-9 fraction was found useful in serological differentiation of microfilaraemics from those with disease manifestations when filarial immunoglobulin G antibodies were detected. The antigenic epitope of CFA2-1 appears to be a carbohydrate, whereas CFA2-9 appears to be protein in nature.     

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.