Sampling method standardization with r<i>Wb</i>SXP-1 assay.

<p>Samples from (<b>A</b>) MF, (<b>B</b>) EN, (<b>C</b>) CP, (<b>D</b>) NEN were analysed. Samples collected as sera, whole blood, on filter paper and by the new slide method are shown. Each data point represents individual absorbance from the four different types of samples. The horizontal bars represents the mean value of each group. The values are expressed as mean ±SD.</p

Expression and purification of r<i>Wb</i>SXP1 protein.

<p>(<b>A</b>). Expression of r<i>Wb</i>SXP-1 was performed by inducing salt inducible <i>E.coli</i> GJ1158 with 250 mM NaCl. The cell lysate was electrophoresed on a 12.5% polyacrylamide gel under reducing conditions and stained with Coomassie brilliant blue. Lane 1: molecular weight markers; Lane 2:vector induced; Lane 3: r<i>Wb</i>SXP-1 uninduced; Lane 4:r<i>Wb</i>SXP-1 induced; Lane 5: r<i>Wb</i>SXP-1 purified protein. (<b>B</b>). Immunoreactivity of purified r<i>Wb</i>SXP-1 with Anti-histidine antibody(dilution 1∶10,000). Lane 1 : molecular weight marker; Lane 2 : Purified r<i>Wb</i>SXP-1.</p

Human immune response to r<i>Wb</i>SXP-1.

<p>Immunoreactivity of r<i>Wb</i>SXP-1 with different clinical sera of lymphatic filariasis. 2 µg (approx) of purified r<i>Wb</i>SXP-1 was electrophoresed on 12% SDS-PAGE and transferred onto nitrocellulose membrane, blots were probed with pooled sera from MF, EN, CP and NEN individuals. High reactivity with pooled MF sera was seen with No reactivity with EN, CP and control NEN sera. Lane 1 : molecular weight marker; Lane 2 : MF; Lane 3: EN; Lane 4: CP; Lane 5: NEN.</p

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

<div><p>Background</p><p>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 r<i>Wb</i>SXP-1 antigen detection assay to our new sampling methodology.</p><p>Methodology</p><p>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 r<i>Wb</i>SXP-1 antigen detection assay using monoclonal antibody raised against r<i>Wb</i>SXP-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 r<i>Wb</i>SXP-1 assay.</p><p>Principal Findings</p><p>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 r<i>Wb</i>SXP-1 assay gave 98 antigen positive results whereas the microscopic method gave only 17.</p><p>Conclusions</p><p>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 r<i>Wb</i>SXP-1 antigen detection assay. The 1106 samples from Polur were collected using the new method. The r<i>Wb</i>SXP-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 r<i>Wb</i>SXP-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.</p></div

Analysis of different sampling method with r<i>Wb</i>SXP-1 assay.

<p>Analysis of different sampling method with r<i>Wb</i>SXP-1 assay.</p

Standardized r<i>Wb</i>SXP-1 assay with slide method samples.

<p>Standardized r<i>Wb</i>SXP-1 assay with slide method samples.</p

Survey details for Polur Village.

<p>Survey details for Polur Village.</p

Standardized r<i>Wb</i>SXP-1 assay for the slide method of samples.

<p>Four different clinical groups MF (n = 20), EN (n = 20), CP (n = 20) and NEN (n = 10) were used for the assay. The MF samples had high significance with a P value of <0.0001. The horizontal bars represents the mean value of each group. The values are expressed as mean ±SD and shown in the graph. The cut off value of 0.3477 is calculated by NEN+3SD. Samples with OD values above the cut off are considered as r<i>Wb</i>SXP-1 positive.</p

r<i>Wb</i>SXP-1 assay for Polur samples collected by new slide method.

<p>The samples collected in Polur, Tiruvannamalai, Tamil Nadu were assayed by the r<i>Wb</i>SXP-1 assay and by thick smear microscopic detection after staining with JSB stain. A total of 98 antigen positive samples were detected by r<i>Wb</i>SXP-1 assay, whereas the microscopic detection gave only 17 positives. The 17 positive samples detected by the microscopic method were positive in the assay also. The Antigen detection was significant with the microscopic detection with a P value of 0.0469.</p