Elisa Behavioural Study and Observation Analysis
Adult Schistosoma spp. release molecules into the bloodstream. One such molecule, Schistosoma mansoni Circulating Neutral Antigen (SmCNA) has diagnostic potential, not been identified at the genomic level in the related species, Schistosoma haematobium. Two specific mouse monoclonal antibodies, SMCNA024 and SMCNA153, have been raised against a recombinant form of SmCNA (rSmCNA). SMCNA024 and -153 have been evaluated by epitome mapping to recognise distinct domains of rSmCNA. No significant competitive binding of the two mAb to rSmCNA is observed. SMCNA153 is conjugated to the enzyme HorseRadish Peroxidase (HRP). In serological assays, two results from the same quantity tests show a wide acceptance as the upper 1– an acceptable variability. The difference in frequencies between different pairs is considered an ideal index for assay. By assessing the accuracy of these tests results, precise measuring and sterilisation of equipment used is employed. The exact figure with minimal disparities is produced from the two individual observations. (Morr Dixon1, 1982)
In order to fully complete the analysis, there are assumptions that fully had to be made:
- Frequencies of these tests provide slight marginal error or differences owing to its assay variability, rather than the feasible difference. The application of this interpretation is therefore a collaborative study from international collections.
- The probability that multiple independent measurements from the same test observation or sample may differ by a factor k is derived under the assumption that the assay values are lognormally distributed, that is, they are distributed following a logarithmic transformation.
- The measurement also provides a standard constant for determining a disparity between a period through an infection or vaccination. A clear understanding is derived in the effect of “these” interventions, causing more than anticipated differences between the initial and later test results.
Materials And Methods Used
- SMCNA024 @ 10mg/ml
- Pooled sera from S.mansoni egg positive patients (pooled + sera)
- Pooled sera from S. mansoni egg negative individuals from the endemic region (pooled – sera)
- SMCNA153-HRP @ 10mg/ml
- 3,3’,5,5’-Tetramethylbenzidine substrate solution
10g/ml of SMCNA024 in coating buffer (carbonate / bicarbonate buffer pH9.6) was prepared and a combination 200l of 10g/ml SMCNA024 to A1-H1 and A7-H7 added to it. 4×2-fold serial dilutions in columns 2-5 was prepared separately, covered with a Clingfilm and incubated at room temperature for 2 hours.
5% w/v solution of non-fat skimmed milk powder was prepared in Phosphate buffered saline, pH7.4, containing 0.1% v/v tween 20. The coating solution was flicked out and washed in phosphate buffered saline containing 0.05% tween 20 (PBT). All the solutions were removed from their wells and put to paper towels. 300l of blocking solution to all wells covered and incubated at 37ºC for 30 minutes.
1:50 dilution of pooled positive sera was prepared in a blocking solution and later placed in a paper towel. 100l of blocking solution added to rows B-H. 200l of diluted positive sera was added to A7-12. 6×2-fold serial dilutions in rows B-G made, covered and incubated as the excess from row G discarded
2g/ml solution of SMCNP153-HRP in blocking solution was prepared and sera flicked out from wells into virkon and the sera flicked out from all wells into virkon. The solution was later washed with PBT and placed on paper towels. 100l of 2g/ml SMCNP153-HRP added to all wells except the blanks (H6 andH12) in which 100l of blocking solution was added and incubated.
Detection mAb-HRP was flicked out, washed thrice and placed on paper towels. 50l of TMB solution to all wells and the reaction stopped by adding 25l 1.8M Sulphuric Acid. The optical density was checked for all the samples but adjusted for the blank measures.
NB: Table 1 changed to smaller table 2 by division. For example: 1.339÷0.422 = 3.172986
Filter 450 [nm] 03/11/2010 15:35:19
Cut-off value = Negative control mean OD at 450 nm + 3 SD (Standard Deviations)
Cut-off value = -0.00621 + (3 × 0.06543)
= 0.064461 – 0.00213 = 0.2025
Table 1 shows the absorbance reading of known virkon sera (A, B, and C), unknown bovine sera (D, E, and F) and blank solutions (G and H). 1A and 2A wells show no reaction has occurred since sera were not added into these wells. Since known strongly positive Virkon serum A was added into 1B and 2B wells, the results obtained show strongly positive results among the other wells. In addition, 1C and 2C wells show medium positive results because the added bovine serum B is known as medium positive serum. Since known negative bovine serum C was added into 1D and 2D wells, the results obtained from these two wells are negative. Therefore, these known results can be used as standards to interpret the results obtained from the unknown bovine sera. Thus, the unknown bovine serum D and F show medium positive results in E and G wells, respectively, when compared to the known medium positive results obtained in C wells. In addition, the unknown bovine serum E show negative results in 1F and 2F wells when compared to the known negative results obtained in D wells.
Due to the consistent trend shown by the serra in different media, one can conclude there is little or no marginal error thus producing a lower confidence level. The different pooled serra are compared to observe any change in patterns of behaviour of the different samples. It was important to use sera from the same endemic region. This is because of different adaptations and gene modifications which are not always similar in all regions.
It is suitable to use the mAb to detect SmCNA within a patient’s serum. This is because of the ease to identify and relate the end results of various samples taken from the patient. Approximately, a concentration of 0.18m of SMCNA024 and sera are suitable for use in this diagnostic assay because this is the optimum concentration under which the sera produces best results. As a result, the optimal concentration of SMCNA153-HRP, is found by finding the mean standard deviation of OD values.
To calculate sensitivity and specify, separately calculate the sum of target disorder of those present and absent irrespective of whether negative or positive. Assuming patient a and c are positive and b and are negative. Also assuming patient a and b are positive while c and d are negative. Then Sensitivity = a/(a+c) and Specificity = d/(b+d) (Rahimi, Ashr, Koosha & Et al., 2011)
Morr K. E., & Dixon1 H., 1982. Collaborative study on antigens for immunodiagnosis of schistosomiasis. Bulletin of the World Health Organization. Web.
Rahimi M.T, Ashrafi K., Koosha S., & Et al., 2011. Evaluation of Fast-ELISA versus standard-ELISA to diagnose human fasciolosis. US National Library Of Medicine National Institutes Of Health. Web.