2.2.3.4.1 HSV1 ELISA
A 100 μl of the 1/101 diluted serum samples were added to the readymade recombinant- gG1-antigen coated wells (Biokit, Spain). The antibodies in the samples were allowed to combine with the antigens attached to the well during a one hour incubation period at 37 °C. The wells were then washed to remove all residual test specimens and then enzyme-
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labelled antibodies to human IgG (conjugate) were added. The wells were incubated for 30 minutes at 37 °C to allow the conjugate to bind immunologically to the anti-HSV1 IgG antibodies that had combined with the antigen in the well during the first incubation period. After another wash to eliminate the unbound material, an enzyme substrate solution containing a chromogen was added. If the sample contained anti-HSV1 IgG, the solution developed a blue colour and the intensity of the colour was proportional to the number of antibodies in the specimens.
The samples were tested in duplicate and seven control samples were tested simultaneously to validate the assay and extract quantifiable data. The mean absorbance of the low positive control was calculated and considered to be the cut-off value, followed by dividing the sample absorbance by the cut-off value to give a ratio absorbance/cut-off, which is proportional to the concentration of the HSV1 IgG antibodies in each tested specimen.
2.2.3.4.2 HSV2 ELISA
HSV2 IgG was detected and quantified using a similar method to the HSV1 IgG ELISA. However, the wells of the HSV2 IgG ELISA kit were coated with recombinant-gG2- antigen instead of recombinant-gG1-antigen (Biokit, Spain).
2.2.3.4.3 VZV ELISA
VZV IgG was detected and quantified using a similar method to the HSV1 IgG ELISA. However, the wells of the VZV IgG ELISA kit were coated with VZV antigen from partially purified extract of human fibroblast infected with VZV, strain ELLEN (ATCC) (Diamedix, USA). To determine the ELISA unit (EU)/ml, the following formula was used
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following the manufacturer’s instructions (absorbance of calibrator x absorbance of sample = EU/ml of sample).
2.2.3.4.4 CMV ELFA
CMV IgG was quantified by an automated quantitative two-step enzyme immunoassay sandwich method with a final fluorescent detection, enzyme-linked fluorescent assay (ELFA) (bioMérieux, France).
All the assay steps were performed automatically by the vitek immuno-diagnostic assay system (VIDAS) instrument, according to the manufacturer’s instructions (Figure 2.19). The solid phase receptacle (SPR) coated internally by the CMV antigen (strain AD169) acted also as a pipetting device for the assay. Fluorescence was measured twice for each sample tested; the first reading was a background reading of the serum and the second one was taken after the incubation period with the antigen. The instrument, using calibration curves that are stored by the machine, automatically calculated the results and expressed them in AU/ml. The samples were tested in duplicate, along with calibrator and control samples to validate the assay.
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Figure 2.19: VIDAS instrument (bioMérieux, France) [source: Virology Unit at Barts and The London NHS Trust].
2.2.3.4.5 EBV CHEMILUMINESCENT IMMUNOASSAY
EBV viral capside antigens (VCA) IgG was quantified in the investigated serum samples by the LIAISON analyzer, following the manufacturer’s instructions. The samples were tested in duplicate, along with calibrator and control samples, to validate the assay (DiaSorin, Italy).
The fully automated LIAISON immunoassay was used with its magnetic microparticle technology, chemiluminescence with flashlight kinetics, and an isoluminol derivative as labels. The p18 synthetic peptide is the major component used for coating magnetic particles (solid phase) and a mouse monocolonal antibody is linked to an isoluminol derivative (isoluminol-antibody conjugate). The cycle has two incubation periods: VCA antibodies in the sample were allowed to bind to the solid phase during the first incubation period, while the antibody conjugate reacted with VCA IgG already bound to the solid phase during the second incubation period. The unbound material was removed in a wash
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cycle after each incubation period. Subsequently, the starter reagents were added and a flash chemiluminescence reaction was induced. The light signal was measured by a photomultiplier as relative light units (RLU) indicating VCA IgG concentration in tested samples.
30 µL of each sample were used for this analysis. All reagents required for the assay (magnetic particles, luminescence-labelled tracer, two calibrators, diluent and assay buffer) were provided ready to use and assembled in one integrated reagent cartridge identifiable by a bar-coded label providing information such as lot number, expiry date and recalibration data (Figure 2.20). The analyzer automatically calculated the antibody concentrations and expressed it as U/ml.
Figure 2.20: LIAISON analyzer (DiaSorin, Italy) [source: Virology Unit at Barts and The London NHS Trust].
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2.2.3.4.6 HHV8 INDIRECT FLUORESCENT ASSAY
An indirect fluorescent antibody staining method was employed to semi-quantify the HHV8 IgG antibodies in the serum samples (Advanced Biotechnologies, Columbia). The 1:40 diluted serum samples were incubated with HHV8 infected cells on a slide, allowing the antibody in the serum sample, if present, to bind with the antigen present in the infected cells, forming immunological complex. Incubation was done in a moist chamber for 30 minutes at 37 °C. After the wash step (5–10 minutes in a wash buffer), the anti-human IgG conjugated to the dye fluorescein isothiocyanate (FITC) was added to each well on the slide and allowed to bind to the antibody-antigen complexes for 30 minutes at 37 °C.
The slides were then washed (5–10 minutes in wash buffer), mounted and blindly evaluated under a fluorescent microscope by two different investigators. Each slide contained ten tests; the positive and negative controls and wash buffer were tested simultaneously with the seven patients’ samples to validate the assay. The positive control was given a semi- quantitative score equal to 4 and the negative control was given a score of 0 (Figures 2.21 and 2.22). Each patient’s samples were scored relative to the positive and negative controls. The results were checked for consistency and re-evaluated if there were any discrepant results between the two investigators.
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Figure 2.21: HHV8 indirect fluorescent assay (positive control sample).
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2.2.4 STATISTICAL ANALYSIS
The statistical power calculation was investigated using StateMate 2 software to determine the minimum number of samples required in each part of the study. This practice ensured avoiding type I statistical error (rejecting the potentially true null hypothesis), and type II statistical error (failure to reject the potentially false null hypothesis) (GraphPad, USA).
Mean, median, range, minimum, maximum, standard deviation (SD), standard error of mean (Sem), and percentage (%) were used in the descriptive statistics. The obtained data were analysed by Mann-Whitney-U when comparing quantitative data of two groups, Kruskal-Wallis when comparing quantitative data of more than two groups, and non- parametric multivariate analysis of variance (MANOVA) with Bonferroni post tests when comparing multiple variants in relation to different groups. Differences in the rate were analysed using Chi-square tests and the correlation was analysed by using Spearman test. The GraphPad Prism® statistical package was used (GraphPad, USA).
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CHAPTER 3
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