Development Of A Microbead Florescent Immunoassay Biology Essay

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Herpes simplex type 1 (HSV-1), Herpes simplex type 2 (HSV-2) and Herpes varicella zoster (HZV) viruses are important human pathogens. The three viruses are classified within the alphaherpesvirinae subgroup of the Herpesviridae because of the similarities in their genetic code and their biological properties. Serological differentiation of HSV-1 and HSV-2 is particularly difficult because the two viruses share more than 95% genome homology. Cross reaction of the serological response between HSV-1, HSV-2 and also with HVZ has been reported. Existing immunoassays for these viruses utilise either virus propagated in cell culture; sub-units of the viruses purified from cell culture grown virus; or virus glycoprotein antigens expressed in bacteria. All of these methods have limitations in terms of production and/or efficacy. In this study the immunodominant protein of HSV-1 and HSV-2 (glycoprotein D, gD) was expressed in insects cells using a baculovirus expression vector. These proteins together with a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) were used to develop a multiplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of these viruses antibody in human sera.

Methods:

Recombinant proteins (HSV-1 gD and VZV gE) were expressed in insect cells using a novel plasmid expression system (InsectDirect, Novagen, Merck, USA). The full open reading frame of each DNA region encoding HSV-1 gD and VZV gE was amplified by PCR and cloned into pIEx/Bac-3 3C/LIC plasmid expression vector using a ligation-independent cloning (LIC) strategy. Insect cells (Sf9) were transfected with the recombinant plasmid and 48h post infection cells were harvested and the recombinant proteins were solubilised, purified and characterised using immunoblot and ELISA. To differentiate between HSV-1 and HSV-2 an immunodominant epitope of glycoprotein G2 presented in a branched chain format (peptide 55) was used (2).

Using a standard modified two step carbidiimide reaction, each antigen (HSV-1 gD, VZV gE and peptide 55) was coupled directly to a set of SeroMAP carboxylated microspheres. A monoplex assay for each antigen was developed and optimised individually and then the three assays were mixed in a triplex assay. Assay reproducibility was determined by measuring intra and inter assay variation using 10 samples tested in duplicate within the same plate on the same day or on two different days. The assay was then evaluated for the detection and measurement of antibody response to these viruses by testing a well characterised serum panel consisting of 218 human serum samples. Assay Sensitivity and specificity, positive and negative predictive value was calculated by comparison with results for the same samples tested by Western blot assays as a reference test for HSV-1 gD Peptide 55, and by Liaison VZV IgG assay for VZV gE.

Results

Results showed that of the 218 samples tested 184 were HSV IgG antibody positive and 34 were negative. A total of 47 of the 184 HSV positive samples were positive for antibody against peptide 55, indicating HSV-2 IgG positivity (Table 1). For VZV, 202 out of 218 samples were VZV IgG antibody positive (Table 2). Comparison of these results with western blot and ELISA assays previously used to characterise the panel showed sensitivities of 100%, 100% and 97.9 % for HSV gD, VZV gE and peptide 55 respectively. Intra and inter-assay CVs were generally less than 10%. Assay results are compared in Figures 1-3.

TABLE 1. Multiplexed Fluorescence Microbead Immunoassay Results in Comparison to Western Blot Assay.

Western

Blot Assay

HSV gD Assay

Peptide 55

Positive

Negative

Equivocal

Positive

Negative

Equivocal

Positive

184

0

0

47

1

0

Negative

0

34

0

0

34

0

Equivocal

0

0

0

0

0

0

Total

218

Sensitivity: 100% and 97.9% respectively, for HSV gD and Peptide 55.

Specificity: 100% and 100% respectively, for HSV gD and Peptide 55.

Positive Predictive Value: 100% and 100% respectively, for HSV gD and Peptide 55.

Negative Predictive Value: 100% and 97.1% respectively, for HSV gD and Peptide 55.

Table 2 Multiplexed Fluorescence Microbead Immunoassay Results in Comparison to DiaSorin VZV IgG Assay.

DiaSorin VZV IgG Assay

HSV-1 gD Fluorescence Microbead Immunoassay

VZV gE Assay

Positive

Negative

Equivocal

Positive

202

0

0

Negative

0

16

0

Equivocal

0

0

0

Total

218

Sensitivity: 100% Specificity: 100%

Positive Predictive Value: 100% Negative Predictive Value: 100%

Fig 1 Comparison of HSV-2 antibody concentration measured by Peptide 55 ELISA (x axis) and Peptide 55 florescence microbead immunoassay (у axis).

Fig 2 Comparison of VZV antibody concentration measured by VZV gE florescence microbead immunoassay (x axis) and Liaison IgG Assay (у axis).

Fig 3 Comparison of HSV antibody concentration measured by HSV gD florescence microbead immunoassay (x axis) and Diamedix HSV IgG Assay (у axis).

Conclusion:

A sensitive and specific multiplexed fluorescence microbead immunoassay was developed and evaluated against commercially available ELISA assays. The developed assay showed improved sensitivity compared to existing assays. The advantage of the develop assay is its ability to measure antibody to the three viruses within the same sample, it is sensitive, faster and requires very low test sample volumes as little as 2µl can be used to detect antibody to the three viruses in comparison to ELISA assays witch require 30µl (10µl for each virus). The latter finding is particularly important when attempting to detect antibody in sample such as CSF where only low test volumes are commonly available.