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Journal of Immunological Methods 387 (2013) 157–166Contents lists available at SciVerse ScienceDirect
Journal of Immunological Methods
journal homepage: www.elsevier.com/locate/jim
Research paper
Designing of novel antigenic peptide cocktail for the detection of antibodies
to HIV-1/2 by ELISA
Ram P. Tiwari a, Anubhav Jain a,1, Zakir Khan b,1, Pradeep Kumar a, Vipul Bhrigu c, Prakash S. Bisen d,⁎
a
Research and Development Centre, RFCL (formerly Ranbaxy Fine Chem Ltd.), Avantor Performance Materials, New Delhi 110020, India
b
Department of Virology, Unit Viral Neuro-Immunology, Institut Pasteur, 75724 Paris Cedex 15, France
c
Roche Diagnostic India Ltd., New Delhi, India
d
Defence Research and Development Establishment (DRDE), DRDO, Ministry of Defence, Govt. of India, Jhansi Road, Gwalior 474002, India
a r t i c l e i n f o a b s t r a c t
Article history: HIV (human immunodeficiency virus) infection has now become endemic worldwide and
Received 12 September 2012 AIDS ranks fourth among the world's top killers of mankind. A rapid and accurate HIV testing
Received in revised form 12 October 2012 assay is a pre-requisite for practical applicability of diagnostic tests. The aim of this present
Accepted 16 October 2012 study was to design peptide cocktail as an antigen and to develop ELISA test for HIV-1/2
Available online 23 October 2012
antibody detection, with enhanced sensitivity and specificity. A novel peptide stretch V3-I,
covering immunodominant epitope corresponding to V3 hypervariable loop of gp120 antigens
Keywords: of selected Indian isolates, has been studied and incorporated in an antigenic cocktail of gp36,
HIV gp41, and rp24 of HIV-1/2. Peptides from these antigens were chemically synthesized and an
Indian HIV isolates
additional cysteine residue was added at both amino- and carboxyl-terminal sequences of each
V3 hypervariable loop
peptide in order to form inter and intramolecular disulfide bond for the folding of peptides. This
Immunodominant antigens
Cysteine bridge generated conformational epitopes with increased oligomericity and stability of peptide sequences;
Oligomeric peptide and attachment of antigen to the solid support of ELISA plates. The use of antigenic cocktail of folded
peptides and recombinant p24 enhanced sensitivity and specificity of the ELISA test. Evaluation of
the test using 1123 serum samples in comparison with Boston Biomedical Incorporation (BBI)
panels showed 100% sensitivity and 99.3% specificity with no cross reactivity tribulation. In
conclusion, “HIV screen test” detects HIV 1/2 antibodies with a high degree of sensitivity and
specificity and could be a promising tool for seroscreening of blood during transfusion, counseling
and diagnosis of HIV-1/2.
© 2012 Elsevier B.V. All rights reserved.
1. Introduction
HIV/AIDS causes morbidity, disability, mortality, with associ-
ated loss of productivity, and medical care costs. Statistics show
Abbreviations: ELISA, enzyme linked immunosorbent assay; Ag, antigen;
Ab, antibody; HIV, human immuno-deficiency virus; HPLC, high pressure liquid
that approximately 34 million people are currently living with
chromatography; AIDS, acquired immuno deficiency syndrome; env, envelope; one of the ten known subtypes of the HIV infection, with
gp, glycoproteins; C, cysteine bridge; TMB, 3,3,5,5 tetra methyl benzedine; PC, 2.7 million new infections worldwide; and an estimated 2.5 mil-
positive control; NC, negative control; MNc, mean of negative control; MPc, lion have died from this disease. Nearly half of the newly infected
mean of positive control; BSA, bovine serum albumin; NRS, normal rabbit
persons belong to the economically active age group of
serum; NHS, normal human serum; DMSO, dimethyl sulfoxide; H2O2, hydrogen
peroxide; DCM, dichloro methane; TFAA, trifluoro acetic acid; PBS, phosphate 15–24 years. The emergence of new variants reflects HIV-1
buffered saline; OD, optical density; HRPO, horseradish peroxidase; WB, prevalence, subtype epidemiology, and risk-behavior patterns in
Western blot; rp24, recombinant protein; μl, micro-liter; nm, nanometer; N, different geographical areas. Genetic differences among HIV-1
normal; mM, milliMolar; M, molar; v/v, volume by volume; WHO, World variants can influence the biological properties of the virus, its
Health Organization.
⁎ Corresponding author. Tel.: +91 751 2462500.
susceptibility to existing and candidate anti-retroviral drugs, and
E-mail address: psbisen@gmail.com (P.S. Bisen). evolution of drug resistance (UNAIDS/WHO, 2011; Kartikeyan
1
Equal contribution. et al., 2007).
0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jim.2012.10.009
, 158 R.P. Tiwari et al. / Journal of Immunological Methods 387 (2013) 157–166
The strains of HIV-1 can be classified into four groups: the benzhydrylamine resin was added in dichloro methane (DCM)
“major” group M, the “outlier” group O and two new groups, for 30 min in reaction vessels followed by washing with
N and P. Within group M there are known to be at least nine dichloro methane. The resin was deprotected with 25% trifluoro
genetically distinct subtypes of HIV-1. These are subtypes A, acetic acid (TFAA) in dichloro methane for 30 min, washed
B, C, D, F, G, H, J, K and CRFs. Subtypes and CRFs are typically with dichloro methane, neutralized with 10% triethyl amine
associated with certain geographical regions. For example, in dichloro methane, and difluro methane. The N-terminal
subtype A and CRF A/G predominate in West and Central and side chain (sulfohydroxyl group) of the first amino acid
Africa, with subtype A possibly also causing much of the residue (cysteine) were protected with butyl oxycorbonyl and
Russian epidemic (Bobkov et al., 2004) and subtype B has acetamino methyl group, respectively. Half gram-protected
been the most common subtype/CRF in Europe, the Americas, amino acid (BOC-Cys-Acm-OH; Bachem, USA) was dissolved in
Japan and Australia while in Southern and East Africa, India 1 ml dichloro methane and mixed with 3 g of pre-swelled
and Nepal subtype C is predominant (Le Vu et al., 2010; 4-methyl benzhydrylamine resin. Equimolar concentration of
Pollakis et al., 2003). dicyclohexyl carbodiimide (coupling agent) was added and
Initial tests for HIV are usually conducted using the ELISA shaken in half-motion for 60–80 min. Coupling efficiency
antibody test or a rapid antibody test. Unlike previous tests, was estimated using the Kaiser test (Kaiser et al., 1970) and
the fourth generation test detects HIV antibodies and antigens deprotection was carried out with 25% trifluoro acetic acid
simultaneously for both acute and recent HIV infections in dichloro methane for 30 min followed by confirmation with
(Pandori et al., 2009). Food and Drug Administration (USA) the Kaiser test. After second amino acid neutralization and
has approved a number of HIV test kits for diagnosis, prognostic washing, the reaction was further continued for coupling of
determination, patient monitoring, and screening of blood and other amino acid (histidine for V3-I) whose amino functional
tissue donors. It has been observed that certain subtypes/CRFs group and side chain were protected with butyl oxycorbonyl
are predominantly associated with the mutations in envelop and Tosyl. After shaking for 30–50 min and washing, Kaiser
glycoprotein which allow specific modes of transmission (Taylor test, and deprotection were carried out to attach the protected
et al., 2008) and variations in disease progression (Baeten et al., amino acid residues sequentially. Synthesized peptide sequence
2007). However, different HIV subtypes may have different was released from the solid support (methyl benzhydrylamine
immunodominant regions on antigenic proteins that may cause resin) by treatment with anhydrous hydrogen fluoride, trifluoro
different immune reaction and consequently reflect different methane, sulfonic acid, or trifluoro acetic acid to obtain crude
antibody titers in the host body. Therefore, specific tests used peptides. Side chain of trifunctional amino acids were protected
may vary from country to country. by group-specific protectants such as cyclehexyl for glutamic
Challenges and prospects of developing a suitable immuno- acid, benzyl for threonine, Tosyl for argenine, para-methyl
diagnostic test for detecting HIV-1/2-tuberculosis co-infection benzyl for cysteine, 2,6 dichloro benzyl for tyrosine, fluorenyl-
have been reviewed (Tiwari et al., 2005, 2007). The immuno- methyl oxycorbonyl or 2-chlorobenzyloxy carbonyl for lysine
genicity of synthetic gp41 tagged with “stealth” liposomes and and deprotected by adding 25% TFAA and DCM. After peptide
serodiagnostic ability of synthetic gag p24 chemically coupled synthesis, the side chain was deprotected.
with BSA has also been reported (Singh et al., 2007). In
high-prevalence and resource-poor settings, sensitivity, speci-
2.3. Oligomerization of peptides
ficity, and cost-effectiveness are pre-requisites for practical
applicability of diagnostic tests to supplement the compre-
A synthetic oligomeric and heterovalent peptide sequence
hensive nationwide AIDS control programs. Uses of antigenic
from immunodominant region of HIV-1 gp41 (25 amino
cocktail further improved specificity and sensitivity of diagnos-
acids), V3-I from hypervariable loop of gp120 (32 amino
tic test which even can differentiate one pathogen subtype with
acids) and another peptide sequence corresponding from
the others (Tiwari et al., 2005). In the present study, to cover
HIV-2 gp36 (16 amino acids) were modified to form inter-
genetic diversity, novel peptide V3-I derived from gp120 of
and intra-molecular disulfide bridges to detect both HIV-1
Indian isolate HIV-1 along with gp41, rp24 and gp36 were
and HIV-2 infections. The cysteine residues were incorporat-
used as antigenic cocktail to develop an indirect ELISA, herein
ed at both carboxyl- and amino-ends to generate an odd
referred to as “HIV screen test” in order to cater to the
number of cysteine residues, which facilitated inter- and
increasing demand for HIV-1/2 diagnostics.
intra-molecular oligomerization, leading to stability of pep-
tides (Tripathy et al., 1992).
2. Materials and methods
2.1. Antigens 2.4. HPLC purification and sequence analysis
Immunodominant regions of gp120/V3-I (HIV-1 Indian 2.4.1. The reverse-phase cation-exchange chromatogram of the
isolate), gp41 (HIV-1), rp24 (HIV-1), and gp36 (HIV-2) were V3-I peptide epitope
used to prepare antigenic cocktail. The ether precipitated peptide was purified by reverse-phase
cation-exchange column chromatography using a Vydac 400
2.2. Synthesis of peptides VHP column (Fig. 1). The purified fractions were subjected to
amino acid sequencing to confirm the sequence of peptides. The
The peptides were synthesized employing the procedure performances of oligomeric peptides were compared with linear
described by Merrifield (1986) and Stewart and Young (1984) peptides in ELISA. Sequences of peptides used for the develop-
with minor modifications in which, 1.0 g of Boc 4-methyl ment of the HIV screen test are outlined in Table 1A.
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