The clear difference between Western Blot (WB) and Line

Immuno Assays (LIA) and the impact on clinical routine

- Clear-cut results - Automation

- Low rate of indeterminate results - C olor-coded and ready-to-use
- Detection & differentiation reagents
What is a Line Immuno Assay (LIA)?

LIA incorporates separate antigens on nylon strips in order to visualize each reaction separately. LIA anti-
gens are “painted” on the strips rather than being electrophoresed from viral lysates. The carefully selected
antigens are recombinant antigens and synthetic peptides applied at an optimal concentration.

Why use an immunoblot compared to Western Blot?

For the confirmation of antibodies, different assays are avail-
able; a choice can be made between a Western Blot and an
immuno­blot. An immunoblot such as INNO-LIA offers several
TM
advantages over Western Blot. [1]
• The antigens are not derived from lymphocyte-cultured viral
lysates, allowing the optimal selection of only the required an-
tigens. With this INNO-LIA avoids the elevated background
TM
from the non-specific host cell proteins observed with WB;
which in turn is one of the reasons for a higher number of
indeterminate WB-results in non-infected individuals.
• The synthetic and recombinant antigens can be better stand-
ardized, which minimizes lot-to-lot variation.
•
The antigens can be applied in optimal concentrations, ­unlike
­Western Blots, where poor expression of some antigens (e.g. gp41)
is always a problem. The presence of optimal antigen concen-
tration leads to an easy and standardized interpretation.
• Interpretation of INNO-LIA™ can be automated.
• INNO-LIA™ offers better quality control because it includes
­different controls on the strips to verify that the sample was
added, that the procedure was performed correctly and to
grade the reactions of the antigen lines.
• The fewer indeterminate results compared to Western Blot are
confirmed by WHO. INNO-LIA™ HIV I/II Score is recommended
by WHO [2]
after technical review of product quality and
­
performance and included in the List of HIV Diagnostics for
procurement by WHO.[3]

Why confirmation of antibodies after serological screening?

Serological screening tests are highly sensitive tests to provide
reliable detection of antibodies/antigens in a specimen. As
screening tests are designed to make sure no positive specimen is
missed (focus on sensitivity), a portion of samples will inherently
react false-positive. Therefore, the reactive screening results must be
confirmed by a confirmatory assay. Only a positive confirmatory test
allows the diagnosis of an infection, and only such a result should be
used to refer the patient for further counseling. Confirmation of the
initial reactive screening assays should be done through antibody
confirmation and nucleid acid ­testing (NAT). Both technologies have
specific applications: NAT is specific for viral and bacterial DNA/
RNA, line immuno assays are specific for detecting antibodies.

Line Immuno Assays have highly standardized band patterns; due to the use of specific synthetic peptides and recombinant proteins.
This advantage allows the laboratory to use automated interpretation of the results. INNO-LIA™ Score is highly recommended in
accredited and reference laboratories due to its high standardization, options for automation and low rate of indeterminates.
For this reason many laboratories use both tests in
parallel. F­ urthermore, lack of commercially avail-
able tests or cases where NAT reacts false-negative
show that confirmation can not rely on NAT only.
NAT negative results do not ensure that there is no virologi-
cal activity and therefore are not equal to seronegative re-
sults. Occult HCV infections and HIV elite controllers are ex-
[4-13]
amples of seropositive patients with undetectable viral load.

What is the added value of antibody confirmation next to

Nucleic Acid Testing (NAT)?
Elite controllers
Elite controllers are a group of patients within the group of
long-term non-progressors of HIV. Elite controllers are rare HIV-
infected individuals who are able to naturally control HIV repli-
cation without anti-retroviral therapy, persistently maintaining
viral loads below 50 copies/ml for more than 12 months (unde-
tectable by currently available commercial assays). Nonetheless,
these individuals are seropositive and infectious. Elite control-
individuals can only be detected by use of serological assays and
not by NAT. In the setting of blood donation, it is possible that an
antibody positive and NAT negative
donation might transmit infection to
the recipient if only NAT testing is
performed.
Long-term nonprogressors

lers represent 3/1000 HIV infected individuals. [14-15] This group of

HIV controllers

Occult infection
Another form of infection with undetectable viral load is an
occult infection, described in HBV and HCV patients.
Occult Hepatitis C (HCV) infection is:
• Characterized by presence of HCV RNA in liver and in peripheral
blood mono­nuclear cells (PBMCs)
• Characterized by absence of detectable viral RNA in serum by
standard assays
• Found in anti-HCV positive patients with normal serum levels
of liver enzymes
• Found in anti-HCV negative patients with persistently elevated
liver enzymes of unknown etiology
• S till infectious
Occult HCV infection is spread worldwide and found for all HCV
genotypes. This occult infection has been reported in persons
at risk, but also in healthy populations without evidence of liver
disease. Occult HCV infection seems to be less aggressive than
chronic Hepatitis C although patients affected by occult HCV may
develop liver cirrhosis and even hepatocellular carcinoma.[16] The
prevalence of occult infections depends strongly on the patients
s­
tatus (hemodialysis, co-infection with HIV, co-infection with
HBV ...). [17] As an occult HCV infection cannot be detected by NAT,
serological testing is the only way to detect the patients with
undetectable RNA, but who are ­anti-HCV positive.
Differences in line patterns

A Line Immuno Assay only has a selection of antigens, which results in different reactivity patterns
­compared to Western Blot. The differences are explained in the next paragraph.

HIV confirmatory testing precursor gp160

gp120
glycoprotein

Western Blots use gel electrophoresis to separate proteins by

molecular weight and these proteins are then transferred (blotted) gp41
sgp120 and HIV-1
to a membrane. This membrane is cut into strips. The HIV-1 viral gp41 p31
HIV-2
p24
antigens are separated as follows: gp160, gp120, p66, p55, p51,
gp41, p31, p24, p17, and p15. It is important to remember that non- p17

viral proteins, derived from the host cells in which the virus was
cultured, will also be present on the nitrocellulose membrane. Figure 1: Differences between Western Blot and INNO-LIATM
This creates indeterminate results and a high background. [1] HIV I/II Score
GENES
INNO-LIA™ HIV I/II Score does not detect all these antigens, but nef vpu nef
5’ pol Env 3’
only a selection: gp41, gp120, p24, p17, p31, gp36, gp105 (Figure 1).
gag PR RT IN SN TM
For example, why does INNO-LIA™ HIV I/II Score not detect LTR MA vpr LTR
CA NC
tat
gp160? The “env” gene in HIV encodes a single protein, gp160. P6
rev
gp160 travels to the cell surface, where cellular enzymes attack
it, chopping it into two pieces - gp120 and gp41 (Figure 2). If and MA CA PR RT IN
Gag-Pol polyprotein
when new virus particles bud off from the host cell, these two MA CA NC P6
(p160)
SU TM
Gag polyprotein Env polyprotein
protease
pieces lie on opposite sides of the virus membrane. gp120 sits (p55) (gp160)
protease MA CA PR RT IN protease
on the outside of the virus particle, forming the virus’ spikes, P17 P24 P10 P66/p51 p32

while gp41 sits just on the inside of the membrane - each gp41 MA CA NC P6 SU TM
P17 P24 P7 P6 gp120 gp41
being anchored to a gp120 through the membrane (Figure 3).
Detection of gp160 is always accompanied by the presence of Figure 2: Transcription and translation of the HIV genome

either gp41 and/or gp120. A single detection of gp160 will result

in an indeterminate result.
gp120

gp160 is present on Western Blot assays because the antigens are gp41
derived from lymphocyte-cultured lysates and therefore cannot
be separated from the other antigens. gp160 does not give added
value to the interpretation, on the contrary it is the cause of a One spike is made of three
gp120+gp41 subunits
higher number of indeterminate results. HIV viral
membrane

Figure 3: HIV membrane with surface glycoprotein 120

and glycoprotein 41
 INNO-LIA™ HIV I/II Score is a simple and reliable confirmatory test, which can be used either by manual or automated test proce-
dure. Firm and rigid strips are very easy to handle during workflow and archiving. Ready-to-use, color-coded working solutions
substantially limit the possibility of handling mistakes and allow monitoring of the test performance during all steps. Concise inter-
pretation also allows to identify the virus type. The automated reading and interpretation of the strips with the LiRAS® interpretation
software limits the subjectivity in test interpretation and reduces the reporting of false results.
Sverdlovkiy blood bank, Russia

Publications have shown that a single gp160, single p24 or both gp160
and p24 on Western Blot, are mostly related to non-specific reactions, for
example due to influenza vaccination. [18]

Bao et al. found that the most common band patterns of indeterminate HIV
antibody results were mainly p24 monoband, gp160 monoband or a com-
bination with p24. Most of these HIV indeterminate patients (95.6%) were
not infected by HIV, the bands showed up in the WB test and demonstrated
as non-specific reactions. [18] TpN47
TmpA

Treponema pallidum confirmatory testing

A Treponema pallidum Western Blot (also called Syphilis or lues) contains TpN17
many lines, but only 4 lines need to be taken into c­ onsideration to proceed
TpN15
with the interpretation (TpN47, TpN17, TpN15, TmpA). None of the other lines Figure 4: Differences between Western Blot
are needed for the interpretation or clinical diagnosis. (Figure 4) and INNO-LIATM Syphilis Score
Also for INNO-LIA™ Syphilis Score, only a selection of the r­ equired antigens
is used resulting in the most optimal result and interpretation.

rgp46-I
rgp46-II
HTLV confirmatory testing
gag
A HTLV Western Blot contains for example 11 different HTLV specific antigen precursor p53
polyprotein gp46
lines, although when considering the assay interpretation criteria, only 5 an-
tigens are taken into account (p24, p19, GD21, rgp46-I and rgp46-II). This gag
p36 gag p19 /II
p32 gag p24 I/II Confirmation lines
intermediate
already confirms that not all lines are relevant. products
p28 env gp46 I/II
p26 env gp21 I/II
p24 gag p19-I
env gp46-I HTLV-I
p19 HTLV-II
INNO-LIA™ HTLV I/II Score detects the four antigens required for a complete env gp46-II
Discrimination lines
interpretation and differentiation: gp46 I/II, p19 I/II, p24 I/II, gp21 I/II. This GD21

results in few indeterminate results and a straightforward interpretation.

(Figure 5) Figure 5: Differences between Western Blot
and INNO-LIATM HTLV I/II Score
Comparing performance of WB and INNO-LIA™

HIV
Earlier diagnosis
When comparing INNO-LIA™ HIV I/II Score with two Western Blot assays, INNO-LIA™ HIV I/II Score is
detecting s­ eroconversion earlier in 4/11 and 7/11 seroconversion panels respectively. INNO-LIA™ HIV I/
II Score provides earlier diagnosis and more clearcut results.

INNO-LIA™ HIV I/II Score 16 h INNO-LIA™ HIV I/II Score 3 h Western Blot 1 Western Blot 2

8
24 24
24 17
35 36 36
21 38
24 14
8 77

Negative Indeterminate Positive

Figure 6: Comparison on seroconversion panels of INNO-LIA™ HIV I/II Score with 2 WB assays36

Less indeterminate results

When measuring samples from anti-HIV low titer panels, INNO-LIA™ HIV I/II is more sensitive and has
significantly less indeterminate results compared to the two Western Blot assays.

Table 1: Comparison between INNO-LIA™ HIV I/II Score and two WB assays for anti-HIV low titer panels

Negative Indeterminate Positive

INNO-LIA™ HIV I/II Score 16 h / 3 h 3/3 0/2 25 / 23
Western Blot 1 1 10 17

Negative Indeterminate Positive

INNO-LIA™ HIV I/II Score 16 h / 3 h 4/4 1/3 35 / 33
Western Blot 2 3 24 13

• Earlier diagnosis ensures patient safety and prompt treatment [18]

• Fewer indeterminate results lead to less repeat testing and fewer patient follow-
up, which reduces costs.

This document contains a comparison of INNO-LIA™ HIV I/II Score and two WB assays. Fujirebio Europe confirms that the latest commercially available WB assays were used at the time of comparative evaluation. Detailed
information can be made available upon request. Fujirebio Europe undertakes no obligation to update this document after issuance date March 2014.
 We recently changed from a Western Blot system (New Lav Blot, Bio-Rad) for HIV confirmation to an immunoblot system (INNO-LIA™ HIV
I/II Score, Fujirebio Europe). A Western Blot assay is often difficult to interpret because the strips have a high background and with New
Lav Blot, typing of HIV sera (HIV-1 versus HIV-2 infection) requires two different kits. INNO-LIA™ has a selection of only the required HIV
antigens, applied in optimal concentration, which makes it possible to visualize each reaction separately and thereby eliminate the non-
specific background. As one INNO-LIA™ HIV I/II Score strip can differentiate between HIV-1 and HIV-2 infection, less indeterminate results
are obtained. With INNO-LIA™, our laboratory finds less HIV seropositive individuals with untypable infection, which means we need to
perform less repeats and therefore benefit from time and cost savings. The automated INNO-LIA™ strip reading procedure by the LiRAS®
interpretation software allows an objective, easy and standardized strip interpretation. Quality control is also guaranteed because each strip
contains 4 control lines.

Dr. Igor Matkovskiy, Vinnica AI

Recency information for INNO-LIA™ HIV I/II Score Definition:

With a Line Immuno Assay, it is possible to indicate a recent
infection. Certain antigens present on the strip can be associated
with an early infection.
Applying INNO-LIA™ HIV I/II Score to get information on the recency
of HIV-infection, has been ­thoroughly investigated in Switzerland
and is being used for monitoring in the national health system
since 2007. [19-22]
The different algorithms allow to distinguish
between a recent/incident infection ( 12 months) vs an older
HIV infection.
Recent/incident infection = infection of HIV ≤ 12 months
Unique advantage of INNO-LIA™ HIV I/II Score related to recency:
• Antibody response to various HIV antigens at the same time
and in a semi-quantitative way [19-22]
• The pattern and intensity of HIV specific antibodies evolve
during the first weeks to months after infection [19-22]
• Allows identification of different patterns that are characteristic
for early infection [19-22]

Syphilis
A total of 135 serum samples from newly diagnosed syphilis INNO-LIATM had a positive agreement with the consensus results
­patients in different clinical stages were tested to determine of 98.5%, whereas the WB assay 68.2%. Therefore, the LIA assay
sensitivities of the treponemal tests. Lam et al. found that the performed significantly better than the WB assay. [23]

Table 2: Comparison of INNO-LIA™ Syphilis Score and MarDx T. pallidum IgG Marblot Test (WB) results. [23]
Negative Indeterminate Positive Total
INNO-LIA™ Syphilis Score 3 5 127 n = 135
MarDx T. Pallidum IgG Marblot Test 9 38 88 n = 135

HTLV
The study of Sabino et al. showed that INNO-LIATM could resolve 153 of the 172 Western Blot-indeterminate results.
This confirms that also for HTLV, INNO-LIATM HTLV I/II gives significantly less indeterminate results. [24]

Table 3: INNO-LIA™ HTLV I/II results compared to WB 2.3 [24]

INNO-LIA™ result
WB 2.3 result
Positive Indeterminate Negative Total tested
Positive 34 1 56 91
Indeterminate 16 3 153 172
Negative 0 2 14 16
Total 50 6 223 279

Ordering Information
Product Description Art. no.
Assays
INNO-LIA™ HIV I/II Score 80540
INNO-LIA™ HCV Score 80538
INNO-LIA™ Syphilis Score 80542
INNO-LIA™ HTLV I/II Score 80541
Software
LiRAS® for Infectious Diseases v3 80361
LIA-Scan reading template 80493
LIA-Scan reading template (economic) 80543
References
[1]
C onstantine N.T., Zink H. Indian J Med Res 2005; 121(4):519-38
[2]
WHO 2009, HIV assays: operational characteristics, report 16:
rapid assays
[3] 
http://www.who.int/diagnostics_laboratory/­
procurement/120524_v7_product_eligible_for_who_­
procurement_2012.pdf
[4]
Najiouallah et al. J Med Virol 2004; 73:347-349
[5]
Phelps et al. Transfusion 2004; 44(6): 929-33
[6]
Stramer et al. Transfusion 2003; 43:Supplement :40-41A
[7]
Delwart et al. Vox Sang 2004; 86(3):171-7
[8]
Ling et al. JAMA 2000; 28: 4:210-214
[9]
Ferreira et al. Transfusion 2006; 46(1):156-7
[10]
Zanetti et al. Transfusion 2007; 47(7):1328-9
[11]
Harritshoij et al. Transfusion 2008; 48:2026-28
Fujirebio Europe N.V.
Technologiepark 6- B-9052 Gent - Belgium
T +32 (0)9 329 13 29 - F +32 (0)9 329 17 75
www.fujirebio-europe.com
© Fujirebio Europe, October 2015, FRE-021,r1
Product Description Art. no.
Automation
Auto-LIA™ 48 80629
AutoBlot 3000 81148
Auto-LiPA™ 48 80628
AutoBlot 3000H 81149
TENDIGO™ 80412
[12]
Schmidt et al. Vox Sang 2008; 95(Suppl 1) 56
[13]
Kalus et al. Transfusion 2009; 49(3):435-9
[14]
Deeks S.G., Walker B.D. Immunity 2007; 27(3):406-16
[15]
Okulicz J.F., Lambotte O. Curr Opin HIV AIDS 2011; 6(3):163-8
[16]
Carreño et al. World J Gastroenterol. 2012 Jun 21; 18(23):2887-94
[17]
Di Bisceglie. Lancet 1998; 351:351-355
[18]
Bao et al. Zhonghua Liu Xing Bing Xue Za Zhi. 2008 May;
29(5):478-81
[19]
Schüpbach et al. PLoS Med. 2007 Dec; 4:12:e343
[20]
Schüpbach et al. BMC Infectious Diseases 2011; 11:254
[21]
Schüpbach et al. BMC Infectious Diseases 2012; 12:88
[22]
Schüpbach et al. PLoS One. 2013 Aug 26; 8(8):e71662
[23]
Lam et al. International Journal of STD & AIDS 2010; 21:110-113
[24]
Sabino et al. J Clin Microbiol. 1999 May; 37(5):1324-8