Heliyon 6 (2020) e04114

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Heliyon
journal homepage: www.cell.com/heliyon

Research article

Rapid and specific detection of oxidized LDL/β2GPI complexes via facile

lateral flow immunoassay
Xian Wen Tan a, Fumiaki Takenaka b, Hironori Takekawa c, Eiji Matsuura a, b, c, d, *
a
Department of Cell Chemistry, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
b
Collaborative Research Center (OMIC), Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, Japan
c
Faculty of Medicine, Okayama University, Okayama, Japan
d
Neutron Therapy Research Center, Okayama University, Okayama, Japan

A R T I C L E I N F O A B S T R A C T

Keywords: β2-Glycoprotein I (β2GPI) forms indissociable complex with oxidized LDL (oxLDL) into proatherogenic oxLDL/
Biological sciences β2GPI complex through a specific ligand known as 7-ketocholesteryl-9-carboxynonanoate (oxLig-1). Recent dis-
Antibody coveries have demonstrated the atherogenicity of these complexes in patients of both systemic and non-systemic
Biochemistry
autoimmune diseases. Hence, serological level of oxLDL/β2GPI complexes may represent one crucial clinical
Lipid peroxidation
Health sciences
parameter for disease prognosis of atherosclerosis-related diseases. Herein, we established a simple, specific and
Oxidized LDL (oxLDL) rapid gold nanoparticle (GNP) based lateral flow immunoassay (LFIA) to quantify oxLDL/β2GPI complexes from
β2-glycoprotein I (β2GPI) test samples. Specificities of hybridoma cell-derived monoclonal antibodies against antigen, optimal conditions
OxLDL-β2GPI for conjugation of antibody with GNP, and sensitivity of oxLDL/β2GPI LFIA in comparison to an ELISA-based
Lateral flow immunoassay (LFIA) detection method were assessed accordingly. The established oxLDL/β2GPI LFIA was capable of detecting
Enzyme-linked immunosorbent assay (ELISA) oxLDL/β2GPI specifically without interference from autoantibodies and solitary components of oxLDL/β2GPI
Point-of-care present in test samples. A significant correlation (R2 > 0.8) was also obtained with the oxLDL/β2GPI LFIA when
compared to the ELISA-based detection. On the whole, the oxLDL/β2GPI LFIA remains advantageous over the
oxLDL/β2GPI ELISA. The unnecessary washing step, short developmental and analytical time support facile and
rapid detection of oxLDL/β2GPI as opposed to the laborious ELISA system.

1. Introduction of the clinical characteristics and prognoses of antiphospholipid syn-

drome (APS) [5, 6, 7, 8]. Monomeric β2-glycoprotein I (β2GPI) or
The pathogenesis of lifestyle disease such as atherosclerosis is closely phospholipid-bound β2GPI is perceived as the major immunogen held
associated with metabolic abnormalities of lipoproteins [1]. Its onset and accountable for the induction of aPL in APS patients [9, 10, 11, 12].
progression have been intimately linked to lipid peroxidation of β2GPI, a 50 kDa endogenous plasma protein [13], notoriously interact
low-density lipoprotein (LDL) within the arterial intima. Nearly 50% with anionic phospholipids such as phosphatidylserine (PS), cardiolipin
composition of LDL is mainly composed of cholesterol and cholesteryl (CL), and oxidized LDL (oxLDL) to form protein-lipid complexes [14, 15,
esters (CEs), thus it is highly susceptible to oxidation by reactive oxygen 16, 17]. The phospholipid-binding site of β2GPI was previously identified
species (ROS) such as superoxide anions (O-2) and hydroxyl radicals (⋅OH) in its domain V, at the sequence of K282NKEKK287 [17]. β2GPI recognizes
[2]. Oxidized LDL (OxLDL), the oxidized form of LDL, acts as a the structural part of 7-ketocholesteryl-9-carboxynonanoate (oxLig-1), a
pro-inflammatory chemoattractant that activate atherothrombotic im- specific ligand in oxLDL, to form indissociable oxLDL/β2GPI complexes
mune response by promoting pro-thrombotic endothelial dysfunction, [18]. In the presence of IgG anti-oxLDL/β2GPI autoantibodies, the uptake
synthesis and secretion of chemotactic cytokines. These abnormalities of oxLDL/β2GPI complexes by macrophages through their Fcγ receptors
promote recruitments of macrophages and their subsequent activation was enhanced significantly and has notably accelerated the formation of
and intracellular lipid accumulation within atherosclerotic lesions [3, 4]. foam cells and progression of atherosclerosis [18, 19, 20, 21, 22]. Aside
The prevalence of serological antiphospholipid antibodies (aPL), such from APS [23], our previous studies have also demonstrated the athe-
as anticardiolipin (aCL) antibodies and lupus anticoagulant (LA), is one rogenicity of these complexes in patients of non-systemic autoimmune

* Corresponding author.
E-mail address: [email protected] (E. Matsuura).

https://doi.org/10.1016/j.heliyon.2020.e04114
Received 7 April 2020; Received in revised form 15 May 2020; Accepted 28 May 2020
2405-8440/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
X.W. Tan et al. Heliyon 6 (2020) e04114

diseases, such as diabetes mellitus [24] and chronic renal diseases [25]. red, Hepes and sodium pyruvate (Fujifilm Wako Pure Chemical Corpo-
Intrinsically, serological level of oxLDL/β2GPI complexes may represent ration, Osaka, Japan), supplemented with 2.5 % (v/v) fetal bovine serum
a crucial clinical parameter for disease prognosis and risk stratification of and 100 units of penicillin-streptomycin. Cells were cultivated at 37
C, 5
atherosclerosis-related diseases. % humidity for 4–5 days before culture supernatant was collected for
Presently, serological levels of oxLDL/B2GPI complexes are measur- antibody purification.
able by enzyme-linked immunosorbent assay (ELISA). We have formerly Cells suspension was gathered and centrifuged at 1,500 rpm, 4
C for
used a lupus associated APS in NZW x BXSB F1 (W/B F1) mouse model to 5 min to collect culture supernatant. Respective antibodies were isolated
establish a monoclonal IgG (known as ‘WB-CAL-1’) that develop speci- from culture supernatants by purifications through rProteinA Sepharose
ficity towards oxLDL/β2GPI complexes. WB-CAL-1 is highly specific to- Fast Flow (GE Healthcare Bio-sciences, Tokyo, Japan). Briefly, rProteinA
wards the open form of β2GPI that has formed complex with oxLDL and Sepharose Fast Flow affinity medium was pre-equilibrated with 5 bed
not the closed form of β2GPI protein [14, 26, 27]. We later fabricated volumes of loading buffer (50 mM Tris/3.5 M NaCl/0.1 % NaN3, pH8.8).
another monoclonal antibody, 3H3, which share similar Then respective culture supernatants were pre-mixed with loading buffer
antigen-specificity as WB-CAL-1, yet with improved affinity and speci- at 1:1 ratio, and subsequently loaded onto the column. Unbound con-
ficity towards β2GPI complexed with oxLDL [28]. The established indi- taminants were eliminated by washing the column with additional 5 bed
rect sandwich (Figure 1) oxLDL/B2GPI ELISA utilizes two different volumes of loading buffer until the absorbance of collected effluent at
antibodies to target on two different epitopes on oxLDL/β2GPI complex. 280 nm read less than 0.01 AU. Bound proteins were eluted by addition
The coated monoclonal 3H3 antibody on ELISA plate acts as the primary of elution buffer (0.17 M glycine-HCl, pH 2.3). The eluate was fraction-
antibody that specifically recognizes β2GPI complexed with oxLDL only ated at 1mL and each fraction was subsequently neutralized by 3 mL
while the secondary antibody, 2E10 binds to apolipoprotein B100 neutralizing buffer (1 M Tris-HCl, pH 8.8). Elution was performed until
(apoB100) on oxLDL of the complex [29]. the absorbance of collected eluate at 280 nm read less than 0.01 AU.
Despite the applicability of our established oxLDL/β2GPI complexes Antibody-containing fractions were then combined and concentrated
ELISA, it incurs common drawbacks as with other conventional ELISA with Amicon® ultra 0.5 mL centrifugal filters (50K cut-off) (Merck Mil-
techniques. High labor-intensiveness, sophisticated operational steps, lipore, Darmstadt, Germany). Concentration of recovered antibody was
long incubation time, and cost-inefficiency are known drawbacks that determined via Pierce™ BCA Protein Assay Kit (ThermoFisher Scientific,
limit the operational efficiency of diagnostic kits especially for high- Rockford, IL). Purity and antibody activity were verified via SDS-PAGE
throughput applications [30]. With the growing demand for rapid and (10–20 % gels) and ELISA respectively.
cost-efficient point-of-care diagnostic means, novel technologies have
been introduced to further improve current clinical diagnostics into
favorable alternatives. Lateral flow immunoassays (LFIA) have gained 2.2. Isolation of LDL from human serum
interest in diagnostic applications due to their numerous advantages that
complies with the World Health Organization (WHO) guidelines. Criteria LDL was isolated from human serum pool (Cosmo Bio, Tokyo, Japan)
such as being cost-efficient, offering high sensitivity and specificity, easy through a single spin density gradient ultracentrifugation as per method
to use, expeditious, sturdy, and being practical are benchmarked as of Swinkels, Hak-Lemmers and Demacker [32] with slight modifications.
essential settings for diagnostic tests in developing countries [31]. Briefly, 3 different density-adjusted solutions (DAS) (DAS 1: 1.006
Herein, we attempted to establish a simple LFIA system that can rapidly g/cm3; DAS 2: 1.019 g/cm3; DAS 3: 1.063 g/cm3), each containing 1 mM
quantify oxLDL/β2GPI complexes within 20 min. EDTA, were prepared by adjusting the solutions’ densities with potas-
sium bromide (KBr). Thawed human serum pool was pre-mixed with KBr
2. Methodology at a volume-to-weight ratio of 13 mL serum to 1.82 g KBr. Mixture was
left to homogenize via gentle low-speed stirring on ice bath for 10 min.
2.1. Production and purification of anti-β2GPI (3H3) and anti-human Solutions were loaded slowly into polycarbonate (80PC bottle [C]) bottle
apoB100 (2E10) antibodies (Hitachi-Koki, Tokyo, Japan) in the order of plasma (23.6 mL) > DAS 3
(18.2 mL) > DAS 2 (18.2 mL) > DAS 1 (10 mL). Sample-loaded bottles
Anti-β2GPI (3H3) antibody- and anti-human apoB100 (2E10) were then subjected to ultracentrifugation via RP45T rotor-equipped
antibody-producing hybridoma cells were cultivated respectively in SCP85H ultracentrifuge machine (Hitachi-Koki, Tokyo, Japan) at 100,
Iscove's Modified Dulbecco's Medium (IMDM) with L-glutamine, phenol 000 x g, 4
C for 24 h. After centrifugation, VLDL- and
chylomicrons-containing fraction was carefully removed and

Figure 1. Schematic representation of oxLDL/β2GPI ELISA workflow and its principle.

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X.W. Tan et al. Heliyon 6 (2020) e04114

subsequently followed by acquisition of LDL-containing fraction. LDL DMSO and the whole volume of reconstituted NH2 reactive biotin was
fraction was later concentrated via amicon ultra centrifugal filter unit added to the filtration tube together with 100 μL Reaction Buffer (from
(10K cut-off) (Merck Millipore, Darmstadt, Germany) and dialyzed kit). The reaction mixture was then mixed thoroughly by gentle pipet-
against PBS buffer (pH 7.4) overnight in preparation for oxidation. ting, then incubated at 37
C for 10 min. Thereafter, 100 μL of WS Buffer
Concentration of acquired LDL fraction of apolipoprotein B100 was then added to the filtration tube, and followed by centrifugation at
(apoB100) equivalent was determined through Pierce™ BCA Protein 8,000 x g, 4
C for 10 min. Content in filtration tube was then washed
Assay Kit (ThermoFisher Scientific, Rockford, IL). twice with 200 μL of WS Buffer and followed by centrifugation at 8,000 x
g, 4
C for 10 min, to remove excess unconjugated biotin molecules. The
2.3. Preparation of chemically oxidized LDL (Cu2þ-oxLDL) and Cu2þ- biotin-3H3 conjugate was later recovered in 200 μL PBS buffer. Con-
oxLDL/β2GPI centration of biotinylated-3H3 was determined via Pierce™ BCA Protein
Assay Kit (ThermoFisher Scientific, Rockford, IL).
Concentration of LDL was adjusted to 100 μg/mL apoB100 equivalent Commercially available gold conjugation kit (Naked Gold Conjuga-
and subsequently chemically oxidized by 5 μM copper (II) sulphate tion Kit (20 nm), Bioporto Diagnostics, Denmark) was used to conjugate
(CuSO4) at 37
C for 16 h. Oxidation was terminated by addition of EDTA 2E10 antibody with GNP. The spectral absorptivity and average particle
(final concentration of 1 mM) to the reaction mixture. For negative size of GNP were characterized via UV-visible spectrophotometer (Bio-
control, EDTA was added to the LDL and CuSO4 mixture at 0 time (t ¼ 0). Spec-Nano, Shimadzu, Japan) and Malvern Zetasizer Nano ZSP (Malvern
Aliquots of the resulting reaction mixtures were taken for thiobarbituric Instruments, Malvern, UK) respectively. 2E10 antibody was conjugated
reactive substance (TBARS) assay and agarose gel electrophoresis while with 20 nm GNP as per protocol specified by the manufacturing company
the remaining was dialyzed against PBS buffer containing 1 mM EDTA with slight modification. Briefly, 2E10 antibody which was originally
overnight. suspended in PBS buffer were exchanged with ultrapure water (Direct-Q®
Complexation of Cu2þ-oxLDL and β2GPI was performed by mixing Water Purification System, Merck Millipore, Germany) via Amicon®
both component at a ratio of 2:1. Final concentration of Cu2þ-oxLDL and Ultra 0.5 mL Centrifugal Filters 10K (Merck Millipore, Germany). The 20
β2GPI were adjusted to 100 μg/mL and 50 μg/mL respectively. The re- nm GNP (Naked Gold Conjugation Kit (20 nm), Bioporto Diagnostics,
action mixture was then incubated at 37
C for 16 h and was terminated Denmark) has an original optical density (O.D) at 15 A.U. and was then
by freezing the samples at -80
C. Complexation of Cu2þ-oxLDL and adjusted to O.D 6 A.U and O.D 1.5 A.U. by diluting the GNP with ultra-
β2GPI was verified through agarose gel electrophoresis and ELISA assay. pure water (Direct-Q® Water Purification System, Merck Millipore,
Germany) respectively. Diluted GNP of O.D 1.5 A.U. was used for opti-
2.4. Thiobarbituric reactive substance (TBARS) assay mization of GNP-antibody conjugation. Different concentrations of 2E10
antibody (0–25 μg/mL) and pH of coating buffer (pH 7.3-pH 9.6) were
Oxidation of LDL was evaluated via TBARS assay by measuring tested to identify the optimal concentration of antibody and pH for
malondialdehyde (MDA), a byproduct of lipid peroxidation [33]. Briefly, successful and stable conjugation of 2E10 antibody to GNP. Concentra-
reference standards comprised of different concentrations of MDA (0–60 tion of 2E10 antibody was adjusted with ultrapure water (Direct-Q®
nmoles), negative control and Cu2þ-oxLDL fraction were respectively Water Purification System, Merck Millipore, Germany) while pH of
mixed with reaction buffer (consisting of 1 part of 0.67 % (w/v) thio- coating buffer was adjusted with 0.2 M K2CO3. The stability of GNP-2E10
barbituric acid and 1 part of 0.2 % (w/v) trichloroacetic acid. Mixtures antibody was verified via visual examination of physical colour change,
were homogenized briefly and incubated at 100
C water bath for 30 spectral absorption profile and salt aggregation assay [with 10 % (w/v)
min. Reaction mixtures were cooled prior to fluorescence spectroscopy NaCl]. The identified and selected optimal antibody concentration was
analysis at excitation and emission wavelengths of 515 nm and 553 nm up-scaled 4x to accommodate the production of stable GNP-2E10 with
respectively. TBARS values of samples were presented as nmol MDA diluted GNP of O.D 6 A.U. and pH of coating buffers was adjusted with
equivalent per mg protein. 0.2 M K2CO3 accordingly. The 2E10 antibody, at a final concentration of
60 μg/mL was added to the pH- and O.D-adjusted GNP (pH 9.2) and set
2.5. Agarose gel electrophoresis of LDL and related components aside to incubate at room temperature for 1 h. The reaction was later
terminated by addition of stabilizing buffer containing bovine serum
Agarose gel electrophoresis of plasma, LDL, Cu2þ-oxLDL, β2GPI, and albumin (BSA) at a ratio of 1:5 and left to incubate further at room
2þ
Cu -oxLDL/β2GPI was performed with TITAN Gel universal plate temperature for 16 h.
(Helena Laboratories, Beamount, TX). All components (except plasma; 2
μL loading volume) were loaded at a fixed protein equivalent content of 8 2.7. Immunoassays
μg to the dedicated wells on agarose gel films. Then gel electrophoresis
was conducted with barbital electrophoresis buffer (containing 10 mM A total of 9 commercial human serum standards were provided by
barbital, 50 mM sodium diethylbarbiturate, 1 mM EDTA, and 15 mM Corgenix Medical Corporation (Broomfield, CO). The standard serum
NaN3) at 90 V for 30 min. The gel films were then fixed in fixing solution samples were acquired from normal and APS/systemic lupus erythema-
[60 % (v/v) EtOH and 10 % (v/v) CH3COOH in H2O] for 15 min and then tosus (SLE) patients. These standard serum samples, alongside with the
dried in drying oven at 60
C for 20 min. The dried gel films were then chemically synthesized Cu2þ-oxLDL/β2GPI complexes were used in the
stained for protein with Amido black 10B and stained for lipids with Fat following immunoassays:
Red 7B respectively.
2.7.1. Anti-β2GPI-, anti-oxLDL- & anti-oxLDL/β2GPI IgG ELISA
2.6. Preparation of biotin-conjugated anti-β2GPI (3H3) and gold ELISA for anti-β2GPI-, anti-oxLDL- & anti-oxLDL/β2GPI IgG were
nanoparticle (GNP)-conjugated anti-apoB100 (2E10) antibodies prepared by coating Cu2þ-oxLDL (50 μg/mL), β2GPI (50 μg/mL), and
Cu2þ-oxLDL/β2GPI (50 μg/mL), in Hepes buffer (10 mM Hepes and 150
Biotinylation of 3H3 antibody was performed using commercially mM NaCl, pH 7.4), onto respective Nunc MaxiSorp™ flat-bottom
available Biotin Labelling Kit-NH2 (Dojindo Laboratories, Kumamoto, microplates (ThermoFisher Scientific, Middletown, VA) and incubated
Japan) as per product's instructions. Briefly, 200 μg of 3H3 antibody was at 4
C overnight. Microplate was then blocked with 1 % (w/v) BSA at
added to the filtration tube (from kit) and final volume was topped up to room temperature for 2 h. Human serum standards (50-fold dilution in
200 μL with WS Buffer (from kit). Mixture was then homogenized thor- Hepes buffer) were loaded to the wells at 100 μL per well and incubated
oughly through gentle pipetting and followed by centrifugation at 8,000 at room temperature for 2 h. Wells were then incubated with HRP-
x g, 4
C for 10 min. Next, NH2 reactive biotin was reconstituted in 10 μL conjugated anti-human IgG (Goat F(ab')2 Anti-Human IgG (Fab')2

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X.W. Tan et al. Heliyon 6 (2020) e04114

(HRP)-ab98535, Abcam, Eugene, OR) in a dilution 1:5000, at room extensively with 0.05 % (v/v) Tween-20 in 0.01 M PBS buffer at each
temperature for 1 h. Wells were washed extensively with 0.05 % (v/v) interval. TMB-H2O2 chromogenic substrate was added to wells (incu-
Tween-20 in 0.01 M PBS buffer at each interval. 3,30 ,5,50 -tetrame- bated for 30 min) for color development and reaction was later termi-
thylbenzidine (TMB)-H2O2 chromogenic substrate was added to wells nated with 0.3 N H2SO4 solution. O.D of samples was then measured at
(incubated for 30 min) for color development and reaction was later 450 nm and reference wavelength at 600nm via microplate reader
terminated with 0.3 N H2SO4 solution. O.D of samples was then (Tecan Sunrise, Switzerland).
measured at 450 nm and reference wavelength at 600 nm via microplate
reader (Tecan Sunrise, Switzerland). 2.7.3. OxLDL/β2GPI LFIA
The workflow for oxLDL/β2GPI LFIA was depicted in Figure 2A.
2.7.2. OxLDL/β2GPI ELISA Briefly two components were prepared separately. Component A con-
ELISA for oxLDL/β2GPI complexes was performed as per method of sisted of a mixture of 2.5 μL biotin-3H3 antibody (100 μg/mL) and serum
Ames, Ortiz-Cadenas, Torre, Nava, Oregon-Miranda, Batuca, Kojima, samples (25 μL; 2x dilution with sample buffer – borate buffer supple-
Lopez and Matsuura [29] with slight modifications (Figure 1). Briefly, 8 mented with 0.5 % (w/v) BSA, pH 7.4) or Cu2þ-oxLDL/β2GPI reference
μg/mL of 3H3 antibody, in Hepes buffer (10 mM Hepes and 150 mM standard (25 μL; concentrations in the range of 0.01–50.0 μg/mL) while
NaCl, pH 7.4), was coated (100 μL per well) onto Immulon 2HB micro- component B consisted of 2.5 μL GNP-2E10 antibody (60 μg/mL) in 25 μL
plate (ThermoFisher Scientific, Middletown, VA) at 4
C overnight. sample buffer. Both prepared mixtures were homogenized by gentle
Microplate was then blocked with 1 % (w/v) BSA at room temperature pipetting and incubated at room temperature for 15 min. Sample buffer,
for 2 h. Reference blank (Hepes buffer), Cu2þ-oxLDL/β2GPI complexes as LDL (2 μg/mL), Cu2þ-oxLDL (2 μg/mL) and β2GPI (2 μg/mL) were used
reference standards (0.01–2.00 μg/mL), and serum samples (100-fold as negative controls to assess the possibilities of these components for
dilution in Hepes buffer) were loaded to the wells at 100 μL per well and non-specific binding. Then, sequential LFIA detection strip incubation
incubated at room temperature for 1 h. Wells were then incubated with began with 1-minute incubation in mixture of component A and later
biotinylated anti-apoB100 antibody (2E10) and subsequently followed followed by 4 min incubation in mixture of component B. Images of
by HRP-labelled streptavidin for 1 h respectively. Wells were washed developed LFIA detection strips were captured by camera (without

Figure 2. Graphical representations of (A) oxLDL/β2GPI LFIA workflow and (B) principle of assay.

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X.W. Tan et al. Heliyon 6 (2020) e04114

flashlight). The intensities of the test and control lines were measurable The existence of Cu2þ-oxLDL and successful complexation of Cu2þ-
via ImageJ software for data analysis. oxLDL/β2GPI were further verified with agarose gel electrophoresis
(Figure 4B). Migrations of Cu2þ-oxLDL, Cu2þ-oxLDL/β2GPI and their
2.7.4. Statistical analysis respective controls (LDL and β2GPI) were observed via Fat Red 7B and
The four parametric logistic (4PL) analytical tool from MyAssays Amido Black 10B staining. Fat red 7B selectively stains the lipids while
Analysis Software Solutions (http://myassays.com) was used to generate Amido Black 10B stains the protein. Due to the staining nature of staining
four parametric logistic (4PL) standard curves and automated calcula- dyes, migration profile of β2GPI in the agarose gel was only detectable in
tions of oxLDL/β2GPI contents in unknown samples of both ELISA- and Amido Black stained gels. Among all loaded samples, the negatively
LFIA-based analyses. Correlation of oxLDL/β2GPI contents in serum charged oxLDL migrated furthest away from the origin of loading point
samples analyzed by both ELISA- and LFIA-based was determined by and its negative charge acquired from CuSO4-mediated oxidation were
interpolating data derived from both analyses. A R2 value >1.6 is neutralized through its complexation with β2GPI. As such the migration
considered as a significant correlation. profile of oxLDL/β2GPI fell in between those of β2GPI and Cu2þ-oxLDL.

3. Results and discussions 3.3. Characterizations of GNP and GNP-2E10

3.1. Purification and validation of 3H3- and 2E10-antibodies Colloidal gold (such as GNP used in this study), carbon nanotubes,
and fluorescent dyes (e.g. quantum dots) are some of the available labels
Hybridoma cells derived 3H3- and 2E10-monoclonal antibodies were applicable for antibody conjugation. The intense colour, homogeneity,
purified through Protein A affinity chromatography. The purities and high stability in both solution and solid forms, and nonessential visual-
stabilities of 3H3- and 2E10-monoclonal antibodies were verified with developmental process are among the advantageous points of colloidal
SDS-PAGE (Figure 3). Under reducing condition, antibody fragments GNP label as compared to the other two counterparts [30, 34].
with band sizes at 50 kDa (heavy chains) and 25 kDa (light chains) were Fluorescence-based labels such as carbon nanotubes and quantum dots
observed. Full size antibodies were still observable at 150 kDa while often encounter drawbacks such as high background noise, photosensi-
majority of the antibodies have been fragmented into their respective tivity, and require additional developmental process with specific visu-
heavy chains (approximately 50 kDa) and light chains (approximately 25 alizer to visualize [30].
kDa). Reactivities of 3H3 and 2E10 antibodies were further tested in The spectral absorptivity of GNP was confirmed through UV-visible
ELISA and LFIA. spectrophotometer. It showed a characteristic wine-red colour with a
maximum spectral absorptivity (λ max) at approximately 520 nm. The
average hydrodynamic size of bare GNP as determined through dynamic
3.2. Cu2þ-oxLDL and Cu2þ-oxLDL/β2GPI light scattering (DLS) was measured at 35 nm (Figure 5A). After conju-
gation with 2E10 antibody, the average hydrodynamic size of GNP-2E10
Lipid peroxidation rate of LDL was evaluated via thiobarbituric acid increased to 43 nm.
reactive substance (TBARS) assay (Figure 4A). The assay quantifies Optimal antibody concentration and pH for stable GNP-2E10 conju-
malondialdehyde (MDA), a fluorogenic byproduct resulting from the gate were determined via UV-visible spectrometer and salt aggregation
hydrolytic reaction of lipid hydroperoxides [33]. Through a quick com- assay. Concentration of 2E10 antibody beyond 8 μg/mL instigated a
parison between negative control (oxidation reaction of LDL halted by slight red shift in λ max of GNP, and thus indicating successful conju-
the addition of EDTA at time ‘0’) and oxLDL (oxidation at 37
C for 16 h), gation of GNP and 2E10 antibody. The interaction between antibodies
a distinguishable difference in MDA content between two samples was and the surface of GNP prompted a slight shift in surface plasmon reso-
observed. The negative control fraction recorded a MDA content of 13.4 nance band of GNP (λ max at ~520 nm) and concurrently increased its
nmol/mg protein while the oxLDL sample recorded a MDA content of average hydrodynamic size [35].
499.5 nmol/mg protein. The high content of MDA found in the Through salt aggregation assay, unstable GNP-antibody conjugate
Cu2þ-oxLDL denoted oxidation of LDL to Cu2þ-oxLDL. with incomplete antibody coating will aggregate and resulted in visually
assessible colour shift of conjugate solution, from its original red colour
to purplish black [36, 37]. In this study, upon the addition of 10 % (w/v)
NaCl, solutions of GNP-2E10 conjugates with concentration of 2E10
antibody below 8 μg/mL documented a colour change from red to
purplish-black with visible black precipitate (Figure 5B). Due to incom-
plete coating of 2E10 onto the surface of GNP, the GNP aggregated and
fell out of the solution while completely coated GNP-2E10 (with con-
centration of 2E10 beyond 8 μg/mL) did not aggregate in high-salt so-
lution. Minimal concentration of 2E10 antibody required to produce
stable conjugate was logged at 8 μg/mL. In order to produce stable
GNP-2E10 conjugate, additional 20 % of minimum 2E10 antibody con-
centration (10 μg/mL) was recommended (as per manufacturer's
recommendation) and hence was selected for up-scaled production of
stable GNP-2E10.
As for the pH factor of GNP dispersion medium, GNP-2E10 conjugates
were mostly stable in dispersion medium of pH 9.2 and above. Although
the UV-visible spectral profile of GNP-2E10 in different pH dispersion
medium showed a slight red shift in λmax of GNP, salt aggregation assay
revealed GNP-2E10 dispersed in medium beyond pH 9.2 did not aggre-
Figure 3. SDS-PAGE gel image of different fractions derived from Protein A gate in high-salt solution (Figure 5C) hence suggesting the formation of
antibody purification of 3H3- and 2E10-monoclonal antibodies. Samples were stable GNP-2E10 conjugates. The pH of GNP dispersion medium has been
reduced with 2-mercaptoethanol at 99
C for 5 min prior to SDS-PAGE. ‘M’ shown to alter the surface chemistry and charge of antibodies and GNP,
denotes protein marker; ‘A’ denotes cell culture supernatant; ‘B’ denotes effluent and immensely affects the stability and robustness of the resulting GNP-
fraction; ‘C’ denotes purified antibody fraction. antibody conjugate [38]. For upscaled production of GNP-2E10, the

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X.W. Tan et al. Heliyon 6 (2020) e04114

Figure 4. (A) LDL was extracted human serum pool through single spin density gradient ultracentrifugation and LDL-containing fraction was subsequently oxidized
chemically with CuSO4. TBARS assay was performed to evaluate the oxidation of LDL and TBARS values were expressed in nmol MDA per mg protein. (B) Elec-
trophoretic migrations of human pool serum, LDL, Cu2þ-oxLDL, β2GPI, Cu2þ-oxLDL/β2GPI on agarose gel. Amido Black 10B dye was used to stain protein while Fat
Red 7B dye was used to stain lipids.

selected optimal concentration of 2E10 antibody (10 μg/mL in GNP O.D mouse-derived monoclonal antibodies were advantageous owing to
1.5 A.U.) was increased 4x to produce stable GNP-2E10 of O.D 6 A.U. their respective specificities and consistent affinities towards specific
With 60 μg/mL 2E10 antibody and a coating buffer of pH 9.2, the epitopes on the antigen (oxLDL/β2GPI). 3H3 antibody only recognizes
GNP-2E10 remained stable and did not aggregate in 10% (w/v) NaCl β2GPI portion of the antigenic oxLDL/β2GPI and not solitary β2GPI while
solution (Figure 5A). 2E10 antibody binds to apoB100 of oxLDL. Although the two monoclonal
antibodies used were affixed with different conjugators, their specific-
ities and affinities towards their respective epitopes remained
3.4. Principles of oxLDL/β2GPI LFIA unhindered.
The LFIA test strips from Bioporto Diagnostics used in this study were
The portability and user-friendliness of LFIA has revolutionized the composed of a nitrocellulose analytical membrane with wicking pad
point-of-care platform for rapid disease diagnosis and risk stratification attached at one end to haul fluids up the membrane by capillarity. On the
[30]. Herein, we demonstrated the use of LFIA-based platform to assess nitrocellulose membrane, two approximately 1 mm line are coated with
serological level of oxLDL/β2GPI in a simple, rapid and specific manners. avidin (test line) and a mixture of polyclonal anti-mouse/rabbit/goat IgG
The principle of oxLDL/β2GPI LFIA (Figure 2B) is based upon the specific antibody (control line) respectively. The avidin coated test line captures
immunoreactions between the antigen (oxLDL/β2GPI) and two anti- biotinylated components while polyclonal anti-IgG antibody coated on
bodies (3H3 and 2E10 antibodies) on a customized nitrocellulose mem- the control line captures excess IgG/gold nanoparticle conjugated IgG.
brane. The oxLDL/β2GPI LFIA in this study utilized two different

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X.W. Tan et al. Heliyon 6 (2020) e04114

Figure 5. Characterization of GNP. (A) Hydrodynamic sizes of bare Au-GNP and antibody conjugated GNP-2E10 were assessed through dynamic light scattering by
using Zetasizer. Inset shows the stable GNP-2E10 that has been optimized with suitable antibody concentration and pH of dispersion medium. The developed GNP-
2E10 remained stable in 10 % (w/v) NaOH solution. Prior to upscaled production of Au-GNP-2E10, optimal (B) antibody concentration and (C) pH of dispersion
medium essential for production stable conjugate were evaluated via UV-visible spectrophotometry and salt aggregation assay respectively.

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X.W. Tan et al. Heliyon 6 (2020) e04114

The visually visible control lines on LFIA strips denote successful capil- curves (Figure 7A), antibody conjugate combination 1 acquired a better
lary flow of LFIA. signal-to-response than antibody conjugate combination 1. From the
In oxLDL/β2GPI LFIA, oxLDL/β2GPI (antigen) formed antigen- structural perspective, oxLDL has only a copy of apoB protein (apoB100)
antibody complex with biotin labelled anti-β2GPI IgG (biotin-3H3) and [39,40] thus offering just an epitope recognizable by anti-apoB100
GNP labelled anti-apoB100 IgG (GNP-2E10). When passing the sample antibody (2E10 antibody) per molecule of oxLDL/β2GPI. Considering
(containing oxLDL/β2GPI) through LFIA detection strip in the direction that multiple β2GPI proteins form complexes with oxLDL through spe-
of the capillary flow, avidin coated on the test line of LFIA captures biotin cific ligand, oxLig-1, in oxLDL [18], the availability of multiple epitopes
portion of the antibody antigen complex while the conjugated GNP- 2E10 (β2GPI) recognizable by biotin-3H3 antibodies offered better contact
on the antibody-antigen complex provided visual qualitative assessment point per molecule of oxLDL/β2GPI complex between biotin and avidin
of oxLDL/β2GPI, presenting a purplish-red colour on both test line and coated on the sample test line as the antibody-antigen conjugate flow
control line. The intensities of the lines were measurable via ImageJ across the lateral flow test strip in the direction of capillarity.
software for data analysis. Data were represented in the form of intensity In addition, β2GPI, nLDL and oxLDL were used as negative controls to
of test line in relative to the intensity of its respective control line on the evaluate the specificity of oxLDL/β2GPI LFIA. The absences of signal on
LFIA detection strip. Semi-quantitation of oxLDL/β2GPI was determined sample test line of oxLDL/β2GPI LFIA tested with β2GPI, nLDL and oxLDL
through a constructed four-parameter logistic curve with respect to (Figure 6B) denoted negative tests and unaffected interference from
calculated relative intensity units of known concentrations of reference nLDL, oxLDL and β2GPI. The absence of non-specific binding from these
standards (Cu2þ-oxLDL/β2GPI). tested negative controls suggested the specificity of oxLDL/β2GPI LFIA is
predisposed to detection of oxLDL/β2GPI only. The detection range and
sensitivity oxLDL/β2GPI LFIA and oxLDL/β2GPI ELISA were also
3.5. Optimization and assessment of oxLDL/β2GPI LFIA assessed in this study. Different concentrations of Cu2þ-oxLDL/β2GPI
complexes were applied as test samples to both detection systems. By
Two different combinations of antibody conjugates for oxLDL/β2GPI comparing the 4PL curves derived from both detection systems for
LFIA systems were attempted to evaluate their respective detection ef- oxLDL/β2GPI (Figure 7B), it was postulated that the oxLDL/β2GPI LFIA
ficiency and effectiveness. The two antibody conjugate combinations offered a wider range of detection as compared to oxLDL/β2GPI ELISA.
were: (i) Combination 1: biotin-3H3 antibody and GNP-2E10 antibody; The 4PL standard curve oxLDL/β2GPI ELISA recorded a plateau pattern
(ii) Combination 2: biotin-2E10 antibody and GNP-3H3 antibody. at a concentration of Cu2þ-oxLDL/β2GPI near 2 μg/mL. Contrarily, the
Different concentrations of Cu2þ-oxLDL/β2GPI complexes were used as oxLDL/β2GPI LFIA was capable of detecting Cu2þ-oxLDL/β2GPI com-
test samples. The images of developed LFIA detection strips of both plexes up to 50 μg/mL. In addition, respective normalized concentration-
systems were depicted in Figure 6A. Based upon the established standard

Figure 6. Respective images of developed LFIA test strips of (A) reference standards, (B) negative controls and (C) serum test samples. β2GPI, LDL and Cu2þ-oxLDL
were tested as negative controls to evaluate specificity of oxLDL/β2GPI LFIA. Antibody conjugate combination 1 consisted of biotin-3H3 antibody þ GNP-2E10
antibody while antibody conjugate combination 2 consisted of biotin-2E10 antibody þ GNP-3H3 antibody.

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X.W. Tan et al. Heliyon 6 (2020) e04114

Figure 7. (A) Comparisons of four parametric logistic (4PL) curves derived from LFIA of two different antibody conjugate combinations: (1) biotin-3H3 antibody þ
GNP-2E10 antibody and (2) biotin-2E10 antibody þ GNP-3H3 antibody. Respective images of developed LFIA test strips were depicted in Figure 6A (B) Detection
sensitivity and specificity of oxLDL/β2GPI LFIA and oxLDL/β2GPI ELISA were compared by using Cu2þ-oxLDL/β2GPI as test sample. *Units (y-axis) for ELISA-based
system were plotted in accordance to absorbance recorded at 450 nm (reference wavelength at 600 nm) while units (y-axis) for LFIA-based system were arbitrary units
of relative intensities derived from ImageJ analysis. (C) OxLDL/β2GPI contents in serum samples were evaluated with oxLDL/β2GPI ELISA- and LFIA-based assay. The
correlation plot showed significant correlation (R2 value >0.826) between ELISA- and LFIA-based assay. Respective images of developed LFIA test strips were depicted
in Figure 6C.

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dependent signals acquired from both ELISA- and LFIA-based detection oxLDL and anti-β2GPI autoantibodies. Correlation plot of O.D acquired
systems showed significant correlation at a R2 value of 0.999. In terms of from each ELISA assay was plotted against the concentration of oxLDL/
sensitivity, oxLDL/β2GPI LFIA was able to achieve relatively similar β2GPI detected in serum samples by means of ELISA and LFIA
detection sensitivity as oxLDL/β2GPI ELISA but with an added advantage (Figures 8A-8C). It was demonstrated in this study that the anti-β2GPI
of offering a wider detection range. IgG titers in tested serum samples were significantly correlated with the
presence of of oxLDL/β2GPI (R2 value >0.5) (Figure 8A). It has been
established that APS/SLE patients attain high serological titre of auto-
3.6. Application of oxLDL/β2GPI LFIA and oxLDL/β2GPI ELISA for anti-β2GPI IgG and develop clinical features of atherosclerotic compli-
quantitation of oxLDL/β2GPI in unknown samples cations [41]. β2GPI acts as a major antigen for antiphospholipid
autoantibodies in APS patients when it binds to anionic phospholipids
In the present study, a total of 9 different human serum standards and the resulting immunogenic complex is thought to augment the
acquired from normal and APS/SLE patients (tested with positive anti- advancement of atherogenic complications of APS [9, 10, 11, 12]. The
β2GPI IgG and oxLDL/β2GPI subgroup) were used to attest the pre- proatherogenic effects of such immune complexes were further evidently
liminary applicability of oxLDL/β2GPI LFIA for quantitation of oxLDL/ asserted when oxLDL/β2GPI, with anti-β2GPI IgG alongside, were
β2GPI in unknown samples. The oxLDL/β2GPI ELISA was used concur- internalized more rapidly by macrophages through their Fcγ receptors
rently with similar test samples for comparisons. Different concentrations [14].
of Cu2þ-oxLDL/β2GPI were used as reference standard for quantitation. There was significant correlation (R2 value >0.16) between anti-
The quantified oxLDL/β2GPI complexes in unknown serum samples oxLDL IgG and concentration of oxLDL/β2GPI (Figure 8B) which sug-
derived from both immunoassays were depicted Figure 7C. The quanti- gested the prevalence of autoantibodies against oxLDL in tested serum
fied oxLDL/β2GPI through LFIA- and ELISA-based immunoassays showed samples. High serological titer of oxLDL is a common prognosis of
significant correlation at R2 value ¼ 0.826 (Figure 7C). autoimmune disease patients and patients with atherosclerotic compli-
In order to further testify the specificity of oxLDL/β2GPI LFIA, ELISA- cations. OxLDL is perceived as an immunogen held accountable for in-
based bioassays were employed to assess autoantibodies species present duction of anti-oxLDL autoantibodies. A positive correlation between
in tested serum samples by using oxLDL and β2GPI as antigens for anti-

Figure 8. The presences of anti-β2GPI, anti-oxLDL, and anti-oxLDL/β2GPI IgG in serum samples were tested via solid-phase ELISA. Correlativity of respective an-
tibodies: (A) anti-β2GPI IgG; (B) anti-oxLDL IgG; (C) anti-oxLDL/β2GPI IgG was plotted against concentration of oxLDL/β2GPI in serum samples as tested with oxLDL/
β2GPI ELISA and LFIA. Correlation curve anti-β2GPI IgG versus anti-oxLDL IgG (D) was plotted accordingly to evaluate the inter-relativity.

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X.W. Tan et al. Heliyon 6 (2020) e04114

serological titers of anti-oxLDL antibodies and severity of atherosclerosis required by the application of ELISA. Owing to its simplificity, oxLDL/
related complications was established previously [42, 43, 44, 45]. β2GPI LFIA supports potential point-of-care application in future,
However, there was no correlation between concentration of oxLDL/ particularly for high-throughput diagnosis and risk stratification of
β2GPI and anti-oxLDL/β2GPI IgG (R2 ¼ 0.01–0.02) (Figure 8C). As atherosclerosis-related complications.
chemically synthesized oxLDL/β2GPI was coated as antigen on ELISA
plate to measure anti-oxLDL/β2GPI IgG titer from tested serum samples, Declarations
it was postulated that the density of β2GPI portion of oxLDL/β2GPI
coated on ELISA plate as epitope detectable by anti-oxLDL/β2GPI IgG in Author Contribution statement
tested serum titers remained limited. The apolipoprotein B-containing
oxLDL is a relatively huge lipid vesicle as compared to β2GPI. Contrary to X. Tan: Conceived and designed the experiments; Performed the ex-
the direct coating of β2GPI only as solid phase antigen for ELISA, it is periments; Analyzed and interpreted the data; Contributed reagents,
postulated that the sheer size of oxLDL portion of the oxLDL/β2GPI materials, analysis tools or data; Wrote the paper.
complex may have incurred stearic hindrance which lowers the absolute F. Takenaka: Conceived and designed the experiments; Contributed
effective coating density of Cu2þ-oxLDL/β2GPI on the surface of ELISA. reagents, materials, analysis tools or data.
The resulting steric hindrance may have caused epitope-masking, and H. Takekawa: Performed the experiments.
hence resulted in underestimation of anti-oxLDL/β2GPI IgG. Nonethe- E. Matsuura: Conceived and designed the experiments; Analyzed and
less, the prevalence of these autoantibodies in tested serum titers did not interpreted the data; Contributed reagents, materials, analysis tools or
affect the specificity of oxLDL/β2GPI LFIA. data; Wrote the paper.
Lipid and lipoprotein metabolic abnormalities are closely associated
with hyperlipidemia and high levels of plasma LDL and oxLDL. These are Funding statement
factored as part of the fundamental contributing factors leading to the
onset and progression of atherosclerotic cardiovascular complication and E. Matsuura was supported by Ministry of Education, Culture, Sports,
mortality [46, 47]. The discovery of atherogenic oxLDL/β2GPI com- Science and Technology.
plexes in sera of APS patients has been considered as one of the quanti-
fiable risk factors for clinical manifestation of arterial thrombosis in APS Competing interest statement
[41]. However, such atherogenic complexes were also discovered in
patients of non-systemic autoimmune diseases, such as diabetes mellitus The authors declare no conflict of interest.
[24] and chronic nephritis [25]. These sightings may warrant attention
concerning the clinical implications of these circulating oxLDL/β2GPI Additional information
complexes as potential diagnostic biomarker for risk stratification of
atherosclerotic complications. No additional information is available for this paper.
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