Validation and evaluation of a common biomarker in human cancers sera protein detected by a monoclonal antibody UNIVmAb

Abstract

Objective:
Management and diagnosis of multiple human cancers remains a challenge and search for a common biomarker is still debatable. In this manuscript we have evaluated the use of monoclonal antibody UNIVmAb, to detect the protein (H11) as a common biomarker for all cancers irrespective of the grade and origin. We have shown by both ELISA and Western Blot that the H11 protein, is a unique hyaluronan binding protein that has not been detected earlier. H11 protein was fractionated in an anion exchange column followed by cibacron blue gel exclusion chromatography. Hyaluronan binding H11 protein reacted with Monoclonal antibody UNIVmAb and b-HA inspite of b-Hyaluronan (biotinylated Hyaluronan) interaction and HA-Oligo (Hyaluronan oligosaccharides) competition from various grades of Human cancers sera.

Results:
ELISA, Western blot and b-Hyaluronan interactions clearly showed an over-expression of UNIVmAb reacted H11 protein in all fifty cancer's sera when compared with seventy normal sera. UNIVmAb reactive H11 protein can be used as a common biomarker. We believe, UNIVmAb detected H11 protein, is a unique hyaluronan binding protein, that can be used as a common biomarker for all cancers.

Full text

Manjunathetal. BMC Res Notes (2019) 12:744
https://doi.org/10.1186/s13104-019-4780-4
RESEARCH NOTE
Validation andevaluation ofacommon
biomarker inhuman cancers sera protein
detected byamonoclonal antibody UNIVmAb
D. Manjunath1, Sunil B. Kumaraswamy1, Shashidhar Aladhi Venkatakrishniah2, Hitesh Nidumanda Appaiah1,
Anil Thomas1 and Shib D. Banerjee3*
Abstract
Objective: Management and diagnosis of multiple human cancers remains a challenge and search for a common
biomarker is still debatable. In this manuscript we have evaluated the use of monoclonal antibody UNIVmAb, to
detect the protein (H11) as a common biomarker for all cancers irrespective of the grade and origin. We have shown
by both ELISA and Western Blot that the H11 protein, is a unique hyaluronan binding protein that has not been
detected earlier. H11 protein was fractionated in an anion exchange column followed by cibacron blue gel exclusion
chromatography. Hyaluronan binding H11 protein reacted with Monoclonal antibody UNIVmAb and b-HA inspite of
b-Hyaluronan (biotinylated Hyaluronan) interaction and HA-Oligo (Hyaluronan oligosaccharides) competition from
various grades of Human cancers sera.
Results: ELISA, Western blot and b-Hyaluronan interactions clearly showed an over-expression of UNIVmAb reacted
H11 protein in all fifty cancer’s sera when compared with seventy normal sera. UNIVmAb reactive H11 protein can be
used as a common biomarker. We believe, UNIVmAb detected H11 protein, is a unique hyaluronan binding protein,
that can be used as a common biomarker for all cancers.
Keywords: ELISA, Western blot, Hyaluronic acid binding protein, H11 (sera antigen), UNIVmAb common biomarker,
Cancers sera
© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License
(http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium,
provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license,
and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/
publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Introduction
e appearance of cancer serum biomarker is a molec-
ular event that indicates the pathological changes that
happen in a particular tissue or cell type during cancer
development. e most important part of screening is
the ability for an early cancer diagnosis. Cancer diagnosis
based on the quantification or localization of a particular
antigen in cells, tissues or body fluids [1–5] is a topic of
interest with human body fluids (serum) as an important
source of minimal invasive biomarkers. e widely used
blood test for early detection of cancer of the prostate
is PSA (prostate-specific antigen) and the proper use of
this test is highly debatable [6]. However, diagnosis using
circulatory serum antigens makes the detection of bio-
markers more feasible, and it is relatively a non-invasive
method to acquire experimental samples [4, 5, 7–11].
e development of monoclonal antibodies (mAb’s)
identifying specific antigens expressed by cancer cells
offers a diagnostic technique for early cancer detection
[12]. mAb’s are widely used reagents in the clinical diag-
nostic laboratories to develop sensitive immunoassays for
the detection of their target antigen in circulation, their
levels relate with the disease progression [11, 13–16].
(Drugs/Guidance Compliance RegulatoryI nformation/
Guidances CM070107.pdf.
Analysis of human embryonic cells, adult tissues and
human serum has revealed the presence of hyaluronan
(HA) and its receptors. Hyaluronan is a nonsulfated,
high molecular weight glycosaminoglycan consisting of
Open Access
BMC Research Notes
*Correspondence: sdbaner@yahoo.com
3 Department of Anatomy and Cellular Biology, Tuft University School
of Medicine, Boston, MA, USA
Full list of author information is available at the end of the article

Page 2 of 7
Manjunathetal. BMC Res Notes (2019) 12:744
the -glucuronic acid and N-acetyl -glucosamine [17].
HA is present in the extracellular matrix of most tissues
and is enriched in many tumours [17, 18]. It is well docu-
mented that HA and its receptors, known as hyaladher-
ins (hyaluronan binding proteins) are involved in matrix
regulation, cell proliferation, migration and malignant
tumour progression [18]. Hyaladherins not only interact
with hyaluronan at the matrix proper but also with hya-
luronan in the plasma membrane as cell surface receptors
and thus influence cell physiology, including secretion of
this protein into the circulatory system [19–21]. Previ-
ously, we have identified H11B2C2 clones reactive hyalu-
ronan binding protein and revealed its association with
tumour progression by immunohistochemistry [13]. e
antigen expression increases during tumour progression
irrespective of cancer origin. Antibody produced by the
clones, detected the antigen in multiple human cancer
tissues/serum and we renamed the antibody UNIVmAb.
We assume that UNIVmAb reactive H11 is a protein bio-
marker in various tumour tissues that may give us cru-
cial diagnostic information in early cancer detection. We
have described the procedures for fractionation of serum
proteins, mainly hyaluronic acid binding protein (HABP),
from various cancer patient’s samples and healthy sub-
jects using single dimension electrophoresis. e detec-
tion of H11 antigen using UNIVmAb in multiple cancer’s
led us to think that H11 antigen can be a common bio-
marker for multiple cancers.
Main text
Materials andmethods
Hyaluronan (Na salt) (Acros Organics, New Jersey,
USA). Guanidium hydrochloride, bovine testicular hya-
luronidase type I S, EDC [1, ethyl 3-(3-dimethyl ami-
nopropyl) carbodiimide hydrochloride, MES buffer
(2-N-morpholino ethane sulfonic acid) Protease inhibi-
tors cocktail and biotinylated goat anti-mouse IgG’s HRP
conjugate were procured from Sigma chemicals, USA.
EZ-Link Biotin LC hydrazide purchased from Pierce,
Rockford, USA. Anti-human CD44 (H-CAM, Clone
1M7.8.1) antibody was purchased from Fisher Scientific,
USA. Streptavidin-peroxidase conjugate (HRP) obtained
from Invitrogen, USA.
Sample collection andpreparation
e study consisted of 70 normal subjects and 50 can-
cer patients (Lymphoma (2 patient samples), Salivary
(1 patient sample), Tongue (4 patient samples), yroid
(1 patient sample), Buccal Mucosa (1 patient sample),
Lung (3 patient samples), Breast (6 patient samples),
Stomach (3 patient samples), Gallbladder (1 patient sam-
ple), Oesophagus (3 patient samples), Colon (5 patient
samples), Pancreas (1 patient sample), Rectal (2 patient
samples), Urinary bladder (2 patient samples), Prostate (4
patient samples), ovary (5 patient samples), endometrial
(4 patient samples) and cervix (2 patient samples). Serum
samples from normal subjects and cancer patients were
accessed from cancer hospitals in Mysore, India (Preethi
Centre for oncology, and KR Hospital, Logic and Clue
Diagnostic center) and the protocols were approved by
the ethical review committee (IHEC-UOM NO 35) and
the patient’s consent were taken. Blood samples were col-
lected from each patient before any treatment. Samples
were centrifuged at 2000×g for 30min at room tempera-
ture and the separated sera were stored at − 80°C. e
H&E stained tumour sections of patients were obtained
from hospitals and were graded using the TNM grading
system. Serum samples treated with 4× lysis buffer, con-
taining 0.2M Tris–HCl (pH 8.0), 80mM EDTA, 4mM
PMSF, 4mM Benzamidine-HCl and 2% Triton X 100 plus
protease inhibitor cocktails were centrifuged at 10,000×g
for 30min at 4°C. e supernatant was stored at − 80°C
until further analysis. e protein estimation was done
at UV 280nm and Bradford reagent assay using Bovine
serum Albumin (BSA) as standard.
Biotinylated hyaluronic acid was prepared according
to Boregowda etal. [13] and Srinivas etal. [24]. In brief,
HA dissolved in PBS-A was dialyzed in MES buffer and
reacted with biotin_LC-hydrazide and EDC in DMSO.
is was Incubated for 16 h and then dialyzed against
PBS-A and stored in glycerol at – 20°C.
Production ofmonoclonal antibody UNIVmAb
Hybridoma and the antibody were prepared according to
Boregowda etal. [18, 22, 23]. In brief, the hybridoma was
grown in DMEM with human serum (pathogen and com-
plement free) that were received from the hospitals. e
antibody production in the presence of human serum
(any blood groups) did not affect UNIVmAb recogntion
of the human H11 antigen. e clones were grown in
DMEM containing 10% (v/v) inactivated human serum.
After 21days, the media was collected and precipitated
with cold saturated ammonium sulphate solution (final
50%) at 4°C overnight and centrifuged at 12,000×g for
30 min. e pellet was dissolved in PBS and dialyzed
against PBS.
Statistical analysis
Statistical differences between groups from ELISA were
analyzed using graphpad prism version 5 software.
Results are expressed as the mean ± SD. A diiference with
P values is defined as follows: P < 0.001 = extremely sig-
nificant. For westerblot, image analysis was done using
Image J software.

Page 3 of 7
Manjunathetal. BMC Res Notes (2019) 12:744
Methods
Detection ofH11 antigen byELISA using UNIVmAb
MaxiSorp flat-bottom high protein binding capacity pol-
ystyrene-96 well plates were used. Serum samples were
diluted with 0.05M carbonate-bicarbonate buffer pH 9.6
to obtain a final concentration of 1µg/ml. 100µl of sam-
ples in triplicate were plated on to the 96well plate and
incubated overnight at 4°C. Following day the plate was
blocked with skimmed milk (prepared in PBS) for 1h and
incubated with UNIVmAb at 1:10,000 overnight at 4°C.
Following day the plate was washed with 0.2% Tween-PBS
followed by incubation with b-goat anti-mouse antibody
at 1:20,000 for 1h and reacted with streptavidin-peroxi-
dase at 1:50,000 for one hour. Plate was washed with 0.2%
Tween-PBS and 100µl of ABTS (1.0mg/mL) in 0.1 M
citrate buffer at pH 4.0 and 5%. Hydrogen peroxide. e
reactions were stopped after one hour with 0.2M citric
acid, and the absorbance was measured at 405nm Fig.1.
Experiments were repeated at least three times. Protein
levels were measured by quantitative ELISA.
Western blot analysis ofserum according toBoregowda
etal. [15] andFekry etal. [16]
50g proteins from serum lysate were resolved on 10%
SDS-PAGE, transferred to PVDF membrane and reacted
with UNIVmAb (1:1000 dilution) or anti HCAM mAb
(1:1000 dilution) or with bHA probe (1:100 dilution)
overnight at 4 °C. Following day, the blot was devel-
oped and the proteins were detected using Enhanced
ChemiLuminescence (ECL). Since the isolation of anti-
gen is vital to understand its property, we used purified
circulating antigen by antibody conjugated CNBR acti-
vated Sepharose and Cibacron blue affinity purification
method. e purified H11 protein was reacted with UNI-
VmAb and was also cross reacted with bHA and HA-
oligo (500 µg Oligo) competition was also performed.
is showed that H11 antigen is a Hyaladherin.
Results anddiscussion
We validated the clones H11B2C2 reactive novel HABP
in human cancer tissues. HABP overexpression was
related to poor tumour outcomes [13, 25]. e antibody
produced by the clones detected the antigen in all human
cancer tissues and cancers sera; we renamed the antibody
as UNIVmAb. We have also identified soluble hyaluronan
binding proteins in colon cancer serum with a molecular
mass of 57 and 30kDa [14]. We believed that the soluble
57kDa H11 protein from colon cancer might have rela-
tion with multiple cancer samples. UNIVmAb reactive
H11 proteins maybe a new class of hyaluronan binding
protein and we did a further investigation in the present
study to understand its expression and the nature of H11
protein in various cancer’s sera to further characterize
by biochemical analysis and its application as a common
cancer biomarker. Table1 shows the detailed results of
H11 expression in various cancers sera.
From ELISA experiment (Fig.1), normal sera showed
a mean O.D: 0.175 from triplicates, However sera from
Grade 1: O.D is 0.25, Grade 2: O.D is 0.46, and Grade 3:
O.D is 0.52 that showed increased expression of the anti-
body reactive H11 antigen. Even though eight samples
from 18 different cancers show H11 antigen activity with
Fig. 1 Detection of normal and Cancer antigen by ELISA using UNIVmAb. a Lane 1, 2, 3 normal serum (each average of three determinations) Lane
4. Ca stomach Grade 1. Lane 5. Ca tongue Grade 1. Lane 6. Ca Colon Grade 1. Lane 7.Ca stomach Grade 2. Lane 8.Ca cervix Grade 2, Lane 9. Ca Cervix
Grade 3. b 1–4, normal serum, 5 and 6 Grade 1, Tongue, 7–9 Grade 2, breast, (10–13 Grade 3 samples) 10: Colon, 11: Lung, 12: Oesophagus, 13:
Ovary. (average of four samples from each serum). There is gradual over-expression of H11 in sera as the tumour progress

Page 4 of 7
Manjunathetal. BMC Res Notes (2019) 12:744
UNIVmAb, similar results were also observed with the
remaining cancer samples.
UNIVmAb reactive H11 proteins were overexpressed
inhuman cancer serum shown inFig.2
To detect whether UNIVmAb reactive antigens were pre-
sent in circulation, we conducted western blot analysis
of normal and various cancers sera samples using UNI-
VmAb. e UNIVmAb showed reduced H11 reactivity in
normal sera at 57kDa (Fig.2 a), the intensity of reaction
is enhanced in tumour samples of high-grade cancers
(Fig.2d vs b, c). We compared this with 70 normal sam-
ples and 50 cancer patient’s samples of different grade.
We have shown H11 expression in 12 different healthy
individuals sera samples in Fig.2 (panel a lanes 1 to 12).
e UNIVmAb reaction with serum proteins of six dif-
ferent cancer sera of different cancer patients are shown
in panel b, c and d. e presence of UNIVmAb reactive
protein in normal and eighteen cancer subjects suggests
that H11 protein might have several biological functions.
Similar results were observed in the tumour/normal tis-
sues [13]. Figure3 Scion image analysis of the western
blot showed that UNIVmAb reacted antigen overexpres-
sion is real and accepted when tested with all other can-
cers sera samples.
Western blot analysis with bHA probe showed that
the detected serum HABP expression is H11 antigen
(Fig not shown) and HA-Oligo competition showed 80%
Table 1 Evaluation ofUNIVmAb reacted H11 expression inmultiple tumours
We also detected H11 expression from astrocytoma glioblastoma and medulloblastoma not included into the table. XXX = overexpression of H11
Xxx very strong; xx moderate; x weak
Tumour types Grade Number oftheexperiment fromeach patient
studied H11 expression
innumber
ofpatients in()
Salivary I 3 X (1)
Tongue II 3 Xx (4)
III 3 xxx
Thyroid (papillary) II 3 Xx (1)
Buccal mucosa II 3 Xx (1)
Lung II 2 Xx (1)
III 3 Xxx (2)
Breast I 4 X (2)
II 3 Xx (2)
III 4 Xxx (2)
Stomach II 3 Xx (1)
III 3 Xxx (2)
Gall bladder I 3 X (1)
Oesophagus II 3 Xx (1)
III 3 Xxx (2)
Colon I 2 X (1)
II 3 Xx (2)
III 4 Xxx (2)
Pancreas II 3 Xx (1)
Rectum II 3 Xx (2)
Ovary II 3 Xx (2)
III 3 Xxx (3)
Endometrium I 2 X (1)
II 3 Xx (2)
III 4 Xxx (1)
Cervix II 3 Xx (2)
Urinary bladder I 3 X (2)
Prostate I 2 X (2)
II 3 Xx (2)
Lymphoma II 3 Xx (2)

Page 5 of 7
Manjunathetal. BMC Res Notes (2019) 12:744
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
1 2 3 4 5 6 7 8 9 10 11 12
A HS
B G-I
C G-II
D G-III
Fig. 2 UNIVmAb reactive human sera antigen expression by western blot. a Healthy controls serum from 12 different individuals. b–d: Grade 1,
Grade 2, and Grade 3. Lanes 1 and 2: Tongue, Lanes 3 and 4: Salivary, Lanes 5 and 6: Breast, Lanes 7 and 8: Colon, Lanes 9 and 10: Cervix, Lanes 11
and 12: Ovary
Fig. 3 Scion image analysis, Normal (a) Grade 2 (b) and Grade 3 (c). Scion Image Analysis of Panel 1: Scion image clearly showed the overexpression
of H11 antigen as the tumour progress to grade three. However, we showed after Western blot analysis that UNIVmAb reacted antigen
overexpression is real and accepted when tested with all other cancers sera samples

Page 6 of 7
Manjunathetal. BMC Res Notes (2019) 12:744
reduction of antigen expression in all cancer’s sera. ese
results indicate that the 57kD, named H11 antigen is a
hyaluronan binding protein (data not shown). We per-
formed immunoprecipitation and affinity purification of
normal and cancer patient’s serum proteins with mAb
to prove that mAb is reacting with 57kDa protein with
high specificity. As expected the normal sera showed low
levels of circulatory 57 kDa by western blot, whereas,
advanced cancer sera showed overexpression of 57kDa
antigen. To show that the cancer antigen 57kDa is associ-
ated with carrier protein such as Albumin we used Ciba-
cron blue gel exclusion method. Eluted fractions were
tested for reactivity with b-HA and UNIVmAb, showing
a strong reaction at 57kDa (data not shown). To investi-
gate UNIVmAb reactive cancer antigen may be related to
a known hyaladherin, CD44 (std); Whether, UNIVmAb
immunoprecipitated protein can pull down CD44 reac-
tive protein, we used stomach grade lll cancer sample,
which overexpressed for mAb reacted H11 antigen and
normal sera were tested. We find no reaction to HCAM
antibody to the H11 antigen (data not shown).
Conclusion
Blood-borne metastasis is the greatest obstacle to cure
patients with cancers. e abundant blood proteins, such
as albumin, immunoglobulin, etc.; may mask the less
abundant proteins, which are usually potential markers.
ere are many known specific serum markers but not a
common biomarker for various cancers sera.
UNIVmAb detected the overexpression of serum spe-
cific H11 antigen in various cancer’s sera, offers signifi-
cant advantages. In the present study, screening serum
samples from 70 normal and 50 cancer patients samples
(18 different cancer types of various grade) of different
grades we predicted that the UNIVmAb might be used
as a antibody to detect a common cancer biomarker. is
Mab detected a protein H11 and is unique HABP not
identified earlier in serum and serum albumin may be the
carrier protein of H11 antigen. Still unanswered question
is, what is the nature of 57kDa H11 antigen. We investi-
gated the H11 antigen by proteomic analysis from grade
ll adenocarcinoma of the colon. We observed the pres-
ence of IgGH1, member of immunoglobulin super family
(data not shown). e present data showed that UNI-
VmAb detected the H11 might be a unique hyaladherin
and can be used as a common biomarker for progressive
human cancers sera.
Limitations
At present, there is no common biomarker for multi-
ple cancer detection. e present study could reflect
the UNIVmAb reactive serum antigen as a common
biomarker for multiple cancers. In addition, future
experiment on the proteomic analysis will help us iden-
tify the nature of the antigen as a potential common
biomarker.
Abbreviations
HA: hyaluronan; HABP: hyaluronan binding protein; b-HA: biotinylated hyalu-
ronan; HA-oligo: hyaluronan oligosaccharides; ABTS: 2.2′-azino-Bis-3-ethylben-
zothiazoline-6-sulfonic acid.
Acknowledgements
The authors acknowledge Preethi Center of Oncology and K. R. Hospital for
supplying the cancer serum samples. We also thank the nurses at the centre
for drawing blood with the consent of the patients and the technicians who
performed western blot and Elisa experiments. The authors are grateful to
Protein Analysis Center, Karolinska Institute, Sweden for technical support and
providing mass spectrometry analysis.
Authors’ contributions
SBK, HNA and SDB made contributions to conception, design and interpreta-
tion. DM performed the experiments. SBK, SHV, AT and SDB reviewed and
commented on the manuscript. All authors read and approved the final
manuscript.
Funding
Not applicable.
Availability of data and materials
All data generated or analyzed during this study are included in this published
article.
Ethics approval and consent to participate
The ethical review committee of Preethi center of oncology approved the
work (MYS-00340-AA-NH) and the patient written consent was taken.
Consent of publication
Not applicable.
Competing interests
The authors declare that they have no competing interests.
Author details
1 Preethi Center of Oncology, Vattavyalil Cancer Trust, Mysore, Karnataka,
India. 2 Logic and Clue Diagnostics, Mysore, India. 3 Department of Anatomy
and Cellular Biology, Tuft University School of Medicine, Boston, MA, USA.
Received: 2 October 2019 Accepted: 1 November 2019
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