Ci a ion: de la Fuen e, M.;
Rod íguez-Agi e xe, I.; Vecino, E.;
As iga aga, E.; Ace a, A.;
Ba eda-Gómez, G. Ele a ion o Tea
MMP-9 Concen a ion as a Bioma ke
o In lamma ion in Ocula Pa hology
by An ibody Mic oa ay
Immunode ec ion Assays. In . J. Mol.
Sci. 2022,23, 5639. h ps://doi.o g/
10.3390/ijms23105639
Academic Edi o s: Phaed a
Ele he iou and A hina Ge onikaki
Recei ed: 24 Ma ch 2022
Accep ed: 16 May 2022
Published: 18 May 2022
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In e na ional Jou nal o
Molecula Sciences
A icle
Ele a ion o Tea MMP-9 Concen a ion as a Bioma ke o
In lamma ion in Ocula Pa hology by An ibody Mic oa ay
Immunode ec ion Assays
Miguel de la Fuen e 1,2 , Iñaki Rod íguez-Agi e xe 3, Elena Vecino 2,4 , Egoi z As iga aga 1,
A an xa Ace a 2,5,* and Gab iel Ba eda-Gómez 1,*
1Depa men o Resea ch and De elopmen , IMG Pha ma Bio ech S.L., 48160 De io, Spain;
[email p o ec ed] (M.d.l.F.); [email p o ec ed] (E.A.)
2Expe imen al Oph halmo-Biology G oup (GOBE), Depa men o Cell Biology and His ology, Uni e si y o
he Basque Coun y UPV/EHU, 48940 Leioa, Spain; [email p o ec ed]
3Depa men o Oph halmology, Donos ia Uni e si y Hospi al, 20014 San Sebas ian, Spain; [email p o ec ed]
4Begike -Oph halmology Resea ch G oup, BioC uces Heal h Resea ch Ins i u e, 48903 Ba akaldo, Spain
5IKERBASQUE, Basque Founda ion o Science, 48009 Bilbao, Spain
*Co espondence: [email p o ec ed] (A.A.); [email p o ec ed] (G.B.-G.);
Tel.: +34-946-018-024 (A.A.); Tel.: +34-944-316-577 (G.B.-G.)
Abs ac :
Ma ix me allop o einases a e a amily o enzymes undamen al in in lamma o y p ocesses.
Be ween hem, MMP-9 is up- egula ed du ing in lamma ion; hus, i s quan i ica ion in non-in asi e
luids is a p omising app oach o in lamma ion iden i ica ion. To his goal, a bioma ke quan i ica ion
es was de eloped o ocula in lamma ion de ec ion using an i-MMP-9 an ibody mic oa ays
(AbMAs). A e alida ion wi h eigh heal hy con ol ea samples cha ac e ized by ELISA, 20 samples
we e es ed om indi iduals diagnosed wi h ocula in lamma ion due o: ca a ac s, glaucoma,
meibomian gland dys unc ion, alle gy, o d y eye. Concen a ion alues o ea MMP-9 we e ob ained
o each sample, and 12 pa ien s su passed he pa hological h eshold (30 ng/mL). A signi ican
ele a ion o MMP-9 concen a ion in he ea s o glaucoma pa ien s compa ed wi h heal hy con ols
was obse ed. In o de o e alua e he diagnos ic abili y, an ROC cu e analysis was pe o med using
ou da a, de e mining he op imal h eshold o he es a 33.6 ng/mL o ea MMP-9. In addi ion,
a con usion ma ix was applied, es ima ing sensi i i y a 60%, speci ici y a 88%, and accu acy a
68%. In conclusion, we demons a ed ha he AbMAs sys em allows he quan i ica ion o MMP-9 in
pa hologies ha in ol e in lamma ion o he ocula su ace.
Keywo ds:
ea MMP-9; enzyme bioma ke ; diagnosis; moni o ing; an ibody mic oa ay; ocula
in lamma ion; glaucoma; poin o ca e; in i o diagnos ics
1. In oduc ion
Bioma ke s can be de ined as biological analy es by which a pa icula pa hological
o physiological p ocess can be iden i ied o cha ac e ized [
1
]. They allow a mo e p ecise
diagnosis and he moni o ing o pa hologies and condi ions. A bioma ke can de e mine
i he pa ien has a pa icula medical s a e, he di e en sub ypes o he pa hology i
applicable, and he bes ea men indica ed, imp o ing he moni o ing o he he apy
esponse, he diagnosis, and he p og ession [2].
Among all ypes o bioma ke s, enzymes a e gaining impo ance in many pa holo-
gies [
3
,
4
]. Enzymes a e chemical ca alys s ha help o ganisms conduc essen ial biochemical
eac ions. De iciency, mal unc ion, educed/inc eased ac i i y, o o e exp ession o en-
zymes and hei inhibi o s can cause a a ie y o clinical condi ions [
5
]. Consequen ly, he
s udy o enzymes and hei inhibi o s is ca dinal o unde s anding disease pa hophys-
iology and de eloping no only he apeu ic op ions bu also diagnos ic and moni o ing
s a egies, as enzymes a e powe ul ma ke s o disease [
5
,
6
]. In his ega d, de ec ion and
In . J. Mol. Sci. 2022,23, 5639. h ps://doi.o g/10.3390/ijms23105639 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 5639 2 o 16
quan i ica ion o enzymes in biological luids is an in e es ing ield o esea ch, as i can
lead o imp o emen s in pa hology p ognosis and pa ien li e.
One o he main p ocesses in which enzymes pa icipa e is in lamma ion, a unda-
men al mechanism o main enance o body homeos asis e sus in ec ions and inju ies.
No el published esea ch has es ablished a ela ionship be ween sys emic in lamma ion
and se e al highly p e alen pa hologies, such as cance [
7
] and neu odegene a i e [
8
],
au oimmune [
9
], ca dio ascula [
10
], and me abolic diseases [
11
]. In addi ion, many ocu-
la pa hologies such as Sjog en’s synd ome [
12
], o ke a oconjunc i i is sicca, commonly
named as d y eye (DE) [
13
], ha e also been co ela ed wi h in lamma ion. Fu he mo e, an-
imic obial p ese a i e compounds such as qua e na y ammonium benzalkonium chlo ide
(BAK), used in an iglaucoma eye d op ea men s, ha e been associa ed wi h ch onic ocula
in lamma ion [
14
,
15
]. Many clinical symp oms o ch onic ocula in lamma ion ha e been
epo ed in pa ien s unde long- e m an iglaucoma ea men [
16
]. I has been de e mined
ha BAK ac s a di e en le els o he cell machine y, in e ac ing wi h cell memb anes and
ecep o s. I a ec s conjunc i al epi helial cells and p o okes ocula in lamma ion signs and
symp oms such as loss o goble cells, conjunc i al squamous me aplasia and apop osis,
dis up ion o he co neal epi helium ba ie , and damage o deepe ocula issues [
16
].
These oxic e ec s igge in lamma ion pa hways ha p ecipi a e he o e exp ession o
ce ain enzymes. Taking his in o accoun , enzymes can be used as bioma ke s, ei he o
diagnosis o o moni o ing he esponse o a ea men and e alua ing he ad e se and
oxic e ec s o he he apy.
Ma ix me allop o einases (MMPs) a e a amily o enzymes ha play impo an oles
in in lamma o y p ocesses [
17
,
18
]. MMP-9, also called gela inase B, is a zinc and calcium
ion-dependen enzyme ha is in ol ed in issue emodeling by deg ading ypes IV and
V collagen o he ex acellula ma ix (ECM) in physiological p ocesses such as wound
healing and bone g ow h [
19
,
20
]. This enzyme plays an impo an ole and is up egula ed
in in lamma o y pa hologies, a h i is, ca dio ascula and pulmona y diseases, as well as
in cance [
18
]. MMP-9, along wi h o he MMPs, is up egula ed du ing in lamma ion in
di e en issues and luids such as se um, sali a, syno ial liquid, o ea , becoming an in-
e es ing enzyme bioma ke . Thus, de ec ion and quan i ica ion o MMP-9 in non-in asi e
luids is a p omising app oach o in lamma ion p e en ion, diagnosis, and disease o
ea men moni o ing. Conc e ely, MMP-9 has been also ex ensi ely s udied as a bioma ke
o in lamma ion in ea samples [
21
–
25
]; his bioma ke is highly o e exp essed in di e en
diseases associa ed wi h ocula in lamma ion and in ocula su ace pa hologies [
22
,
26
,
27
].
In he co neal epi helium, bo h TGF-
β
and IL-1 cy okines, key playe s in he egula ion o
in lamma o y p ocesses, s imula e MMP-9 o e exp ession [28].
Cu en ly, he diagnosis o he main ocula su ace in lamma ion pa hologies is mos ly
subjec i e and is based on he knowledge o he oph halmologis and he signs and symp-
oms o he pa ien s [
29
,
30
]. Howe e , he disco e y and use o bioma ke s, such as MMP-9,
ha e opened new lines o esea ch aiming o de elop new diagnosis ools [
31
]. These
bioma ke s a e ex emely use ul in he clinic because hey e lec he pa hological s a e
o he pa ien , as well as he e olu ion o he disease in molecula e ms; hus, hey can
be used, no only o diagnosis bu also o e alua ing he p ognosis and o moni o ing
he p og ession o he pa hology and he esponse o ea men s. Va ious s udies ha e
alida ed ea MMP-9 as one o he main bioma ke s o ocula in lamma ion diseases [
23
].
Di e en comme cial diagnosis poin o ca e (PoC) and
in i o
diagnos ics (IVDs) es s
ha e been de eloped o e alua ion o ea MMP-9, such as In lammaD y [
24
,
27
]; howe e ,
hese ypes o es s ha e he main d awback o gi ing only a posi i e/nega i e esul , no
allowing he quan i ica ion o he bioma ke , no a p ecise e alua ion o he pa hological
s a us o he pa ien , no he moni o ing o he disease, due o hei a iabili y [
32
]. Ad-
di ionally, his hampe s he co ela ion be ween symp oms and bioma ke concen a ion,
p ecluding he s a i ied diagnosis o pa ien s.
Al e na i ely, mic oa ay echnology can be applied as a pla o m o bioma ke -based
diagnos ics o moni o ing. Cell memb anes, whole cells, an ibodies, enzymes, nucleic acids,
In . J. Mol. Sci. 2022,23, 5639 3 o 16
and o he p o eins can be immobilized on di e se su aces using mic oa ay echnology
wi hou losing hei unc ional s uc u e. As a esul , hey a e used in immunochemis y,
au o adiog aphy, adioligand and binding in es iga ions, mi ochond ial oxici y assays, as
well as o he echniques such as colo ime y and mass spec ome y [
33
–
37
]. Mic oa ays
allow he educ ion o he numbe o samples, medica ions, chemicals, and esidues.
Among hem, an ibody mic oa ays (AbMAs) a e used simila ly as a minia u ized enzyme-
linked immunoso ben assay (ELISA) o he de ec ion o analy es. Howe e , AbMAs show
highe sensi i i y o he iden i ica ion o bioma ke s han adi ional ELISA, demons a ing
hei imp o emen s in clinical si ua ions when aking also in o accoun he p e iously
men ioned ad an ages [
25
]. Cu en ly, AbMAs a e widely used o disease diagnosis in
di e se pa hologies such as cance [38], o ocula condi ions [39], among o he s [40,41].
Hence, he aim o his wo k was o de elop an AbMA es o ocula in lamma ion
de ec ion by quan i ying ea MMP-9 bioma ke (Figu e 1). Fo his pu pose, an ibodies
agains human MMP-9 we e immobilized o e glass slides whe e he sample was incu-
ba ed and he bioma ke was cap u ed. Then, he bioma ke was de ec ed using a labeled
an ibody cock ail ha p oduced a luo escen in ensi y signal di ec ly p opo ional o he
concen a ion o MMP-9 in he sample. Fo he alida ion o he es , eigh non-pa hological
ea samples we e used. Enzyme MMP-9 bioma ke concen a ion was con i med using
con en ional ELISA as he gold s anda d o cha ac e ize he samples and assess he eliabil-
i y o he es . Subsequen ly, ea samples om 20 indi iduals clinically diagnosed wi h
ocula in lamma ion we e assayed. Using a calib a ion line, p o ein bioma ke p esence
was quan i ied in each o he samples employing AbMAs, ea ly alida ing he de eloped
echnique as an MMP-9 in lamma ion- ela ed de ec ion ool.
In . J. Mol. Sci. 2022,23, 5639 4 o 16
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 o 18
Figu e 1. De ec ion and quan i ica ion o MMP-9 enzyme in lamma ion bioma ke in human ea
samples using AbMAs. Fi s , he selec ed an ibodies we e immobilized on o glass slides ha we e
incuba ed wi h he sample. Then, MMP-9 was cap u ed by he men ioned an ibody and de ec ed
wi h a labeled an ibody cock ail. Finally, he in ensi y o he signal was quan i ied and he da a
acqui ed, allowing he analysis o he MMP-9 bioma ke in he samples.
Figu e 1.
De ec ion and quan i ica ion o MMP-9 enzyme in lamma ion bioma ke in human ea
samples using AbMAs. Fi s , he selec ed an ibodies we e immobilized on o glass slides ha we e
incuba ed wi h he sample. Then, MMP-9 was cap u ed by he men ioned an ibody and de ec ed
wi h a labeled an ibody cock ail. Finally, he in ensi y o he signal was quan i ied and he da a
acqui ed, allowing he analysis o he MMP-9 bioma ke in he samples.
In . J. Mol. Sci. 2022,23, 5639 5 o 16
2. Resul s
2.1. Subjec s
A coho o samples om bo h heal hy con ols and pa ien s su e ing ocula in lam-
ma ion was used o alida e cus omized AbMAs as an MMP-9 quan i ica ion assay o
ocula in lamma ion e alua ion in human ea luid.
Fi s ly, ea samples we e ob ained om olun ee s as de ailed in he Ma e ials and
Me hods Sec ion. Tea samples we e di ided in o wo g oups: heal hy con ols, named
as HC, and pa ien s, named as P. The second one was composed o indi iduals su e -
ing ocula in lamma ion due o di e en pa hological condi ions such as ca a ac s, glau-
coma, meibomian gland dys unc ion (MGD), alle gy, o DE. Pa ien s we e e alua ed using
a Schi me ’s es ; no mal alues we e conside ed
≥
10 mm we ing o he pape a e
5 min, whe eas ea de iciency alues we e
≤
5 mm. All pa ien s, excep om P 9, P 11,
and P 18, p esen ed ea de iciency. Glaucoma pa ien s we e all unde p os aglandin
eye d op ea men ; hese d ugs we e p ese ed wi h BAK. P 9 was unde wo di e en
BAK-p ese ed p os aglandin analogue ea men s. No Schi me ’s es was pe o med o
heal hy olun ee s o a oid he in e en ion since hey did no p esen clinical condi ions.
In addi ion, he gende and age o he pa ien s we e de ailed (Table 1).
Table 1.
Baseline cha ac e is ics o he pa ien s and heal hy con ol indi iduals. The heal hy con ols
we e collec ed om olun ee s wi hou any ocula pa hology diagnosed.
Tea Sample G oup Age Gende Condi ions Schi me ’s Tes (mm)
HC 1 Heal hy Con ol 25 Male n/a n/a
HC 2 Heal hy Con ol 26 Female n/a n/a
HC 3 Heal hy Con ol 30 Female n/a n/a
HC 4 Heal hy Con ol 23 Female n/a n/a
HC 5 Heal hy Con ol 40 Male n/a n/a
HC 6 Heal hy Con ol 23 Female n/a n/a
HC 7 Heal hy Con ol 24 Female n/a n/a
HC 8 Heal hy Con ol 29 Female n/a n/a
P 1 Pa ien 79 Female Ca a ac s 5
P 2 Pa ien 73 Female Ca a ac s 5
P 3 Pa ien 66 Female Ca a ac s 3
P 4 Pa ien 81 Female Ca a ac s 5
P 5 Pa ien 89 Female Ca a ac s 2
P 6 Pa ien 62 Female Ca a ac s 0
P 7 Pa ien 70 Male Ca a ac s 1
P 8 Pa ien 73 Female Ca a ac s 3
P 9 Pa ien 68 Female
Glaucoma
6
P 10 Pa ien 60 Male
Glaucoma
5
P 11 Pa ien 75 Female
Glaucoma
7
P 12 Pa ien 70 Female
Glaucoma
4
P 13 Pa ien 82 Male
Glaucoma
5
P 14 Pa ien 82 Male
Glaucoma
5
P 15 Pa ien 52 Male MGD 5
P 16 Pa ien 49 Female Alle gy 4
P 17 Pa ien 29 Female DE 5
P 18 Pa ien 30 Female DE 6
P 19 Pa ien 49 Female Alle gy 5
P 20 Pa ien 38 Female MGD +
DE 3
2.2. An ibody Mic oa ay Valida ion
The eigh samples om he heal hy olun ee s we e cha ac e ized using an an i-
human MMP-9 ELISA ki . In o de o assess he eliabili y o his echnique, in con as
wi h he gold s anda d, he ob ained alues we e compa ed wi h he quan i ica ion o
ea MMP-9 using he de eloped AbMAs. When compa ing he concen a ion o MMP-9
ob ained wi h each echnique, simila esul s we e ob ained (Figu e 2). Addi ionally, a simple
In . J. Mol. Sci. 2022,23, 5639 6 o 16
bi a ia e co ela ion was calcula ed, se ing he signi icance a
α
= 0.05 using a wo- ailed es .
A Pea son co ela ion coe icien ( ) o 0.9918 was ob ained wi h a signi icance o (****),
p- alue < 0.0001.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 6 o 18
2.2. An ibody Mic oa ay Valida ion
The eigh samples om he heal hy olun ee s we e cha ac e ized using an an i-
human MMP-9 ELISA ki . In o de o assess he eliabili y o his echnique, in con as
wi h he gold s anda d, he ob ained alues we e compa ed wi h he quan i ica ion o ea
MMP-9 using he de eloped AbMAs. When compa ing he concen a ion o MMP-9
ob ained wi h each echnique, simila esul s we e ob ained (Figu e 2). Addi ionally, a
simple bi a ia e co ela ion was calcula ed, se ing he signi icance a α = 0.05 using a wo-
ailed es . A Pea son co ela ion coe icien ( ) o 0.9918 was ob ained wi h a signi icance
o (****), p- alue < 0.0001.
MMP−9 (ng/mL)
Figu e 2. Concen a ion o MMP-9 in he collec ion o ea samples om heal hy con ols, wi hou
any ocula diso de diagnosed, using ELISA (blue) as he gold s anda d echnique and AbMAs
(o ange). MMP-9 concen a ion is ep esen ed as ng/mL o each indi idual. A g ay line is plo ed
a 30 ng/mL o MMP-9 enzyme in ea , he h eshold alue a which highe concen a ions a e
conside ed a sign o ocula in lamma ion.
2.3. Analysis o Pa hological Samples
Enzyme bioma ke MMP-9 was also e alua ed in he 20 pa ien samples using he
AbMA de eloped echnology. Concen a ion alues o MMP-9 we e ob ained o each
ea sample. P 1, P 3, P 4, P 6, P 7, P 8, P 9, P 10, P 11, P 13, P 14, and P 16 samples su passed
he pa hological h eshold es ablished a 30 ng/mL o MMP-9 in he luid. These 12
samples ep esen 60% o he ea collec ion om pa ien s diagnosed wi h an ocula
pa hology used in his s udy (Figu e 3).
Figu e 2.
Concen a ion o MMP-9 in he collec ion o ea samples om heal hy con ols, wi hou any
ocula diso de diagnosed, using ELISA (blue) as he gold s anda d echnique and AbMAs (o ange).
MMP-9 concen a ion is ep esen ed as ng/mL o each indi idual. A g ay line is plo ed a 30 ng/mL
o MMP-9 enzyme in ea , he h eshold alue a which highe concen a ions a e conside ed a sign
o ocula in lamma ion.
2.3. Analysis o Pa hological Samples
Enzyme bioma ke MMP-9 was also e alua ed in he 20 pa ien samples using he
AbMA de eloped echnology. Concen a ion alues o MMP-9 we e ob ained o each ea
sample. P 1, P 3, P 4, P 6, P 7, P 8, P 9, P 10, P 11, P 13, P 14, and P 16 samples su passed he
pa hological h eshold es ablished a 30 ng/mL o MMP-9 in he luid. These 12 samples
ep esen 60% o he ea collec ion om pa ien s diagnosed wi h an ocula pa hology used
in his s udy (Figu e 3).
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 o 18
MMP−9 (ng/mL)
Figu e 3. ng/mL o MMP-9 in ocula in lamma ion pa ien ea samples quan i ied using AbMA. A
g ay line is plo ed a 30 ng/mL o MMP-9 enzyme in ea , he h eshold alue a which highe
concen a ions a e conside ed a sign o ocula in lamma ion.
In addi ion, he MMP-9 concen a ion in each ea sample was compa ed be ween
heal hy and pa hological subg oups (Figu e 4). The no mali y o he samples was
e alua ed using a Shapi o–Wilk es ; se ing he signi icance a α = 0.05 using a wo- ailed
es , he g oups did no ollow a Gaussian dis ibu ion. The pa ien g oup was di ided
in o a glaucoma g oup, a ca a ac g oup, and he o he pa hologies g oup, englobing
MGD, alle gy, as well as DE. Cli ’s del a alues we e calcula ed o quan i ying he
amoun o di e ence be ween con ol and pa hological g oups. The Cli ’s del a alue
when compa ing he heal hy g oup and he o he pa hologies g oup was δ = −0.208; o
ca a ac s pa ien s e sus heal hy indi iduals, i was δ = 0.438; and o glaucoma pa ien s e sus
heal hy indi iduals, i was δ = 0.583.
Figu e 3.
ng/mL o MMP-9 in ocula in lamma ion pa ien ea samples quan i ied using AbMA.
A g ay line is plo ed a 30 ng/mL o MMP-9 enzyme in ea , he h eshold alue a which highe
concen a ions a e conside ed a sign o ocula in lamma ion.
In . J. Mol. Sci. 2022,23, 5639 7 o 16
In addi ion, he MMP-9 concen a ion in each ea sample was compa ed be ween
heal hy and pa hological subg oups (Figu e 4). The no mali y o he samples was e alua ed
using a Shapi o–Wilk es ; se ing he signi icance a
α
= 0.05 using a wo- ailed es ,
he g oups did no ollow a Gaussian dis ibu ion. The pa ien g oup was di ided in o
a glaucoma g oup, a ca a ac g oup, and he o he pa hologies g oup, englobing MGD,
alle gy, as well as DE. Cli ’s del a alues we e calcula ed o quan i ying he amoun
o di e ence be ween con ol and pa hological g oups. The Cli ’s del a alue when
compa ing he heal hy g oup and he o he pa hologies g oup was
δ
=
−
0.208; o ca a ac s
pa ien s e sus heal hy indi iduals, i was
δ
= 0.438; and o glaucoma pa ien s e sus
heal hy indi iduals, i was δ= 0.583.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 8 o 18
MMP−9 (ng/mL)
MMP−9 (ng/mL)
MMP−9 (ng/mL)
Figu e 4. Tea MMP-9 concen a ion di e ences in he g oups o pa ien s su e ing ocula
in lamma ion e sus he g oup o heal hy con ols. Cli ’s del a alues a e displayed o each
compa ison. (A) Di e ences be ween heal hy con ols and MGD, DE, and alle gy pa ien s (o he
pa hologies g oup). (B) Di e ences be ween heal hy con ols and ca a ac s pa ien s. (C) Di e ences
be ween heal hy con ols and glaucoma pa ien s.
Figu e 4.
Tea MMP-9 concen a ion di e ences in he g oups o pa ien s su e ing ocula in lam-
ma ion e sus he g oup o heal hy con ols. Cli ’s del a alues a e displayed o each compa ison.
(
A
) Di e ences be ween heal hy con ols and MGD, DE, and alle gy pa ien s (o he pa hologies
g oup). (
B
) Di e ences be ween heal hy con ols and ca a ac s pa ien s. (
C
) Di e ences be ween
heal hy con ols and glaucoma pa ien s.
In . J. Mol. Sci. 2022,23, 5639 8 o 16
No di e ences we e obse ed when compa ing ea MMP-9 concen a ions be ween
age, gende , and Schi me ’s es esul s (da a no shown).
When analyzing he in lamma ion bioma ke in he samples based on he es ab-
lished pa hological h eshold (30 ng/mL), di e ences we e obse ed be ween he g oups
(Figu e 5). Bo h heal hy con ols and o he pa hologies g oups p esen ed MMP-9 concen a-
ions mainly below he h eshold; con a ily, he ca a ac s and glaucoma g oups p esen ed
ea MMP-9 alues mos ly o e 30 ng/mL. In summa y, 88% o he heal hy con ols and
83% o he o he pa hologies g oup samples we e unde he h eshold; 75% o ca a ac s and
83% o glaucoma ea samples we e o e he h eshold.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 9 o 18
No di e ences we e obse ed when compa ing ea MMP-9 concen a ions be ween
age, gende , and Schi me ’s es esul s (da a no shown).
When analyzing he in lamma ion bioma ke in he samples based on he es ablished
pa hological h eshold (30 ng/mL), di e ences we e obse ed be ween he g oups (Figu e
5). Bo h heal hy con ols and o he pa hologies g oups p esen ed MMP-9 concen a ions
mainly below he h eshold; con a ily, he ca a ac s and glaucoma g oups p esen ed ea
MMP-9 alues mos ly o e 30 ng/mL. In summa y, 88% o he heal hy con ols and 83%
o he o he pa hologies g oup samples we e unde he h eshold; 75% o ca a ac s and
83% o glaucoma ea samples we e o e he h eshold.
(>30 ng/mL) 1165
(≤30 ng/mL) 7521
Figu e 5. Numbe o indi iduals o pa ien s o e and below he pa hological h eshold
in he di e en g oups.
2.4. E alua ion o he Diagnos ic Pe o mance o he Tes
Finally, in o de o e alua e he diagnos ic abili y o he de eloped AbMA es , a
ecei e ope a ing cha ac e is ic (ROC) cu e analysis was pe o med [42,43]. The op imal
h eshold alue o he es was de e mined using his analysis, esul ing in 33.6 ng/mL o
ea MMP-9 using ou da a. In addi ion, a con usion ma ix was se up (Figu e 6) ollowing
he indica ions o he guide The Fi ness o Pu pose o Analy ical Me hods o Eu achem
[44], assessing he sensi i i y a 60%, he speci ici y a 88%, and he accu acy a 68%.
Figu e 5.
Numbe o indi iduals o pa ien s o e and below he pa hological h eshold in he di e en g oups.
2.4. E alua ion o he Diagnos ic Pe o mance o he Tes
Finally, in o de o e alua e he diagnos ic abili y o he de eloped AbMA es ,
a ecei e ope a ing cha ac e is ic (ROC) cu e analysis was pe o med [
42
,
43
]. The
op imal h eshold alue o he es was de e mined using his analysis, esul ing in
33.6 ng/mL o ea MMP-9 using ou da a. In addi ion, a con usion ma ix was se up
(Figu e 6) ollowing he indica ions o he guide The Fi ness o Pu pose o Analy ical
Me hods o Eu achem [
44
], assessing he sensi i i y a 60%, he speci ici y a 88%, and he
accu acy a 68%.
In . J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 10 o 18
Figu e 6. Con usion ma ix o ea MMP-9 analysis o e he di e en samples. T ue posi i e (TP), alse
posi i e (FP), alse nega i e (FN), and ue nega i e (TN) a es a e de ailed. Sensi i i y is calcula ed as
TP/(TP + FN), speci ici y as TN/(TN + FP), and accu acy as (TP + TN)/(TP + FP + FN + TN).
3. Discussion
We de eloped an AbMA es immobilizing an an i-MMP-9 IgG an ibody and
es ablishing a de ec ion p o ocol o he quan i ica ion o MMP-9 in ea s. The pu pose o
his es is he de ec ion and quan i ica ion o human MMP-9 in ea samples as an
ins umen o ocula in lamma ion e alua ion. The da a ob ained demons a ed once
again ha MMP-9 is a good bioma ke o in lamma ion in a ious ocula pa hologies [45],
as well as alida ing mic oa ay immunode ec ion echnology as a diagnos ic ool in he
de ec ion o MMP-9 and he moni o ing o pa ien s wi h in lamma ion- ela ed pa hologies
such as glaucoma.
Ou esul s alida ed he de eloped AbMA es o he de ec ion and quan i ica ion
o human MMP-9 in ea samples as an ins umen o ocula in lamma ion e alua ion.
Fo his pu pose, eigh ea samples om heal hy indi iduals we e collec ed, as well as 20
ea samples om pa ien s su e ing a ious ocula in lamma o y condi ions: ca a ac s,
glaucoma, meibomian gland dys unc ion, alle gy, and DE. All 28 ea samples we e
de ined in e ms o dono age, gende , condi ion and Schi me ’s es esul s. Fi s ly, MMP-
9 concen a ion was de e mined wi h he cu en ly used gold s anda d in his a ea, he
ELISA echnique, in o de o e alua e i he e is a posi i e co ela ion be ween his
echnique and he AbMA es , since he AbMA es aims o be a new me hod o bioma ke
quan i ica ion. The concen a ion o MMP-9 in he ea collec ion om heal hy dono s was
quan i ied by ELISA and AbMAs acco ding o hei speci ic p o ocols. A s a is ically
signi ican co ela ion in he ob ained alues was obse ed, wi h a Pea son co ela ion
coe icien o 0.9918 and a p- alue < 0.0001. These esul s a e in ag eemen wi h p e ious
s udies using his echnology in human ea [25], poin ing ou ha AbMAs a e a use ul
and eliable ool o MMP-9 quan i ica ion in human ea samples.
Once he es was alida ed, ea samples o 20 pa ien s wi h ocula in lamma ion
we e s udied. These pa ien s we e su e ing om di e en diseases ela ed o
in lamma ion (ca a ac s, glaucoma, MGD, alle gy, and DE) [46]. MMP-9 bioma ke was
quan i ied using he de eloped AbMAs, which esul ed in 60% o he samples su passing
he pa hological h eshold. This was es ablished a 30 ng/mL based on he li e a u e [47];
highe concen a ions o he enzyme bioma ke in ea samples we e associa ed wi h
ocula su ace in lamma ion [26]. The obse ed global ele a ion o MMP-9 in he
pa hological samples was easonable due o he in lamma o y cha ac e is ics o his
bioma ke , which inc eases in esponse o s ess when cy okine o chemokine pa hways
Figu e 6.
Con usion ma ix o ea MMP-9 analysis o e he di e en samples. T ue posi i e (TP), alse
posi i e (FP), alse nega i e (FN), and ue nega i e (TN) a es a e de ailed. Sensi i i y is calcula ed as
TP/(TP + FN), speci ici y as TN/(TN + FP), and accu acy as (TP + TN)/(TP + FP + FN + TN).
In . J. Mol. Sci. 2022,23, 5639 9 o 16
3. Discussion
We de eloped an AbMA es immobilizing an an i-MMP-9 IgG an ibody and es ablish-
ing a de ec ion p o ocol o he quan i ica ion o MMP-9 in ea s. The pu pose o his es
is he de ec ion and quan i ica ion o human MMP-9 in ea samples as an ins umen o
ocula in lamma ion e alua ion. The da a ob ained demons a ed once again ha MMP-9 is
a good bioma ke o in lamma ion in a ious ocula pa hologies [
45
], as well as alida ing
mic oa ay immunode ec ion echnology as a diagnos ic ool in he de ec ion o MMP-9
and he moni o ing o pa ien s wi h in lamma ion- ela ed pa hologies such as glaucoma.
Ou esul s alida ed he de eloped AbMA es o he de ec ion and quan i ica ion
o human MMP-9 in ea samples as an ins umen o ocula in lamma ion e alua ion.
Fo his pu pose, eigh ea samples om heal hy indi iduals we e collec ed, as well as
20 ea samples om pa ien s su e ing a ious ocula in lamma o y condi ions: ca a ac s,
glaucoma, meibomian gland dys unc ion, alle gy, and DE. All 28 ea samples we e de ined
in e ms o dono age, gende , condi ion and Schi me ’s es esul s. Fi s ly, MMP-9
concen a ion was de e mined wi h he cu en ly used gold s anda d in his a ea, he ELISA
echnique, in o de o e alua e i he e is a posi i e co ela ion be ween his echnique and
he AbMA es , since he AbMA es aims o be a new me hod o bioma ke quan i ica ion.
The concen a ion o MMP-9 in he ea collec ion om heal hy dono s was quan i ied
by ELISA and AbMAs acco ding o hei speci ic p o ocols. A s a is ically signi ican
co ela ion in he ob ained alues was obse ed, wi h a Pea son co ela ion coe icien o
0.9918 and a p- alue < 0.0001. These esul s a e in ag eemen wi h p e ious s udies using
his echnology in human ea [
25
], poin ing ou ha AbMAs a e a use ul and eliable ool
o MMP-9 quan i ica ion in human ea samples.
Once he es was alida ed, ea samples o 20 pa ien s wi h ocula in lamma ion
we e s udied. These pa ien s we e su e ing om di e en diseases ela ed o in lamma ion
(ca a ac s, glaucoma, MGD, alle gy, and DE) [
46
]. MMP-9 bioma ke was quan i ied using
he de eloped AbMAs, which esul ed in 60% o he samples su passing he pa hological
h eshold. This was es ablished a 30 ng/mL based on he li e a u e [
47
]; highe concen-
a ions o he enzyme bioma ke in ea samples we e associa ed wi h ocula su ace
in lamma ion [
26
]. The obse ed global ele a ion o MMP-9 in he pa hological samples
was easonable due o he in lamma o y cha ac e is ics o his bioma ke , which inc eases
in esponse o s ess when cy okine o chemokine pa hways a e ac i a ed. When MMP-9
alues we e analyzed o each subjec wi hin he ou s udy g oups, he MMP-9 h eshold
was exceeded by only 12% and 17% o indi iduals in he con ol and o he pa hology
g oups, espec i ely. Howe e , 75% and 83% o pa ien s in he glaucoma and ca a ac
g oups, espec i ely, had a ea MMP-9 concen a ion abo e he pa hological h eshold
o 30 ng/mL, deno ing ocula su ace in lamma ion. Simila esul s we e desc ibed by
Kim and cowo ke s when hey epo ed ha app oxima ely 72% o glaucoma pa ien s
displayed a high concen a ion o ea MMP-9 (o e 40 ng/mL); howe e , when s udy-
ing a con ol g oup o 47 heal hy subjec s, only abou 32% o hem showed an inc ease
in his bioma ke [
48
]. Again, his alida ed he AbMA esul s in conco dance wi h
he li e a u e [
25
], highligh ing he impo ance o MMP-9 as a ea bioma ke o ocula
su ace in lamma ion.
In o de o assess he diagnosis capabili y o he es when s udying each pa hology,
ea MMP-9 concen a ion was compa ed among g oups and Cli ’s alue was calcula ed
as a use ul complemen a y analysis o he co esponding hypo hesis es ing [
48
]. When
compa ed wi h he heal hy con ols, he o he pa hologies, ca a ac s, and glaucoma g oups
ob ained
δ
alues o
−
0.208, 0.438, and 0.583, espec i ely. Values o e a
δ
= 0.474 mean
a la ge di e ence be ween he wo g oups [
48
]. Taking his in o accoun , he glaucoma
g oup was he one wi h he highes
δ
when compa ed wi h he con ols, which indi-
ca ed a majo di e ence in he p esence o his bioma ke , p elimina ily poin ing ou
ha he de eloped echnology was able o de ec ocula in lamma ion pa hology- ela ed
s a es. Likewise, he ca a ac s g oup p esen ed a
δ
= 0.438, meaning a medium (de ined
In . J. Mol. Sci. 2022,23, 5639 16 o 16
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