.

Qui trnh ELISA trc tip dng xc nh Staphylococcus Aureus:

Phng php trc tip Elisa c chng minh l rt ph bin chun on cc bnh truyn
nhim nht nh: ngi vim gan siu vi, HIV v ng vt nh: vim v, nhim khun xut huyt
(Nickerson v cc cng s, 1989. Rehmant et al, 2002).
Vt liu v phng php:
+ Chun b cc KN: S. aureus c nui cy trn mi trng Staph.110 37 0C trong 24h. Thu
chng vi khun vi nc mui m phosphate (pH 6.8) c cha formalin 0.3% v un nng 100 0C trong
1h (Heddleston et al, 1972; Zia v cng s, 2000).
+ Sn xut KT: ba con th c tim dch huyn ph ni trn di da vi lng 0.5;1;1.5 v 2ml
khong 4 ngy. Sau 7 ngy tip tc tim 1ml S.aureus c nui cy trong mi trng canh tht vo th.
Sau 14 ngy tim mi trng ging nh trn, thu nhn mu v huyt thanh khi git th (Relman v
cng s, 2002).
+ Tinh ch v c lng Protein: KT th c phn lp v tinh khit mt phn thng qua phng
php kt ta ammonium sulfate (Hudson v Hay, 1980) v hm lng protein c c tinh bng
phng php Biuret sau khi tiu bn theo ng chun ca huyt thanh b albumin (Zia v cng s,
2001).
+ S tip hp: Enzyme peroxidase t u tng (10mg) c tip hp vi mt phn KT tinh
khit ca th, s dng bc hai trong phng php glutaradehyde (Zia v cng s, 2000).
+ Lp v KT: 100l KT th (pha long 1/10 trong dung dch m ph) c vo tng ging,
polystyrene, tm microtitration gm 96 ging 4 0C trong 24h. Sau , tm c ra nm ln vi m
ra. Cc tm c gi c nh bi 100l PSB vo tng ging v 37 0C trong 24h. Sau khi , tm
c ra sch bng m ra (Horadagoda et al, 1993)
Elisa trc tip:
+ Elisa trc tip c thc hin pht hin KT tip hp enzyme. Cc tip hp enzyme ny pha
long trong PBS l 1:100; 1:200; 1:400 v 1: 800. 100l PBS c thm vo mi ging. Huyt thanh th
c pha long trong PBS t l 1:10.
+ l 2 ln tun t pha long t 2 ln n 11 ging, nh
100l c thm vo. Ging 12 c s dng kim sot. Cc a c 37 0C trong 2h. Sau ra
li 5 ln bng dung dch ra. Tip tc pha long 100l mi hng, u tin pha long A+B, th 2 l pha
long C+D, th 3 l pha long E+F, th 4 l Pha long G+H trn tm microtitration. 37 0 C trong 2h.
Sau khi ra li 5 ln bng dung dch ra. Sau thm 100l guaiaclo (guaicol + H2O2) v 370C
trong 20 pht. Tip tc thm 50l H2SO4 1M (thuc dng) vo mi ging. O.D c ghi li ngay lp
tc trong cng vi a c Elisa, c bc sng 450nm (Kemney v Challacombe, 1989).
+ Phn tch thng k: cc d liu c phn tch qua Duncans Multiple Range (DMR) kim tra
hon ton theo thit k ngu nhin (CRD) (Steel v Torrie,1984).
Kt qu v tho lun:
+ Cc xt nghim huyt thanh khc nhau c s dng cho cc KN pht hin nh l th nghim
nhanh chng lm ngng kt, th nghim agar gel pht hin, nhng tt nht l phng php ELISA pht
hin KN. N tng ca phng php ELISA l da trn s thay i mu sc, c li th hn cc xt nghim
khc nh ngng kt, hunh quang hoc phng x trong phn ng KN-KT c th c o u n khch
quan trong sc k n gin. Ngoi ra, s dng cc tm microtitre ELISA cho php mt s lng ln
phn ng c c trong thi gian ngn (Dawkins et al,1990).
+ C nhiu enzyme khc nhau c s dng trong phng php ELISA nhng peroxidase c u
tin hn nhng loi khc bi s tinh khit, hot tnh c hiu, nhy cm ca cht nn thm d, d tip hp,
tnh hiu qu khi tip hp v s n nh v s tip hp (Kemeny v Challacombe, 1989). Peroxidase l
mt cht oxy ha sinh hc quan trng v ph bin trong cc vt liu t thc vt. Trong thc vt th n c
1

mt trong c chua (Zia et al,2001), ci nga, u tng (Ambreen et al, 2001), khoai ty, c ci, c rt, la
m, l, chui (Reed v cng s, 1975).
+ Peroxidase c chit xut t u tng do tnh hot ng cao v d dng tm, kim. Hot ng
ca peroxidase th c chng t 146.12U/ml. u tng l mt ngun phong ph ca peroxidase
c bo co bi Ambreen et al, 2000. Sau peroxidase mt phn c tinh khit bng cch s dng
phng php lm kt ta ammonium sulfate. K thut ny s dng v n ph bin nht, s dng thuc th
c mui trong cc protein, ha tan cao v phn gii ion cao (Voet et al, 1999). KT c sn xut
bng cch tim cc KN S. aureus vo th v s dng phng php kt ta ammonium (Hudson v Hay,
1980). Lng protein c c tnh bng cch s dng phng php biuret cng nh phng php
U.V. v kt qu ca hai phng php c ghi trong bng .
Bng : D on protein ca mu th:
Sample
Biuret Method (mg/ml)
UV Method (mg/ml)
A
1.6
1.544
B
1.024
0.917
C
1.0791
0.932
+ C nhiu phng php khc nhau tip hp ging nh phng php meleimide, phng php
oxy ha preiodate, bc mt ca phng php glutaraldehyde v bc hai ca phng php
glutaraldehyde nhng bc hai c a chung hn cc phng php khc v kt qu ca vic tip hp p
dng hai phng php glutaraldehyde, cho kt qu ng tin cy v hiu qu hn (Baker, 1989). Khng th
c kt hp vi 10mg ca peroxidase v s kt hp c th nghim thng qua ELISA trc tip. Cc
kt qu ch ra rng trong pha long 1:100 ca OD mu A dao ng t 1,18-0,972. Trong 1:200 OD pha
long trong khong 0,97-0,93. Trong 1:400 gi tr OD dao ng 0,714-0,72. Trong 1:800 gi tr pha long
c hp th l 0,5-0,6.
+ Trong trng hp mu B, pha long 1:100 OD t 1,406-1,205 trong khi pha long 1:200 OD
dao ng t 1,185-1,103. Trong 1:400 OD dao ng t 0,714-0,72. Trong 1:800 gi tr pha long c hp
th t 0,99-0,93.
Mu C, t l 1:100 OD pha long t 1,74-1,61. Trong pha lang 1:200 OD t 1,36-1,23.
+ Trong 1:400 OD t 1,21-1,40. Trong 1:800 gi tr pha long hp th l 1,26-1,39.
C mt s gim ng k ca mu v gi tr OD pha long 1:100 so vi dung dch pha long 1:200,
1:400 v 1:800 l pha long 1:100 cho kt qu tt nht.
+ Kt lun v kin ngh: 1:100 l tt nht cho ELISA trc tip chng li S.aureus. Kt lun tng
t c rt ra khi thng k phn tch thng qua th nghim DRM theo thit k hon ton ngu nhin