High-quality deionized water (see Note 3).
2. Lummol stock solution: 1.25 mMlumino1 in O.lMTris-HCI buffer, pH 8.6 Store
at 4’C m the dark. Make up fresh each week. Lummol should be recrystallized as
the sodium salt before use (see Note
.
3 Hydrogen peroxide: 30% (w/v). Store at 4°C.
4. p-Iodophenol stock solution: p-iodophenol, 1 mg/mL m dimethyl sulfoxrde
(DMSO). Make up fresh each day.
14. Working enhanced chemlluminescence substrate solution (see Note 9)-either:
a. p-iodophenol-enhanced substrate: luminol 1.25 mM, p-iodophenol 4 мкМ,
H202 2.7 mM). Add 15 мкL of stock hydrogen peroxide and 40 mL of
p-iodophenol stock solunon to 50 mL of stock substrate solution,, and mix
well. Make up dally using high-quality deionized water (see Note 3), and
store in the dark when not in use, or
b p-Hydroxycinnamic acid-enhanced substrate (luminol 1.25 mM, p-hydroxy-
cinnamic acid 30 мкM, H202 2.7 mМ). Add 15 mL of stock hydrogen peroxide 202 stott
and 1 mg ofp-hydroxycinnamrc acid (alternative names p-coumaric acid or
4-hydroxycmnamtc acid, e g., Aldrich [Gillmgham, UK] cat. no. 2,320-7) to
50 mL of stock substrate solutton. Mix for 30 min before using Make up
dally using high-quality deromzed water (see Note 3), and store m the dark
when not in use
For best possible sensitlvtty, the final assay wash (step 6) and all substrate reagents
should be made up in water of the highest posstble purtty Trace contaminatton
with bacteria, algae, organic compounds, and chlorine IS a particular problem. Enhanced Chemilum/nescence 203
Laboratory-grade distilled or reverse osmoses water should be further treated
using a deionization cartridge. The water plant must be well maintained to avoid
bacterial growth in deionization columns, plumbing, and storage tanks. Alterna-
tively, commercial HPLC-grade water has been found to be satisfactory.
Lummol is available as the free base under alternative chemical names from sev-
eral chemical suppliers, mcludmg
a 5-Amino-2,3-drhydro-1,4-phthalazinedione (Sigma [St Louis, MO], cat no.
A 851 IP).
b 3-Ammophthalhydrazide (Aldrich cat. no 12,307-2).
There is considerable batch to batch variability in commercial lummol. The
purity of the original material IS only tmportant m determining how many recrys-
tallization steps are required. Recrystallization gives a consistently high activity
product and may be performed as follows (for further details, see ref 7):
a Dissolve lummol in 5% (w/v) sodium hydroxide at room temperature until
close to saturation (about 200 g/L). The color of this solution will depend on
the original lummol. Cool the solution in an ice bath and allow to crystallize
for 4 h. Recover the sodium lummol crystals using suction filtration on a
glass fiber filter disk (Whatman GFA or similar), and wash using a small
volume of ice-cold 5% sodium hydroxide The crystals should be white or
only slightly discolored. Sodium luminol should be recrystallized at least
twice after a white product is obtained
b. Dissolve the sodium lummol in a minimum volume of 5% sodium hydroxide
at room temperature Allow to crystallize on an ice bath for 18 h A refrigera-
tor can be used as an alternative, although crystallization may be slower and
yield may be low Recover the crystals by filtration as above
The initial crystalline form is the hexa-hydrate, which converts to an anhydrous
powder on drying over silica gel. Sodium lummol is stable to heat (meltmg point
>400°C), but undergoes photochemical reactions and should be stored in the dark
Sodrum lummol has recently become available fro
he author has no experience with this product
Thorpe, G H. G and Kricka, L. J. (1986) Enhanced chemilummescent reactions
catalyzed by horseradish peroxtdase. Methods Enzymol. 133,33 l-354.
Kricka, L. J., Stott, R A W., and Thorpe, G. H. G (1991) Enhanced chemilumi-
nescent detection of horseradish peroxtdase labels m hgand binder assays, m
Lumtnescence Techniques tn Chemical and Biochemtcal Analysis (Bayens, W R.
G., De Kekelene, D , and Korkidis, IS , eds.), Dekker, New York, pp 599635
Thorpe, G. H. G , Stott, R A. W., Sankolh, G. M , Catty, D , Raykundaha, C.,
Roda, A and Kricka, L J (1987) Solid supports and photodetectors m enhanced
chemiluminescent immunoassays, in Btolumtnescence and Chemtlumtnes-
cence New Perspectives (Scholmerich, J., Andreesen, R., Kapp, M., and Woods,
W. G., eds.), Wiley, Chichester, UK, pp. 209-213.
Matthews, J A., Batki, A., Hynds, C., and Kricka, L. J. (1985) Enhanced chemi-
luminescent method for the detection of DNA dot-hybridization assays. Analytt-
cal Biochem 151,205-209
Thorpe, G H. G and Kricka, L. J. (1986) Enhanced chemilummescent reactions
catalyzed by horseradish peroxtdase. Methods Enzymol. 133,33 l-354.
Kricka, L. J., Stott, R A W., and Thorpe, G. H. G (1991) Enhanced chemilumi-
nescent detection of horseradish peroxtdase labels m hgand binder assays, m
Lumtnescence Techniques tn Chemical and Biochemtcal Analysis (Bayens, W R.
G., De Kekelene, D , and Korkidis, IS , eds.), Dekker, New York, pp 599635
Thorpe, G. H. G , Stott, R A. W., Sankolh, G. M , Catty, D , Raykundaha, C.,
Roda, A and Kricka, L J (1987) Solid supports and photodetectors m enhanced
chemiluminescent immunoassays, in Btolumtnescence and Chemtlumtnes-
cence New Perspectives (Scholmerich, J., Andreesen, R., Kapp, M., and Woods,
W. G., eds.), Wiley, Chichester, UK, pp. 209-213.
Matthews, J A., Batki, A., Hynds, C., and Kricka, L. J. (1985) Enhanced chemi-
luminescent method for the detection of DNA dot-hybridization assays. Analytt-
cal Biochem 151,205-209
Так! Прошу извинить за ошибки распознавалки. Текст изобилует очепятками.
Если возникнут конкретные вопросы по концентрациям из-за опечаток спрашивайте, уточню.
Так же прошу обратить внимание, что данные субстраты ииспользуются для определения концентрации фермента пероксидазы, а не перекиси водорода. Те в данном случае фермент находится в насыщенном субстратами(субстраты в избытке, фермент в недостатке) состоянии. Далее: измерение фактически производятся по интегралу выделевшися во время реакции света. В вашем же случае фермент должен быть в избытке, а перекись в недостатке и измерение концентрации нужно вести по изменению интенсивности испускания во времени или по интенсивности через строго опредленное время после добавления перекиси в реакцию. это так мои мысли вслух.
), потом помещает в стеклянную кювету, и потом уже я помещаю кювету в хемилюминометр, запускаю запись сигнала, затем прикапываю в кювету пероксид и наблюдаю изменение кинетики ХЛ.
)