Analytica ChimicaActa, 266 (1992) 193-199

193

Elsevier Science Publishers B V, Amsterdam

Rapid enhanced chemiluminescent enzyme immunoassay

for ferritin monitored using instant photographic film

Angela O'Toole

Department of Clinical Endocrinology, Women's Hospita4 Birmingham (UK)

Larry J Kncka

Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, PA 19104 (USA)

Gary H G Thorpe and Thomas P Whitehead

Wolfson Applied Technology Laboratory, Wolfson Research Laboratories, Queen Elizabeth Medical Centre, Edgbaston,

Birmingham B1S 2TH (UK)

(Received 14th January 1992, revised manuscript received 24th February 1992)

Abstract

A rapid chemiluminescent enzyme immunometnc assay (EIA) for serum femtin has been developed which

combines a halophenol-enhanced chemilummescent assay for the peroxidase label with photographic detection

(20000 ASA instant film) of light emission Assays were performed using a microtitre plate as the solid support and a

specially designed camera lummometer to measure light emission from individual antibody coated wells The assay

operates as a threshold test above a certain serum femtm concentration the film was fully or partially exposed whilst

below this level the film remained unexposed The effect of incubation time and temperature were investigated and

two rapid mimunoassays were developed In one, a 10-mm incubation with sample and with conjugate detected 1 ng

(10 g 1 -1 ) of ferntm, whilst in the other, 3-mm incubations at ambient temperature detected 40 ng (400 lAg I- ') of

femtin These assays were reliable, produced a permanent photographic record of the results and the fastest assay

was completed in less than 10 min

Keywords Chenulummescence, Enzymatic methods, Immunoassay, Camera lummometer, Ferntin, Serum femtm

The chemilummescent assay for horseradish

peromdase based on the 6-hydroxybenzothiazole

or halophenol-enhanced lummol-peroxide reac-

tion is both rapid and sensitive and can be used

as a replacement for the conventional colonmet-

nc assays for peroxidase labels [1-5]

Correspondence to L J Kncka, Department of Pathology and

Laboratory Medicine, Hospital of the University of Pennsylva-

nia, William Pepper Laboratory, 3400 Spruce Street, Philadel-

phia, PA 19104-4283 (USA)

0003-2670/92/$05 00 0 1992 - Elsevier Science Publishers B V All rights reserved

An advantage of an enhanced chemilummes-

cent assay for peroxidase is that it is rapid (1 min

as compared to typically, between 15 and 30 nun

for a colorimetric assay) thus the tune taken to

quantitate peroxidase labels is trivial in compari-

son with incubation times with sample and with

conjugate m a typical enzyme unmunoassay Since

it is unlikely that a significantly faster chemilumi-

nescent peroxidase assay will be developed, any

further reduction in the tune to complete a

chemilummescent enzyme immunoassay can only

194

be achieved by reducing incubation tunes For

sandwich immunoassays such reductions are pos-

sible but have not been extensively investigated

For example, Yolken and Leister [6] showed that

in an assay for H influenza type b polysaccharide

it was possible to reduce incubation times with

sample and with conjugate to as little as 10 min

This assay was, however, less sensitive (by a fac-

tor of 10) than an assay using 2-h incubations It

was also possible to eliminate one of the mcuba-

tions by combining the sample and conjugate in a

single 10-min incubation

A further advantage of the enhanced chemilu-

mmescent peroxidase assay is that the magnitude

of the light emission is sufficiently high to make

possible its detection using photographic film

The combination of chemiluminescent assays

and photographic detection of light has formed

the basis of some very simple and rapid assays for

analytes such as ATP, NADH and primary bile

acids [7-9] These operate as threshold tests

above a certain analyte concentration the film is

exposed whilst below this concentration it re-

mains unexposed

The major objective of this study was to com-

bine together the enhanced chemilummescent as-

say for peroxidase, an enzyme immunoassay, and

photographic detection of light emission to pro-

duce a rapid and simple chemilummescent en-

zyme immunoassay which would operate as a

threshold test An enzyme immunoassay for serum

ferritin was chosen for study since diagnostically

it is important to know if a patient has levels

either lower than 10 g 1 -1 (iron deficiency

anaemia) or higher than 400 g 1 -1 (iron over-

load) Thus two immunoassays were constructed

in one the threshold was set such that the film

was only exposed when specimens with ferritin

concentrations greater than 10 Ag 1 -1 were as-

sayed, whilst in the other assay the threshold for

exposure of the film was set at 400 g 1 -1

p-Iodophenol was chosen as the enhancer since it

is particularly effective in enhancing the light

emission from the lummol-peroxide-peroxidase

reaction Initially assay conditions were optimised

using a conventional lummometer which used a

photomultiplier tube to detect light emission The

assays were then transferred to a camera lumi-

A O'Toole et al /Anal Chun Acta 266 (1992) 193-199

nometer which used instant photographic film as

the light detector [9-11]

EXPERIMENTAL

Hydrogen peroxide (30%, w/v) was purchased

from Sigma (Poole) p-Iodophenol was obtained

from Aldrich (Gillingham) An antiferritin-per-

oxidase conjugate was supplied by Dako Im-

munoglobulms (Copenhagen)

Luminol

This was purified and converted to its sodium

salt by recrystalization from hot sodium hydrox-

ide (1 M) and decolorization with charcoal [12]

Measurement of light emission

Photomultiplter tube A lummometer designed

and built in the laboratory based on a side-window

photomultiplier tube (EMI Type 9781A, 94 AA

lumen') having facilities for automatic injection

of reagents into reaction cuvettes was used to

measure light emission [13]

Photographic film Polaroid Land film Type

612 (ASA 20000) (Polaroid, Cambridge, MA)

served as the light detector The camera lumi-

nometer consists of a light-tight box mounted on

top of a Polaroid film camera back Inside the

light-tight compartment is a mask into which fits

a 10 x 6 well piece of a microtitre plate This

serves to isolate light emission from individual

wells of the microtitre plate The mask is sepa-

rated from the Polaroid film by a shutter With-

drawal of the shutter allows the mask to drop

onto the film thereby producing a contact print of

glowing solutions contained in individual wells of

the microtitre plate housed in the mask At the

end of the exposure period the shutter is in-

serted, thus terminating the exposure of the film,

and the instant film is developed Degree of

exposure of the film is proportional to the inten-

sity of the light emission

Solid support

Polyvinylchlonde (PVC) M24 microtitre plates

(Dynatech, Billmghurst) were coated with the IgG

fraction of anti-human ferntin (Dako) by adding

A O'Took et aL /Anal Chum. Acta 266 (1992) 193-199

100 l of a solution of the antibody (10 g 1 - ')

diluted 1 m 2000 with carbonate-bicarbonate

buffer (0 05 M, pH 9 5) to each well and mcubat-

ing the plate at 4C overnight

Horseradish peroxidase assay

Colonmetnc end-point The substrate solution

comprised a mixture of hydrogen peroxide (25

mM) and 2,2'-azino-di-(3-ethylbenzthiazoline

sulphonate) diammomum salt (ABTS, Boeh-

ringer, Mannheim) 3 0 mM in sodium acetate

buffer (0 1 M, pH 4 5) containing sodium dihy-

drogen phosphate (0 05 M) Substrate solution

(250 l) was added at timed intervals to each well

and colour allowed to develop for 0 5 h A sample

(200 l) of solution from each well was diluted

with 1 ml of PBS [phosphate buffer (0 015 M, pH

7 2)-sodium chloride (0 15 M)] and the ab-

sorbance measured at 415 rim

Enhanced chemilummescent end-point The

substrate solution comprised sodium lummol (25

mg) in 50 ml Tris buffer (0 1 M, pH 8 5) contain-

ing 31 l hydrogen peroxide (30%, w/v) and

p-iodophenol (13 6, 27 2, or 40 9 M) in dimethyl

sulphoxide (DMSO) Substrate solution (200 l)

was added to each well and the light emission

recorded using a luminometer

Ferntin standards These were prepared from a

sample of purified human spleen ferritin as de-

scribed previously [14]

EM for serum ferntan Ferritin standards (100

l) or serum samples (100 l) were incubated in

the antibody coated wells for 1 h at room temper-

ature The wells were then emptied and washed 3

times using PBS-Tween (phosphate buffer 0 015

M, pH 7 2, 0 15 M sodium chloride containing 0 5

ml 1 - ' Tween 20) A 1 500 dilution of an antifer-

ritin-peroxidase conjugate (100 l) in PBS-

BSA-Tween (2 g 1 - ' BSA) was added to each

well and incubation continued for 1 h at room

temperature The wells were washed and dried as

described above Bound peroxidase activity was

then determined using either the colorimetric or

chemiluminescent assay procedure

In order to measure light emission using the

photomultiplier tube based lummometer, individ-

ual wells were cut from the plate and lodged in

the bottom of a plastic cuvette (Sarstedt, Leices-

195

ter) Substrate was then pipetted directly into the

well and the light emission measured

Modified chemdummescent EIAs for ferntm

Incubation tunes The effect of varying the

incubation period was studied by assaying ferntm

standards (0-100 g 1 - ') where the incubation

period with sample and with conjugate were re-

duced to 10, 5, 3 and 1 min Timing errors in the

assays with short incubations were minimized by

using a multi-pipette (8 channel Titertek Flow

Laboratories, Irvine) to dispense samples and

reagents

Incubation temperature The above series of

experiments were also carried out at 37C in

order to assess the influence of incubation tem-

perature on the assay

Enhancer concentration Total and peak light

emission in a chemiluminescent assay for peroxi-

dase depend upon the concentration of the en-

hancer (p-iodophenol) Thus the influence of dif-

ferent concentrations of p-iodophenol (13 6, 27 2,

40 9 M) in the substrate solution on the detec-

tion limit for ferritin was investigated

Kinetics of light emission The time taken to

reach peak light emission in the halophenol-en-

hanced chemilummescent peroxidase assay is de-

pendent on the peroxidase concentration Thus

the kinetics of light emission were studied by

assaying a series of ferritin standards (0-100 g

1 - ') and monitoring light emission for 5-10 min

Precision studies Assessment of analytical im-

precision is problematical for assays which pro-

duce semi-quantitative results as in the case of

the luminescent EIA for ferritm with photo-

graphic detection of light emission Thus an esti-

mate of the reliability of the assay was deter-

mined by repeatedly analysing the series of fer-

ritin standards and scoring the results for each

standard (exposure of the film) on a detected/

undetected basis

Clinical specimens A series of serum speci-

mens representing a range of ferntm concentra-

tions and which had been previously assayed us-

ing a Fernzyme kit (Abbott Laboratories, North

Chicago, IL) were reassayed using the chemilumi-

nescent EIA (photographic detection of light)

The ferritin standards used in the Fernzyme kit

196

and the standards prepared from human spleen

were cross-analysed in order to determine the

comparability of the ferntin values assigned to

the two sets of standards

RESULTS

Colonmetnc EIA for femtin

An EIA based on PVC MTPs as the solid

support was required since only these plates

would fit in the camera lumiometer Thus a

colonmetric EIA for ferntm based on polystyrene

MTPs described earlier [12] was adapted to PVC

plates Acceptable analytical performance was

achieved on plates coated with a 500-fold dilution

of antiferritin and two 1-h incubations at room

temperature with sample and conjugate respec-

tively This assay formed the basis of the rapid

chemiluminescent EIA described in the following

sections

Chemiluminescent EIA for ferntm using a pho-

tomultiplier tube to detect light emission

The feasibility of performing luminescent

EIA's with short incubation tunes was initially

investigated using a conventional lummometer to

detect light emission, since a photomultiplier tube

TABLE 1

Enhanced chemiluminescent enzyme immunoassay for serum femtin using a photomultipher tube to detect the light emission The

assays were performed at room temperature

a D = Detected (light emission twice the blank value), ND = not detected

A OToole et al. /Anal. Chum Acta 266 (1992) 193-199

is more sensitive than photographic film Only

standards with high femtin concentrations were

detected using shorter incubations (two 10-mm,

two 5-min, two 3-mm, or two 1-mm) and these

results are summarized in Table 1 Increasing the

concentration of p-iodophenol up to 40 9 M

increased peak light emission and hence the sen-

sitivity of the assay For example, using 27 2 M

p-iodophenol the 15 Ag 1 -1 ferritin standard was

detectable whilst using 409 M the 10 g 1 -1

standard was detectable above background [i e ,

the signal was twice the background (blank) light

emission] (Table 1) High concentrations of p-

iodophenol were not studied, since they are

known to inhibit light emission in a chemilumi-

nescent peroxidase assay [2] Thus in subsequent

studies using the camera lummometer a p-

iodophenol concentration of 40 9 M was used in

the peroxidase assay

The time taken to reach peak light emission

varied with femtin concentration The slowest

kinetics (time to peak light emission, ca 3 min)

were observed with the 10 g 1 -1 femtin stan-

dard Thus in subsequent studies using the cam-

era lummometer, the influence of a delay be-

tween the initiation of the luminescent reaction

and detection of light emission was investigated

Since this study was directed primarily towards

Ferritin

(g l- ')

Assay result

a

100 D D D D D D D D

50 D D D D D D D ND

30 D D D D D D D ND

20 D D D D D D ND ND

15 D D D D D D ND ND

10 D ND D D ND D ND ND

Incubation time (min)

Sample 60 10 10 10 5 5 3 1

Conjugate 60 10 10 10 5 5 3 1

p-Iodophenol (M) 272 136 272 409 272 409 272 272

A O'Toole et al /Anal Chun Acta 266 (1992) 193-199

the use of photographic film as a detector, the

influence of a delay on the sensitivity of the

chemilummescent femtin assay using a photo-

multiplier tube as detector was not studied

Chemiluminescent EM for ferntin using photo-

graphic film to detect light emission

The various steps in the assay are listed in

Table 2 and typical examples of results from a

luminescent EIA for ferritm carried out using the

camera lummometer are shown in Fig 1 The

blank, which consisted of p-iodophenol-lumi-

nol-peroxide, was not detectable The exposure

latitude of the film is such that generally, a graded

response was not seen and assessment of results

was on a detected (film exposed) undetected (film

unexposed) basis hence this assay worked as a

threshold test

Assay speed and sensitivity

The best combination of speed and sensitivity

was achieved in the assay using 10-mm mcuba-

tions with sample and with conjugate This assay

allowed detection of the 10 g 1 - i ferritin stand-

ard (1 ng in the well) and took ca 25 min to

complete (Table 3) Detection of higher concen-

trations of ferritm was not problematical and a

400-g 1 -i standard could be detected using 3-

min incubations with sample and conjugate at

ambient temperature (data not shown)

Precision

In a limited study of precision the assay was

found to be reliable, e g, in nine consecutive

TABLE 2

Flow chart for an enhanced chemilummescent EIA with pho-

tographic detection of light emission using a camera lumi-

nometer

(a)

197

Fig 1 Photographs of light emission from enhanced chemilu-

minescent EIAs for serum ferntin using a camera lummome-

ter (a) Placement of serum ferntin standards (g l -1 ) on the

microtiter plate Results are for successive 10-mm incubations

with the standard and conjugate, (b) no delay between initia-

tion and measurement of light emission, and (c) a 30-s delay

analyses of the ferritin standards (10-min incuba-

tions) all of the standards produced similar de-

grees of exposure of the photographic film Un-

like other assays a different detector (photo-

graphic film) is used for each of the series of

assays which comprise the precision study, thus

minor variations in film sensitivity will contribute

towards any imprecision observed

Temperature

The rate of antigen antibody reactions in-

creases with temperature, thus sensitivity in sand-

wich immunoassays can be improved by perform-

Step Time (nun)

Incubate with sample 1-30

Wash < 1

Incubate with conjugate 1-30

Wash < 1

Add chemiluminescent substrate < 1

Delay period 0-2

Expose film 025

Develop film 0 5

198

A O'Toole et al /Anal Chun. Acta 266 (1992) 193-199

TABLE 3

Enhanced chemilummescent enzyme immunoassay for serum femtin using a photographic film to detect the light emission The

assay was performed at room temperature and p-todophenol (40 9 AM) was used as the enhancer

a

D = Detected (film exposed), F = famt (film partially exposed), ND = not detected

mg the assay at elevated temperatures Increasing

the incubation temperature from room tempera-

ture to 37C improved the sensitivity of the assays

using very short incubation times (e g , 2 x 3 min)

(Table 4)

Clinical studies

In a batch of 31 specimens the rapid enhanced

chemilununescent EIA for serum ferritm (thresh-

old, 400 g 1 -1) correctly identified all specimens

with ferritin concentrations above 400 g 1 -1

TABLE 4

Effect of coating and incubation temperature on the perform-

ance of an enhanced chemilummescent enzyme immunoassay

for serum femtin p-Iodophenol (409 AM) was used as the

enhancer and instant photographic film was used to detect the

light emission The incubation times with sample and with

conjugate were each 3 min

a

For definitions see Table 3

Likewise the EIA with a threshold set at 10 g

1 -1 correctly identified all specimens with ferritin

concentrations above 10 g 1 -1 Initially, some

difficulties were encountered because of differ-

ences between the values for the ferntm stan-

dards used in the enhanced chemilununescent

EIA and in the commercial ferntm EIA used to

assay the specimens Cross-analysis of standards

however, established a conversion factor for com-

parison of results from the two assays

DISCUSSION

This study has established the enhanced

chemilummescent EIA for ferntm using instant

photographic film as a viable alternative to simi-

lar assays monitored colorimetrically The assay is

sensitive (10 g 1 -1 ), a permanent record of the

results is obtained and these can easily be read

visually In its present format the assay operates

as a threshold test and assay conditions can be

adjusted so that only samples above a certain

ferntin concentration will produce sufficient light

to expose the instant film The chemiluminescent

detection method is much quicker than conven-

tional colorimetric assays (less than 1 mm for

exposure and film development versus 15-30 min

for incubation and measurement of absorbance in

a colorimetric assay) and the total assay time can

Femtm (g I - t) Assay result

100 D D D D D D D D D D D D

50 D D D D D D D ND D D D ND

30 D D D D D D D ND D D ND ND

20 D D D DD D D ND ND D ND ND

15 D D D D NDF D ND ND ND ND ND

10 D F D D ND ND F ND ND ND ND ND

Incubation time (mm)

Sample 60 10 10 10 5 5 5 3 3 3 3 1

Conjugate 60 10 10 10 5 5 5 3 3 3 3 1

Delay time (s) 0 0 30 120 0 30 120 0 0 0 120 0

Exposure time (s) 30 30 30 15 15 15 15 15 120 300 15 300

Femtin (g I - t) Assay result

a

100 D D D D

50 ND D D D

30 ND ND D D

20 ND ND F D

10 ND ND ND ND

Delay time (s) 0 120 0 120

Exposure time (s) 15 15 15 15

Coating temperature (C) 4 4 4 4

Incubation temperature (C) 20 20 37 37

A O'Toole et al /Anal Chum Acta 266 (1992) 193-199

be further reduced by shortening the incubation

times with sample and with conjugate This is,

however, only achieved at the expense of assay

sensitivity for example a photographic assay us-

mg two 10-min incubations will detect 1 ng of

ferntin whilst an assay using two 1-min incuba-

tions can only detect 10 ng of ferritm

In immunoextraction assays incubation times

are usually an order of magnitude longer than the

time taken to dispense samples or reagents This

ensures that, for large batches, the effect of any

discrepancies in timing between dispensing sam-

ple or reagents for the first and last assay tube

are minimal As incubation periods are short-

ened, the time taken to dispense reagents eventu-

ally becomes significant compared to the incuba-

tion time, and timing errors can only be mini-

mized by limiting the size of the batch of speci-

mens tested In this study only small batches of

specimens were tested and timing errors further

minimized by using an eight channel multiple

pipette It is however, possible to simultaneously

dispense reagents to all wells of a microtitre plate

using a 96-place multiple dispenser and this type

of device should facilitate the development of

rapid assays with large batches of samples

Instant photographic film has proved a very

convenient and sensitive detection method for

light emission in the enhanced chemiluminescent

EIA for ferritin Further improvements in sensi-

tivity would be obtained by thermo-kinetic en-

hancement of the immunological reaction and by

using a faster film, but to-date, 20 000 ASA is the

fastest instant film available An advantage of the

combination of an enhanced chemiluminescent

assay and photographic detection of light is that

the final step in the assay can be carried out in a

simple instrument (camera luminometer) which

requires no power supply This is a totally chemi-

cal assay in the peroxidase assay, chemical en-

ergy is transformed into light emission which is

then detected by the light-induced chemical reac-

tions m the photographic film emulsion These

advantages suggest that enhanced chemilummes-

cent EIAs monitored using instant film would be

ideal for rapid clinical laboratory and extra-

laboratory screening tests which do not require

high analytical sensitivity

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