Evaluation of two photometers used in enzyme linked immunosorbent assays

0095-1137/82/070168-06$02.00/0

Evaluation of Two Photometers Used in

Enzyme-Linked

Immunosorbent

Assays

V. M.GENTAtANDJ. H. BOWDRE*

Clinical Microbiology Laboratories, North Carolina MemorialHospital, University ofNorth CarolinaSchool

of

Medicine,

Chapel

Hill, North Carolina 27514

Received27August 1981/Accepted13April1982

The performance of two commercially available high-speed photometers, designed for through-the-plate reading, was evaluated. Linearity of instrumental reading and reproducibility of same-day and2-day measurementswere assessed by least-squares analysis and analysis of variance, respectively. For both instru-ments,the photometric error was on the order of thousandthsofan absorbance

unit andwasmuch smallerthan theerrorof thecurrentlyavailableenzyme-linked

immunosorbentassays.

High-speed photometers designed for

through-the-plate

reading

insure a

rapid

turn-over time for

measuring

colorimetric reactions

in microtrays.

Consequently,

the

quality

of

theseinstruments is an

important

consideration

in the adoption of

enzyme-linked

immunosor-bent assays(ELISA) and similarassays

by

labo-ratories with large numbers of

specimens.

Un-like conventional photometers, these

photometers use a vertical light path for

through-the-plate reading. Oneresultisthat the

length of the light path is notfixed by cuvette

thickness,sothat theabsorbance varies withthe

volumeof solutioncontained in each well. For a prototype ofone through-the-plate photometer (Titertek Multiskan; Flow Laboratories, Inc.,

McLean, Va.), Ruitenberg et al. (5) evaluated

intrarun imprecision and concluded that

photo-metricinaccuracy is probably minor in

compari-son with biological variability and dispensing

errorin theperformance of ELISA.We

expand-ed these studies and evaluated the linearity of

instrumental reading and the reproducibility of

measurementswithinthe samedayandbetween

2consecutive days fortwocommercially

avail-able photometers, Titertek Multiskan (Flow

Laboratories)andMicroelisaAutoreader

(Dyna-techLaboratories, Inc., Alexandria, Va.).

MATERIALS AND METHODS

Photometers. TheTitertek Multiskan was equipped with 405-, 414-, 450-, and 492-nm filters with a speci-fied half-band pass of 8 to 12 nm. We estimated the half-band pass of the 405-nm filter to be 10 nm. The Microelisa Autoreader MR580 was equipped with 410-, 455-, 490-, and 570-nm filters; the half-band pass

ofthesefilters is specified as 10 nm but could not be

t Present address: General Hospital Pathologists, Ltd., General Hospital ofVirginia Beach, Virginia Beach, VA 23454.

assessed byusbecause of thedesign of the instrument. Both photometers make an automatic background absorbance correction by means of a selected cup (Microelisa Autoreader)or aselected column of cups (TitertekMultiskan) filled with anappropriate buffer solution.

Testchromophoresolution. Aconventional chromo-genic solution commonly used in ELISA (8) was

prepared byincubatingtheenzymealkaline phospha-tase (from calfintestine, type VII; Sigma Chemical Co., St.Louis, Mo.) with its substrate, p-nitrophenyl-phosphatedisodium(Sigma104phosphatase substrate tablets) in diethanolamine buffer (pH 9.8) at room

temperaturefor 30 min. Thereactionwasthenstopped by the addition of 3 N NaOH. This chromophore solution was diluted with diethanolamine buffer to

produce test solutions in the range of 0.050to 1.400 absorbance units. The absorbanceof thetestsolution

at405 and 410nm wascheckedby repeated measure-mentswithacalibrated dual-beamspectrophotometer (Beckmanmodel 25; Beckman Instruments, Inc., Palo Alto, Calif.) before and after assays with each of the twophotometers.

Microdispenser. The test solution was dispensed into the cups ofmicroplates with a calibrated micropi-pette(Gilson Pipetman; Rainin Instrument Co. Inc., Woburn, Mass.). The coefficient of variation of this micropipettewaslessthan1%.

Microplates.Flat-bottomed polystyrene microplates (Linbro EIA microplates; Flow Laboratories) with 12 columnsof eight wells each were used.

Experimental designs. (i) Linearity of instrumental

reading. (a) For each microplate, the first column of cups wasfilled withbuffer, and columns 2 through 6 were filled with 200

RId

of test solution containing increasing amounts of chromophore. (b) For each microplate, the first column of cups was filled with buffer, and columns 2 through 6 were filled with 50, 100, 150, 200,and250,u1of a test solution containing a

constantconcentration of chromophore.

(ii) Effect of adding solvent to a determined amount of

chromophore.Toassess the effect ofdispensing error inaddingasolvent to a determined amount of chromo-phore, thefirst column of cupsof a microplate was

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EVALUATION OF TWO PHOTOMETERS IN ELISA

0.6[

E

C

It)

0 04

0.2

0.6I

E

C

0

0.41

0.2

0.4 0.8 1.2

A 41On (BECKMAN, MODEL 25)

FIG. 1. Comparison of the Multiskan and Mi-croelisa Autoreader with a dual-beam spectrophoto-meter.Abscissas: Absorbancesat405nm(A) and410 nm(B)determined withaBeckman model 25

spectro-photometer for 200 ,ulofsolutionscontaining

increas-ingamountsofchromophore. Ordinates: Absorbance of thesamesolutions determined at 405nm with the Multiskan(A) and 410nmwith the Microelisa

Auto-reader (B). (-) Least-squares regression lines; (0)

meansofeight independent measurements; (---)95%

predictionintervals.

filled with 50 Fl of buffer, and columns 2 through 5 werefilled with 50,ul of chromophore solution. Then tocolumns 3through5wereadded50,100,and150,ul

of buffersolution, respectively.

(iii) Intrarunimprecision. The intrarunimprecision

of instrumentalreadingswas assessedby performing eight repeated measurements of 200 ,ul of solutions with absorbancereadingsin therangeof 0.050to0.850 absorbance unit.

(iv) Imprecision intradayand betweendays.The first column ofamicroplatewasfilled with 200,ulof buffer solution. Columns 2through12werefilled with 200,u1

oftestsolution.Groupsofindependentmeasurements weretakenthesamedayorover2 consecutivedays.

To minimizeevaporation, theplateswere sealed and

keptinarefrigeratorat4°C.

Statistical analysis. Least-squares regression

analy-sis (3) and one- and two-way analyses of variance

(ANOVA) (3) were employed to study linearity and

reproducibilityof instrumentalmeasurements,

respec-tively. Data were analyzed by using the Statistical

Package for the Social Sciences (4) or the Minitab

Statistical Package (6), which were available to us

through the University of North Carolina and the

NorthCarolinaMemorial Hospitalcomputer systems.

The graphical display ofmeanscomparisonswas

con-structed by the method of Andrewsetal. (1).

RESULTS

Sinceboth of these photometersuse avertical light path for through-the-plate reading, the lin-earity of instrumental reading was verified by regression analysis of the absorbance curves generated by either increasing the amounts of chromophore in afixed volume oftest solution

orincreasing the volume oftestsolution contain-ing a fixed concentration of chromophore.

Fig-ure 1 shows the linearity of readings when increasing amounts of chromophorewere

pres-entin afixed volume oftestsolution. Readings

obtained with a dual-beam spectrophotometer

were used for comparison. For the Titertek Multiskan, the regression equation was y =

0.6

E

C

In)

0

0.41

0.21

0.41

E

C

0ci

0.2

50 100 150 200 VO LU ME (p

I1)

FIG. 2. Linearity ofreadings withincreasing vol-umesof the same chromophore solution. Abscissas: Volumes ofchromophoresolution. Ordinates: Absor-bancesdetermined at405 nmwith theMultiskan(A)

andat410nmwith the Microelisa Autoreader(B). (-)

Least-squares regression lines; (0) means of eight independentmeasurements;(---)95%prediction

inter-vals. A.MULTISKAN FLOW

yz0.002+0.542X/X

r2-0.999

¼~~~~~~~~~~~~~

A405nm

B.MICROELISA AUTOREADER DYNATECH

_ A,

9=O0.004+O0.584X

r2.0. 999 /

I,

/I

X

A.MULTISKAN FLOW 7

y 0.052+0.002X ---I r2 O0.999

I

B.MICROELISAI

AUTOREADER DYNATECH

A ,

y:0.002+0.002X

r2.0.999

I,,

- I

-I | I

B.MIREIS VOL. 16,1982

F

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TABLE 1. Effect ofdilutingaconstantamountofchromophore: ANOVA resultsfor theMicroelisa Autoreader andTitertek Multiskan

Photometer Source df _squares

Sum

of Mean_{of squares}

sum

Fratio Fprobability

Microelisa Autoreader Between 3 0.0001361 0.0000454 0.76 P = 0.526 Within 28 0.0016617 0.0000593

Titertek Multiskan Between 3 0.001839 0.000613 P = 0.029

Within 28 0.004916 0.000176 3.49

0.002+0.542x, and the coefficientof determina-tion, r2,was0.999. For the Microelisa Autoread-er, the regression equation was y = 0.004 +

0.584x, and r2was0.999. Thelarge coefficient of

determination indicates a strong linear relation-ship between readingswithadual-beam spectro-photometer and with each of the two

photo-meters.Itwasapparentbyvisualinspectionthat both regression lines were linear; thiswas

con-firmed by thetestfor lack offit(P < 0.05) (3). The dashed lines delimit the 95% prediction interval, whichwas narrowandwasabout 0.010

absorbance unit over the range of

measure-ments. These data indicate that the spread around the regression line was minimal, since for each level ofx,95% ofthe observed values of

yfell withinanintervalof 0.020 absorbance unit. Thelinearity of instrumental readingwasalso checked against volumetric increments of the

same chromophore solution (Fig. 2). For both

photometers, least-squares analysis generated straight regression lines with narrow 95%

pre-diction intervals of about 0.015 absorbance unit and r2 = 0.999. These results confirmed the

linearity of instrumental readings and indicated that,asexpected, these photometers differ from

conventionalphotometers in that the volumetric

errorin dispensing a chromophore solution

af-fects the overallerror.Theslopes of both

regres-sion linesindicate thatavolumetric increment of

1 ,ul determined a photometric increment of

0.002absorbance unit.

Conversely, for a through-the-plate-reading

photometer, the volume in which a given

amount of chromophore is diluted should not

affect themean absorbance measurements. We verified this hypothesis under extreme condi-tions. To four columns of eightwells ofa

micro-plate containing 50,ul of chromophore in dieth-anolamine bufferweadded 50, 100,or150

RI

of

thesamebufferor nobufferatall. The hypothe-sisofequality ofmean absorbancesamong col-umns was then tested with one-way ANOVA. The ANOVA summary tables forthe Titertek

MultiskanandMicroelisaAutoreaderareshown in Table 1. For both photometers, the sums of

squares(estimates of the variance) between and

within groups (columns) were very small. For theTitertekMultiskan, the F ratio is 3.49 (P =

0.029). For the Microelisa Autoreader, the F

ratio is 0.76 (P = 0.526). The results indicated

thatdilution ofagivenamountofchromophore

in anonabsorbing solution did not significantly

affectthe mean absorbance measurements.

The imprecision of instrumental measure-ments was assessedby comparingthe means of groupsofmeasurements taken either in the same

run, in different runs the same day, or over 2

consecutive days. Table 2 shows the mean,

standard deviation, and coefficient of variation

(CV) forgroups ofmeasurementstaken from the

same microplate. The CV for bothinstruments

wasless than

12%,

evenfor the smallest

absor-bance value in therangestudied.TheCVvalues

obtained with the Microelisa Autoreader were

somewhat smaller than those with the Titertek

Multiskan.

The one-way ANOVA for the means of groupsof eightmeasurementstaken on the same

day with the Microelisa Autoreader did not

reveal statistically

significant

differences

be-tweenthesemeans(P=

0.468).

Similarly,

statis-tically significant differences were not found

betweenmeansofgroupsofmeasurements

per-formedover2consecutive

days (P

= 0.779).

Since the light beam for the Titertek

Multi-skan isdivided intoeight

independent

pathways,

TABLE 2. Intrarunimprecisionofphotometer

measurementsfromasingle microplate

Photometer Intrarun imprecision

group Mean SD cv

Microelisa Autoreader

1 0.052 0.006 11.5

2 0.106 0.004 3.8

3 0.217 0.002 0.9

4 0.430 0.002 0.5

5 0.844 0.007 0.8

Titertek Multiskan

1 0.061 0.007 11.5

2 0.108 0.010 9.2

3 0.210 0.008 3.8

4 0.413 0.008 1.9

5 0.805 0.025 3.1

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TABLE 3. TitertekMultiskan: imprecision of same-day results two-way ANOVA between columns and rows

(D

Source df Sum of Meansumof Fratio Fprobability

squares squares

Rows 7 0.00083909 0.00011987 13.26 P<0.0001

Columns 10 0.00017861 0.00001786 1.98 P =0.0491

Error 70 0.00063266 0.00000904

which are aligned with corresponding rows of

cups of a microplate, the imprecision was

as-sessed by simultaneouslytesting theequality of

mean measurements for columns and rows by two-way ANOVA (Table 3). There were no

statistically significant differences between

col-umns (P= 0.0491), but the differences between

rows(eightseparatelight paths) were

statistical-ly significant(P < 0.0001). This large F ratio is

explained by the very small mean sum of

squares error (0.00000904). This statistic is an

unbiased estimate of the variance of the entire

population

of

wells andwas one orderof

magni-tude smaller than the mean sum of squares

between rows (0.00011987). Thus, it was the

very precision of the measurements which

al-lowed asmallsystematic error betweenrows to

be detectedasstatistically significant. The

actu-al range of mean readings for rows was very narrow(0.557to 0.565 absorbance unit)(Fig. 3),

andtherewas partial overlapping of95%

confi-dence intervals (bars). Differences onthe order

0.567

-

0.564-E

C

0

0.61

0.558

{-A B C D E F G H

ROWS

FIG. 3. Imprecisionof the Multiskan readings be-tween rows (eight separate light paths). All wells contained 200 Fl of the same chromophore solution. Abscissa: Plate coordinates for the rows. Ordinate: Absorbances at 405 nm. The means of11

measure-mentsareindicatedasdots and their95% confidence intervals asbars.

of thousandths of an absorbance unit are of no practical significance in results of ELISA, in

which the inherent variability is much greater

(7).

The means of 11 groups of eight measure-ments taken on 2 consecutive days were

com-pared (Fig. 4). The differences withindayswere

minimal, and the 95% confidenceintervals(bars)

were largelyoverlapping. The absorbance

read-ings of day 2 were consistently greater than

those ofday1. Theone-wayANOVAgaveanF

ratio of 5.02 (P < 0.0001). However, again the

pooledstandard deviation was very small(0.004

absorbance unit), and the range of the means was narrow(0.550to0.561 absorbanceunit). In

this case also, very small differences were

de-tectable as statistically significant because the

overallvariabilityofrepeated readingswasvery

small. Differences ofthis magnitude in ELISA

results are of minimal practical significance.

Further studies todetermine the relative

impor-tanceof the various componentscontributingto

thesumofsquareerrorsforsame-dayand2-day

assays were not

performed. However,

the

re-sults of the dilution

experiment

indicated that

evaporation

of the solvent would have atrivial

effect.

i -I I 2

DAY

FIG. 4. ImprecisionoftheMultiskanover2days.

Allwells contained200 ,ul ofthe same chromophore solution. Abscissa: 22groups ofmeasurementstaken

on2consecutivedays. Ordinate: Absorbances deter-minedat405nm.Themeansof five

repeated

measure-ments are represented as dots and their95% confi-denceintervalsasbars.

VOL.16,1982

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0

0.5 0-0.4

Aw

sobc at44100 a -A455nm

wavelngthode o opertion)Well 455,aine 200

-j Y=0.0044-0.584X Y=0.006+0.450X

m r250999rsom O :

length, 410nm) or in thedual-wavelength mode(0.3E

003 0.

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FIG. 5. MicroelisaAutoreader, comparisonof

ab-sorbance at410nmwiththatat410 455nm (dual-wavelengthmodeofoperation). Wells contained 200

pd of solution with increasing amounts of

chromo-phore.Abscissa:Absorbancedeterminedwitha

Beck-man model25 spectrophotometer. Ordinates:

Absor-bance determined with the Microelisa Autoreader

operatingeither in thesingle-wavelengthmode (wave-length,410nm)or inthedual-wavelengthmode

(wave-lengths,410and 455 nm).

The Microelisa Autoreader, as a means of

correctingfor scratchesorotherartifacts onthe

microplates, cancomparereadings at two

wave-lengths selected from the four standard filters

supplied

with the instrument. One of these

wavelengths is selected to be near the

absorp-tionmaximum ofthechromophoreused,andthe other is selected to serve as a reference

wave-length. Ifthereference wavelength is absorbed

to some extent by the chromophore, the

dual-wavelength mode gives absorbance values whichare smaller than those measured with the

one-wavelengthmode. Figure5shows thiseffect

by comparing the regression lines obtained for

the same chromophore solutions at the

wave-length of 410 nm (this is the regression line shown inpB)Fig. and atthe wavelength of410 455 nm (dual-wavelength mode). The two

regression lines were significantly different by

visual inspection, and this difference was

con-firmed by the statistical test for equality of

regressionlines (P< 0.0001) (3).

DISCUSSION

These studies indicated that for these two

commercially available through-the-plate-read-ing photometers, linearity in measuring

absor-bance of solutions with increasing amounts of

chromophoreandsame-dayand2-day

reproduc-ibilitywere satisfactory.

Theimprecision of instrumental readingswas

verysmall,ontheorder of thousandths ofaunit

over arange of 0.050 to 1.400absorbanceunits.

Theseestimatesaresimilartothosereported by

Ruitenberg etal. (5). Asmight be expected, the

CV forboth instruments was smaller for

read-ings ofatleast0.2absorbance unit than formore

dilute solutions. Therefore, the CVcanbe

mini-mized by designing ELISAtests togive

absor-bance values greater than 0.2 for samples of

interest.

The Titertek Multiskan showed statistically

significant differences among mean absorbance

readings of the eight individual

light paths

and

between mean absorbance readings of the 2

consecutive days. However, these statistically

significant differences were very small (0.003

and 0.004 absorbance unit,respectively) and of

nopractical clinicalimportance for the available

ELISAtests, in which differences of 0.003

ab-sorbance unit are notcritical in

evaluating

test

results.

Forboth

through-the-plate-reading

photome-ters, there was a linear

relationship

between

volumes of chromophore solution and

absor-bance

readings,

and the

slope

was 0.002

absor-bance unitfor both the Titertek Multiskan and

the Microelisa Autoreader(Fig. 2). This clearly

indicates, as do the results ofRuitenberg et al.

(5), that volumetric inaccuracy in dispensing

chromophore is amajorsource oferror.

Thevolume in whichadeterminedamountof

chromophore

was diluted did not affect the

instrumental

readings.

Consequently, in ELISA,

volumetric errors inadding buffer and inhibitor

solutions do not affect the overall error of the

assay.

Toobtain comparable results of ELISA

per-formed in laboratories with different

through-the-plate-readingphotometers, the photometers

should be calibrated

against conventional

spec-trophotometers. Furthermore, for the

Mi-croelisa Autoreader, the performance of the

dual-wavelengthmodeshould becompared with

thatof theone-wavelength mode. Itisimportant

tonote thatreadings taken in these two modes

were not comparable, particularly for the larger amountsofchromophore (Fig. 5).

These studies were addressed to aspects of

the evaluation of two commercially available

through-the-plate-reading photometers. The

re-sults of this study confirm and extend those of

Ruitenbergetal. (5),who studied aspects of the

performance ofaprototype oftheTitertek

Mul-tiskan. For anexhaustive evaluation ofthe

per-formance ofspectrometers, thelisting of

specifi-cations proposed by the International

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EVALUATION OF TWO PHOTOMETERS USED IN ELISA 173

Federation of Clinical Chemistry should be

con-sulted (2).

LITERATURE CITED

1. Andrews, H. P., R. D. Snee, and M.H. Sarner. 1980. Graphical display ofmeans.Am.Stat. 34:195-199.

2. Haeckel, R., C.H.Collonbel, T. D.Geary, F. L. Mitchell, R.G. Nedeau,andK.Okuda. 1980. Provisional guidelines (1979) for listing specifications ofspectrometersin clinical chemistry. Clin. Chim. Acta 203:249-258.

3. Neter, J., and W. Wasserman. 1974. Applied linear statisti-calmodels. Richard D.Irwin, Inc., Homewood, Ill. 4. Nie, N. H., C. H.Hull, J. G.Jenkins, K.Steinbrenner,and

D. H. Bent. 1970. Statistical package for the social

sci-ences,2nd ed. McGraw-Hill Book Co., New York.

5. Rultenberg, E. J., V.M.Sekhuis, and B. J. M. Brosi. 1980. Some characteristics ofa newmultiple-channelphotometer forthrough-the-plate reading of microplatestobeused in enzyme-linked immunosorbentassay. J. Clin. Microbiol.

11:132-134.

6. Ryan, T.A., Jr., B. L. Joiner, and B. F. Ryan. 1976. Minitab:astudent handbook.Duxbury Press, North

Scitu-ate,Mass.

7. Vejtorp,M.1978.Enzyme-linked immunosorbentassayfor determination of rubellaIgG antibodies. Acta Pathol. Mi-crobiol.Scand. Sect. B 86:387-392.

8. Voller, A., D. Bidwell, and A. Bartlett. 1980. Enzyme-linked immunosorbent assay, p. 359-371. In N.R. Rose and H.Friedman (ed.), Manual of clinical immunology, 2nd ed. AmericanSociety forMicrobiology, Washington, D.C.

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