J BlOLUMlN CHEMILUMIN 1995; 10: 35-40

Enhanced C hemi luminescent Sandwich Enzyme lmmunoassay for Hen Egg Lysozyme

Pave1 Rauch, Martin Poplstein, lgor Hochel and Ladislav Fukal Department of Biochemistry and Microbiology, Institute of Chemical Technology, 166 28 Prague, Czech Republic

Elida Ferri, Carlo Alberto Abagnato and Stefan0 Girotti” Institute of Chemical Sciences, University of Bologna, Via San Donato 15, 40127 Bologna, Italy

Aldo Roda Department of Pharmaceutical Sciences, University of Bologna, Bologna, Italy

A sensitive chemiluminescent sandwich-type enzyme immunoassay for hen egg lyso- zyme was developed. The assay was performed on polystyrene microtitre plates using immobilized specific polyclonal rabbit antibody against lysozyme, a peroxidase conju- gate and the H202/luminol-enhanced chemiluminescence detection reagent. The che- miluminescent signal was detected using either a microplate luminometer, or photographic f i lm in a camera luminometer. The detection l imit for lysozyme was 0.3ng/mL, and this was three times lower than that obtained using a colorimetric method w i th H202 and o-phenylendiamine as substrates. Recovery o f the assay was 97-112% and the relative standard deviation ranged from 3.6% t o 10.3%. The immu- noassay overcame interference from the food sample matrix when lysozyme, used as a bacteriostatic agent, was measured.

Keywords: lysozyme; chemiluminescence; enzyme immunoassay; food samples

INTRODU CTlON

Lysozyme (EC 3.2.1.17) occurring in hens’ eggs is a hydrolytic enzyme which splits the mucopoly- saccharides of bacterial cell walls. This effect can be utilized in the preservation of foods, mainly those that are heat labile, during so-called ‘cold sterilization’. Lysozyme is used, for instance, in the preservation of spices, sausages, unpasteurized milk, fresh fruits and vegetables (1). The addition of lysozyme to prevent the unwanted butyric acid fermentation in cheese (2) is the most important technological application nowadays.

* Author for correspondence.

The determination of lysozyme, present as a bac- teriostatic agent at concentrations ranging from 0.05% to 0.3% (w/w), requires very sensitive assays. Moreover, a high sensitivity is needed to overcome the interference problems frequently encountered in this assay, owing to the presence of several interfering substances in food samples.

Several methods have been developed, based on the catalytic activity of this enzyme (3), chromato- graphic procedures (4), or immunoassay (5). Che- miluminescent detection methods allow the development of sensitive assays (6) , mainly if lumi- no1 with suitable enhancers is applied to enzyme immunoassays which use peroxidase as label (7). Here, we report the development and applications

CCC 0884-3996/95/010035-06 0 1995 by John Wiley & Sons, Ltd.

Received 10 December 1993 Revised 21 February 1994

36 P. RAUCH ET AL.

of an enhanced chemiluminescent immunoassay of lysozyme. It is compared with a conventional colorimetric enzyme immunoassay in terms of analytical performance and applicability to the real food matrix (5).

buffer, were pipetted into appropriate wells of the polystyrene microplates. The immobilization of antibody on the surface of the polystyrene wells occurred overnight at 4°C. The unbound IgG frac- tion was then removed by aspiration and the wells washed three times with the 50 mmol/L carbonate

MATERIALS AND METHODS

Materials

Egg-white lysozyme (EC 3.2.1.17) was supplied by Fluka (Switzerland), horseradish peroxidase (EC 1.11.1.7, HRP), RZ 3, was supplied by Boehringer Mannheim, (Germany). Enhanced chemilumines- cent immunoassay signal reagent (ECL) from Amersham Italia (Milan, Italy) was used for chemi- luminescent detection.

The other chemicals, all of analytical reagent grade, were purchased from Lachema (Prague, Czech Republic) and Carlo Erba (Milan, Italy).

Transparent polystyrene microplates for the enzyme immunoassay (Nunc Immunoplate Maxi- sorp), used both for colorimetric and for chemilu- minescent detection on Amerlite TM Analvser

buffer, pH 9.6. 100 pL of sample or standard solu- tions of lysozyme were pipetted into the wells together with human serum albumin (HSA) to decrease the non-specific binding (1 % final concen- tration) and incubated for 2 h at 37°C. The well contents were removed by aspiration, and the wells washed three times with 10 mmol/L phosphate buffer containing 0.8% (w/v) NaCl and 0.05% (v/v) Tween 20, pH 7.4 (PBS solution). The IgG- peroxidase conjugate (100 pL), diluted 1 : 1000 with PBS solution containing 1% (w/v) HSA was then added to the wells and incubated for 2 h at 37°C. The unbound conjugate was removed by aspiration. Wells were washed three times with PBS-1% (w/v) HSA solution and the bound HRP labelled antibody detected either by chemilumines- cent or by colorimetric assay procedure.

luminometer, were from Nunclon (Denmarki In the case of chemiluminescent detection we pre- Chemiluminescent detection pared a proper aluminium grid, fitting perfectly to the microplate wells, to minimize carryover from well to well. The camera luminometer, Microlite, and the suitable microplates were from Dynatech (Billingshurst, UK). Instant black and white films (667) with 3.000 ASA sensitivity were obtained from Polaroid (Milan, Italy). All food samples were obtained from a local market.

Antibody and labelling procedure

The procedure for the preparation of the rabbit antibody against lysozyme and the isolation of IgG fraction from the immune serum has been described elsewhere (8). The same IgG fraction was used to coat the microplates and to prepare the peroxidase conjugate. The IgG-peroxidase conjugate (protein content, 1.4 mg/mL; peroxi- dase:IgG ratio, 1.8) was synthesized according to Wilson and Nakane (9).

A freshly prepared ECL reagent (1OOpL) was added to each well using a multiple pipette, and the light emission immediately measured in the Amerlite luminometer, over a 30-s time interval for the whole microplate, and expressed in relative luminescent units (RLU).

For photographic detection 1OOpL of a freshly prepared ECL reagent was added to each well of the microplate. The microplate was inserted into the grid, and the light emission recording in the camera1 luminometer, on black and white film, started by immediately removing the metallic slide dividing the microplate from the Polaroid film. For each microplate successive exposure periods of 30 s, 1, 2, and 5 minutes were performed, without an interval between successive exposures, because the light emission is constant for at least 30min. The film pack arrangement permits removal of the exposed film and initiation of the new exposure, without removing the microplate from camera luminometer.

lmmu noassay procedure Colorimetric detection

100 pL of the antibody (2.4 pg/mL IgG fraction) in the 50mmol/L carbonate buffer, pH 9.6, coating 0.1, mL of a freshly prepared colorimetric substrate

CH EM ILU M I N ESCENT LYSOZYM E I M MU NOASSAY 37

reaction was stopped after 30 minutes incubation at 37°C by the addition of 50pL of 2mol/L $ 'O0-' / sulphuric acid (5 ) . The absorbance of the reaction 500-- /

Labsystems (Helsinki, Finland), at 492 nm.

o /O-Q _ _ 0 -- 0

0 /"

l o o c ~ - o ~ O 0

mixture was measured directly in the wells 400-

using a microplate reader Multiscan MCC 340, 300-

/Q 0

.- 5 200.- - .- 0 ;

E

8

(T, 2.0 (\1

1.6

1.2 $

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0.8 u _ _ 0.4 0

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With liquid samples (apple juice, salad dressing, white wine and light beer) only the pH was adjusted to between 6-8 with 66 mmol/L glycine- NaOH buffer pH 10.0. Cheese was gently grated and a sample (1 g) was suspended in lOmL of PBS buffer solution. After mixing for 30 minutes at 20°C, the suspension was filtered using chroma- tographic paper (Whatman No. 1). If necessary, the pH of the filtrate was adjusted to between 6 and 8. This extract, further diluted in PBS, was directly analysed using the immunoassay.

RESULTS AND DISCUSSION

We previously developed a colorimetric sandwich enzyme immunoassay for hen egg lysozyme (5 ) , but the detection limit was inadequate to accu- rately evaluate hen egg lysozyme in food. An immunoassay using a peroxidase-IgG conju ate is simpler than procedures using other labels ( I or streptavidin-biotin), but does not have the low- est detection limit (10). A low detection limit can be reached by the application of the streptavidin- biotin system (six times) based on IgG-biotin and a streptavidin-peroxidase conjugate (1 0), but this procedure is more complicated than enzyme immu- noassay using peroxidase with colorimetric detec- tion. In order to reach a lower detection limit in our immunoassay, and avoid the complications of the streptavidin-biotin system, we used a chemilu- minescent instead of a colorimetric substrate for peroxidase detection.

%5

Standard curves

The standard curves for the lysozyme immuno- assay using chemiluminescent and colorimetric substrates are shown in Fig. 1. The standard curve

Lysozyme (ng/mL)

Figure 1. Standard curves for hen egg lysozyme obtained using an enzyme immunoassay with a chemiluminescent or colorimetric endpoint

extended from 0.8 to 15ng/ml and from 0.2 to 12 ng/ml using colorimetric and chemilumino- metric detection, respectively. The chemilumines- cent immunoassay was three times more sensitive than the colorimetric assay (0.3 vs. 1.1 ng/mL; detection limit based on background + 3SD) (Table 1).

Although chemiluminescent detection using a camera is a semiquantitative method, it has a detection limit, based on a 2 min exposure time, about three times lower than the colorimetric assay (0.4 ng/mL and 1.1 ng/mL, respectively) (Fig. 2) .

Some wells, in the four parallel determinations (A to D), show differences in the recorded light intensity, although the detection limit and the over- all pattern are the same. These differences can be ascribed either to minor differences in the well coat- ing and conjugate saturation, or to specific features of the film, e.g., non-uniform sensitivity or non- uniform processing after exposure.

Assay performance

The accuracy of the chemiluminescent immuno- assay was verified by the method of standard addi- tion to food samples that had not been pretreated with lysozyme (Table 2). Lysozyme was added to different types of samples at final concentrations of 2 and 20ng/mL. The recovery varied between 97% and 112Y0, with the highest values for the salad dressing. Relative standard deviation ranged from 3.6% to 10.3%. Using the colorimetric detec-

3% P. RAUCH ET AL.

~ ~~

Table 1. Comparison of detection limits of hen egg lysozyme enzyme immu- noassays based on luminometric and colorimetric detection of peroxidase

Substrate Non -spec if ic bindingfSDa of the assay

Detection limit

(RLU or AU) (RLu or AU) (ng/mL)

Luminol 1043112 125f4 0.3 o-Phenylendiamine 0.1 42 =t 0.01 8 0.1 70 3I 0.007 1.1

a Standard deviation. RLU: relative luminescence units. AU: absorbance units.

tion system we obtained 93-104% recovery (9, showing that both results are in good agreement.

Interference of food matrix

Interfering substances, frequently found in food samples, especially solids, may cause significant inaccuracy ( 1 1). Undiluted extracts of cheeses (Edam, Emmental), not treated with lysozyme, caused interference in the described immunoassay and can produce false positive results. This matrix effect can be reduced by sample dilution. Fig. 3 c!early shows that only diluted cheese extracts have lower luminescent emission or absorbance values than the respective detection limits (hori- zontal lines). For the colorimetric assay an analyti- cal signal statistically different from noise was observed at an Emmental and Edam dilution of 1 : 8 and 1 : 16 v/v, respectively. In contrast, for

the chemiluminescent immunoassay the Edam extract only interfered (Fig. 3) at dilutions less than 1 : 2, while the Emmental extract did not inter- fere at all, even if directly analysed.

The dilution of the cheese extracts easily over- comes the matrix interference problem in both assays. Moreover, dilution is a very effective solu- tion, because it does not involve significant extra time, cost, or clean-up procedures. However, a drawback to the universal application of this method is the high detection limit of the available colorimetric assays. The limitation of colorimetric as opposed to chemiluminescent detection is a high matrix interference associated with poor detectability which impairs the overall perfor- mance of the assay.

The enhanced chemiluminescence enzyme immunoassay is less affected by matrix interfer- ences. The use of a camera luminometer, which does not require electricity, means this assay can

A B C

1 2 3 4 5 6 7 8 9 Figure 2. Enhanced chemiluminescent immunoassay of hen egg lysozyme detected using a camera luminometer. A, B, C, and D parallel determinations: 1 blank, 2-9 different concentrations of lysozyme (0.2; 0.4; 0.8; 1.6; 3.2; 6.4; 12.8; 25.6 ng/mL)

CH EM1 LUMlN ESCENT LYSOZY ME I M MU NOASSAY 39

Table 2. Analytical recovery of hen egg lysozyme added to food samples using the chemiluminescent immunoassay

Sample Added Found RSDa Recovery (N = 6) (ng/mL) (ng /mL fSD) (%I (%)

Apple juice 2 20

Light beer 2 20

Salad dressing 2 20

White wine 2 20

a Relative standard deviation.

2.1 0 f 0.1 2 19.8 i 1.6 2.04 5 0.1 0 20.6 i 0.8 2.24 * 0.1 8 21.4 * 2.2 1.96 f 0.1 1 19.4 f 0.7

5.7 8.0 4.9 3.9 8.0

10.3 5.6 3.6

105 99

102 103 112 107 98 97

also be performed as a field test. Another advan- tage is that immediately after addition of the chemiluminescent substrate the light emission can be measured repeatedly as the light signal is stable for at least 60 minutes. In the case of colorimetric detection it is necessary to wait for colour develop- ment (20-40 min), stop the reaction, and then mea- sure the absorbance. The sandwich enzyme immunoassay with peroxidase as a label, com- bined with the chemiluminescent detection of the enzyme, is the method of choice, because it is simple, quick, sensitive, and can be applied to different food samples.

Acknowledgement

This work was partly supported by a grant from the Consiglio Nazionale delle Ricerche N. AI9300198.04.

5 150f n I

0 E d a m ex t rac t ESTl E m m e n t h a l ex t rac t

Detect ion limit n 3

n !’” 1.6 5 2 (u V

T!

1.2

0.8 6 +?

0.4 o v) n

0.0 Q. L -

O 1:16 1:8 1:4 1:21:1.3 E 1:16 1:8 1:4 1:21:1.3 E

Dilution ratio

Figure 3. Interference of lysozyme-free cheese extracts a t various dilutions in chemiluminescent and colorimetric lyso- zyme immunoassays; E: undiluted extracts

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