An enzyme-linked immunoassay (ELISA) for measurement of lactoferrin

Journal of Immunological Methods, 65 (1983) 183-190 Elsevier

183

JIM 02867

An Enzyme-Linked Immunoassay (ELISA) for Measurement of Lactoferrin * Seth V. H e t h e r i n g t o n 1, J o h n K. Spitznagel 2 a n d Paul G. Q u i e 1 i Department of Pediatrics, Division oflnfectious Diseases, University of Minnesota, Mayo Memorial Bldg., 420 Delaware ST, S.E., Minneapolis, M N 55455, and 2 E m o ~ University School of Medicine, Atlanta, GA, U.S.A. (Received 18 May 1983, accepted 6 July 1983)

An enzyme-linked immunosorbent assay has been developed for quantitation of lactoferrin (LF) in body fluids. An indirect double-sandwich method was used which allows a sensitivity of 3 ng L F / m l in samples of polymorphonuclear cell lysates and serum. Mean LF content of serum was 0.307+0.066 /Lg/ml (n = 18). Mean LF content of polymorphonuclear cells was 4.90+ 1.48/~g/106 PMN. Concentrations of LF were similar in serum and in plasma of EDTA anticoagulated blood. Advantages of this method include its rapidity, and radioactivity is not required. Key words: immunoassay - lactoferrin - polymorphonuclear cells

Introduction

Lactoferrin (LF) is an iron-binding protein found in breast milk, plasma, secretory granules of polymorphonuclear cells (PMNs) and secretions which bath mucosal surfaces. While precise function(s) is not known, there is evidence implicating its role in (1) chemotaxis of PMNs (Boxer et al., 1982), (2) oxidative metabolism of PMNs (Ambruso and Johnson, 1981) and (3) direct bacteriostatic and bactericidal activity (Bullen and Armstrong, 1979; 1980; Arnold et al., 1982). Rare individuals who possess lactoferrin-deficient PMNs are susceptible to pyogenic infections, underscoring the role of LF in normal host defenses (Breton-Gorius et al., 1980). Previous methods for measurement of LF are (1) single radial diffusion (Masson et al., 1969), (2) Laurell rocket electrophoresis (Tabak et al., 1978) and radioimmunoassay (Bennett and Mohla, 1976). The enzyme-linked immunoassay has many advantages over these methods. The sensitivity of the ELISA is comparable to that

* Supported by a grant from the Minnesota Medical Foundation. 0022-1759/83/$03.00 © 1983 Elsevier Science Publishers B.V.

184

of RIA methods, yet no radioactivity is required. The assay is economical and can be completed in a few hours using plates which have been sensitized overnight. Lactoferrin is particularly adaptable to ELISA measurement since a standard curve (optical density vs. lactoferrin concentration) can be generated to calculate the LF content of samples. Purified LF and all reagents for this assay are commercially available. An indirect double-sandwich method was developed for greater sensitivity since conjugate binding to antibody occurs at several sites (Yolken, 1982).

Materials and Methods

Ninety-six-well polystyrene plates were used for the assay ( N U N C Immunoplate I, Gibco Laboratories, Grand Island, N.Y.). Goat antiserum to human lactoferrin and rabbit anti-human lactoferrin were obtained from Nordic Immunologic Laboratories (El Toro, CA) and Cappel Laboratories (West Chester, PA), respectively. Goat anti-rabbit peroxidase-labeled immunoglobulin (IgG fraction) was also obtained from Cappel Laboratories. Lactoferrin (98% purity) obtained from Sigma Chemical Co. (St. Louis, MO) was suspended in phosphate-buffered saline, pH 7.4 (PBS) at a concentration of 1 mg/ml. Carbonate/bicarbonate buffer, 0.1 M, pH 9.6, was used as a binding buffer. All other reagents were diluted in 0.01 M PBS (0.5 M NaC1), pH 7.4 with 0.5% bovine serum albumin (BSA) added. The plate was washed between steps with a N U N C Immunowash plate washer using PBS with 0.5 M NaC1 and 0.05% Tween 20. Three washes each of 3 min were performed. All incubations were carried out at room temperature for 30 rain unless otherwise noted.

The binding antibody The IgG fraction from goat antiserum to human lactoferrin was prepared by the method of Steinbuck and Audran (1969). Briefly, 2 ml acetate buffer, 0.06 M, pH 4.0, was added to 1 ml of antiserum. Four drops of octanoic acid (Sigma Chemical Co., St. Louis, MO) was added while stirring. After 20 min, the solution was centrifuged, and the supernatant, containing the IgG fraction, filtered. The IgG fraction was then dialyzed against 1 liter of PBS overnight at 4°C. The parent antiserum and IgG fraction were compared for activity as binding antibodies. For each dilution of antiserum, equivalent activity by the IgG fraction was obtained at one less 2-fold dilution. This indicates little competition to IgG binding from other antisera proteins. More important, the background optical densities (OD in the absence of lactoferrin) were not different. Thus, non-specific binding of other ELISA sandwich components to goat serum proteins was insignificant. For all subsequent experiments, whole antiserum was used as the binding antibody, diluted 1:2000 as determined by chessboard dilutions. To each well was added 100 #1 of the diluted antiserum. The plate was incubated overnight at 4°C or for 2-3 h at room temperature.

185

Blocking non-specific binding A major problem of the ELISA method is non-specific binding of other components of the sandwich to the polystyrene plate or antibody-to-antibody interactions. For this assay, background was defined as the optical density measured with normal goat serum substituted for the binding antibody. BSA (4%), gelatin (1%), and poly-L-lysine (100 /~g/ml) were investigated as potential blocking reagents in a separate 1 h blocking step following adsorption of the binding antibody. None of these reagents lowered background OD significantly. The blocking step was, therefore, eliminated from the procedure. The addition of BSA to the diluent and plate washing as described above lowered background OD to an acceptable level (Bullock and Walls, 1977; Hendry and Mclntosh, 1982). Rabbit anti-human lactoferrin was added to the wells in 100/~1 vols. and incubated for 30 rain at room temperature. The optimal dilution of 1 : 4000 was determined by chessboard dilutions. Background OD measured in the absence of rabbit anti-LF were very low (less than 0.030), indicating low non-specific binding by the conjugate.

Conjugate Peroxidase-labeled goat anti-rabbit IgG was used at a dilution of 1 : 8000. The IgG fraction was compared to an affinity-purified IgG product by means of double reciprocal plots ( 1 / O D vs. 1/concentration) as per Masseyeff and Ferrua (1980). The resulting plots are straight lines, the slopes being inversely correlated with non-specific binding. The Cappel peroxidase-labeled IgG fraction gave a less steep slope.

The ELISA assay Goat antiserum to human LF was adsorbed to the polystyrene wells of the microtiter plate as described above. The plate was then washed 3 times. Serum dilutions of 1 : 16 or PMN lysate dilutions of 1 : 200 were added to the wells in 100 #1 aliquots. Following incubation, the plate was washed again. Rabbit anti-human lactoferrin antiserum at a dilution of 1 : 4000 was added next. After incubation and washing, 100 /~1 of diluted conjugate was added, and incubated. The plate was washed again and shaken vigorously to remove excess fluid. Orthophenylene diamine HC1 (Kodak Laboratories, Rochester, NY), 0.4 mg/ml, and H202, 0.025% were combined in fresh citrate buffer, 0.05 M, pH 5.0. Two hundred microliter aliquots were added to the wells and the reaction stopped after 30 min with 50 /tl of 2.5 N H2SO 4. The optical density was read on a Titertek Multiscan plate reader. For each ELISA plate, a standard curve was generated by plotting the optical density against concentration of purified lactoferrin added to wells (3-200 ng/ml; Fig. 1).

Purification of polymorphonuclear cells (PMNs) This was achieved by Dextran sedimentation for 1 h followed by centrifugation of the PMN-rich plasma over lymphocyte separation media (Litton Bionetics, Kensington, MD). Contaminating RBCs were lysed by the addition of 0.87% NH4C1 and the pellet washed twice in Hank's balanced salt solution with 0.1% gelatin. Five million

186

PMNs were pelleted by centrifugation at 800 rpm for 5 min, and LF extracted by the addition of 1 ml of PBS containing 0.5 M NaC1 and 0.1% Triton-X.

Results Measurement o f L F in serum

Serum samples collected from normal healthy donors were measured at serial dilutions of 1 : 4 to 1:64. Table I shows the results of several samples with the lactoferrin content calculated at each serum dilution. The close dilution-to-dilution values suggest that serum proteins do not interfere with the assay. For subsequent experiments, the 1 : 16 dilution was used to determine LF values in serum. To assess the method of collection, the LF of serum was compared to that measured in plasma obtained from E D T A and heparin anticoagulated blood. The results are shown in Fig. 2. For the six samples tested, mean serum LF measured 0.196 _+ 0.066, mean E D T A plasma LF 0.171 _+ 0.075, and mean heparin plasma LF 0.519 _+ 0.215/~g/ml. The effect of storage and separation of serum or plasma from whole blood collection was examined. Blood anticoagulated with heparin (1 U / m l ) and E D T A anticoagulated blood was collected from donors and samples stored at 4°C. LF levels determined in serum or plasma separated at times 0, 2, 4, 8 and 24 h were similar (data not shown). 1,0-

0.90.80.70.60.5->~ 0.4--

a

0.3--

0

0 0.2-

0

/ I 3.12

O

I 6.25

I 125

[ 25

I 50

I 100

I 200

Lactoferrin (ng/ml)

Fig. l. Standard curve for the lactoferrin ELISA. Optical density of the peroxidase reaction measured at 492 nm vs. concentration of purified lactoferrin added to the plate. For each ELISA plate run, a similar standard curve is generated.

187 TABLE I SERUM LACTOFERRIN CONTENT MEASURED AT SERUM DILUTIONS OF 1:4 TO 1:64 (t~g/ml SERUM) Sample

Serum dilution

1 2 3

1:4

1:8

1:16

1:32

1:64

0.699 0.188 0.368

0.740 0.155 0.333

0.770 0.169 0.340

0.617 0.185 0.357

0.640 0.288 0.467

Transferrin is an iron-binding protein in plasma with molecular weight and protein composition similar to those of lactoferrin. Immunologically, however, these two serum proteins are distinct. The serum concentration of transferrin (based on measured total iron-binding capacity of 300 mg F e / 1 0 0 ml) is 103-104 times that of lactoferrin, and so a small amount of cross-reactivity would alter the results of the ELISA. No precipitin lines were visible when goat and rabbit anti-human LF were tested for reactivity with transferrin in dilutions of 1 . 5 - 5 0 / z g / m l by Ouchterlony double diffusion. Transferrin in concentrations of 0.06-0.5 m g / m l was added to purified lactoferrin in concentrations of 3-200 n g / m l . Slight increases in optical density were apparent at transferrin concentrations greater than 0.125 m g / m l in the polystyrene well (Fig. 3). The effect of serum proteins on LF measurement was further explored by recovery experiments. Lactoferrin was added to three sera from normal donors at final concentrations of 25, 50 and 100 # g / m l and recovery was calculated as follows:

% Recovery =

LF measured in sample - LF in serum amount of LF added × 100

As shown in Table II representing the average of triplicate samples of three different serum samples, reproducible results were obtained. 0.9 0.6; 0.5 .E

0.4

0.5 o ,--I

0.2 0.1

l

•

-" !

I

I

Serum

EDTA

I Heparin

Fig. 2. Lactoferrin content of serum and plasma from EDTA or heparin anticoagulated blood.

188 1.00.90.80.70.6-

>~

o,5-

c

0.4-N

•

O 0.2 e~-o 0,1

--

3, 12

6.25

12.5

25

50

050 100

200

L a e t o f e r r i n (ng/ml)

Fig. 3. The effect of transferrin on the measurement of lactoferrin. Purified transferrin in concentrations of 0.125, 0.25 and 0.50 mg/ml was added to wells containing 3-200 ng/ml of lactoferrin. Optical density is plotted against LF concentration. Eighteen serum samples f r o m n o r m a l d o n o r s were m e a s u r e d in triplicate. T h e coefficient of variation a m o n g triplicate samples was less than 5% for 1 5 / 1 8 samples. Assay-to-assay variation, d e t e r m i n e d by m e a s u r i n g L F c o n t e n t of a single se r u m sample in d u p l ic a t e on 10 different occasions, was 13%. M e a n L F c o n t e n t of the 18 serum samples was 0.307 ___0.066 / ~ g / m l with 95% c o n f i d e n c e intervals of 0.2-0.4.

Measurement of L F in P M N s P M N extracts p r e p a r e d as described a b o v e were diluted 1 : 200 in PBS and 100 g l samples a d d e d to the wells. Extractions by freeze-thawing five times in a c e t o n e - d r y ice were also tested but gave inconsistent results. Th e final c o n c e n t r a t i o n of T r i t o n - X was 0.0005% and h a d no effect on optical density when a d d e d to s t a n d a r d L F

TABLE II RECOVERY EXPERIMENT. PERCENTAGE RECOVERY OF PURIFIED LACTOFERRIN ADDED TO THREE SERUM SAMPLES IN PARENTHESES Sample ~

1 2 3

Lactoferrin content measured (ng/ml) for each concentration of LF added (ng/ml) 0

25

50

100

9.4 7.4 48.4

30.2(83) 27.4(80) 75.9(110)

57.3(96) 50.9(87) 90.6(84)

95(86) 94(87) 135(87)

a Serum samples were added to the polystyrene wells at a 1 : 16 dilution.

189 TABLE III SERUM AND PLASMA LACTOFERRIN LEVELS REPORTED BY OTHER AUTHORS Author

Method

Sample

Lactoferrin (/t g/ml)

Bennett and Mohla (1976)

RIA

EDTA-plasma

Boxer et al. (1982) Hansen et al. (1976) Olofsson et al. (1977) Method described

RIA RIA RIA ELISA

EDTA-plasma EDTA-plasma Serum Serum

1.62 + 0.077 (male) 1.07 _+0.067 (female) 1.5 _+1.4 0.13-0.42 0.385 __+0.153 0.307 _ 0.066

concentrations. Measurements of LF content of PMNs from 8 healthy donors revealed a mean of 4.90 _+ 1.48/xg/106 PMNs. Since the ELISA utilizes a peroxidase-labeled antibody for antigen detection, th~ possibility of interference from endogenous myeloperoxidase was assessed. Whe/a plates were washed with methanol and H202 after incubation with P M N extract to inactivate any P M N peroxidase which might bind non-specifically to the plate, no change was found in the calculated LF content. Thus, endogenous peroxidase does not interfere with the assay.

Discussion The method described is a double antibody sandwich ELISA (Voller et al., 1980). It is an economical, rapid, and simple method for the measurement of lactoferrin in serum and P M N lysates. Intra-assay variation is generally less than 5% and inter-assay variation 13%. Little or no interference can be expected from serum transferrin or, in the case of P M N lysates, endogenous peroxidase. The small cross-reactivity seen with transferrin might have been decreased by solid phase absorption of the anti-human lactoferrin antisera with transferrin, unless both LF and transferrin are reacting with the same antibody. In any event, we have accepted this small error to preserve the simplicity of the assay. We did not find the time-dependent variation in serum LF described by Bennett and Mohla (1976). Furthermore, LF measurements of serum and E D T A anticoagulated blood did not differ significantly. Heparin anticoagulated blood, however, gave higher values, an effect also seen with radioimmunoassays. Serum lactoferrin from 18 normal donors measured 0.307 + 0.066/~g/ml. Comparative values from radioimmunoassays are shown in Table III. P M N lysates obtained from incubation with Triton-X had a lactoferrin content of 4.90 ___1.48 /ug/106 PMNs (n = 8). This agrees with values published by several other investigators (Masson et al., 1969; Leffel and Spitznagel, 1972; Boxer et al., 1982). Lactoferrin is a protein found in the secondary granules of PMNs. It has been implicated in the P M N functions of adherence, chemotaxis, antimicrobial killing, and oxidative metabolism (Bullen and Armstrong, 1979; Boxer et al., 1982; Gallin et

190

al., 1982). Serum levels probably reflect both the absolute neutrophil count as well as PMN secretory activity. Olofsson et al. (1977) found that serum LF varied with the PMN count in cyclic neutropenia. High serum levels are found during the initial phase of pneumonia (Hansen et al., 1976) and in chronic myelogenous leukemia (Bennett and Mohla, 1976). Synovial fluid LF is elevated in inflammatory joints and the lysozyme to LF ratio decreases with increasing neutrophilia (Bennett and Skosey, 1977). Measurement of LF may provide a marker for inflammation as well as evidence for lactoferrin deficiency in the patient with recurrent infections. Finally, since LF is found only in the specific granules (Leffel and Spitznagel, 1972; Pryzwansky et al., 1978), it is a useful marker for studies of the role of specific granules in PMN function (Wright and Gallin, 1979).

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