BJ Bentwood

G. Gunzer

H. Fritz

H. Lang

I Olsson

JL Snick Van

K Nemet

KH Duswald

M Baggiolini

Marianne Jochum

MS Leffell

NE Hansen

S Avrameas

S. Neumann

W. Rautenberg

English

Crossref

Quantifizierung von Human Lactoferrin als Entzündungsparameter mittels Enzymimmunoassay vom Sandwich-Typ (ELISA)

Rautenberg, W.

Neumann, S.

Gunzer, G.

Lang, H.

Jochum, Marianne

Fritz, Hans

Open Access LMU ( Ludwig-Maximilians-Univ. München)

Quantitation of human lactoferrin as an inflammation marker by an enzyme-linked immunosorbent assay (ELISA) W. Rautenberg 1, S. Neumann 1, G. Guozer l , H. Lang I , Marianne J ochum 1, and H. Fritz l I Biochemische Forschung E. Merck, Frankfurter Straße 250, 0-6100 Oannstadt I , Fecleral Republic ofGermany 1 Abteilung für Kliniscbe Chemie und Klinische Biocbemie, Chirurgische Klinik der Universität München, Nußbaumstraße 20, 0-8000 München 2, Federal Republic of Germany Quantifizierung , 'on Human Laclorerrin als Ent.tündungsparameter mittels Enzymlmrnunoassay vom Sand",·ich-Typ (ELISA) Human lactoferrin (Lac) is an iron·binding glycoprotein of Mt - 76,000 and is found especially in milk: and many other body fluids, such as tcars, saliva. nasal and bronchial secretions, urine, seminal fluid, and in pancreatic flu.id. Altbough ilie bio-logical significance of Lac is still undcr discussion there is cvj-dencc for its anribacterial activity in milk and blood. Olhcr biologieal roles of Lac include regulatory funetions during in-testinal iron absorption. in myelopoiesis, in neutrophil aggrega-tion and adherence and in inOammation [11. Lac appears lO be synlhesized mainly by polymorphonuelear leukocyles (PMN). 11 15 localized intraccllularly in the seoondary or "spccific" granulcs of these ceHs [2]. The ce[[ular comcnt of Lac is around 3 - 5 )1g/1 06 cells. Nonnally Lac occurs in plasma only in trace amounts. Ouring inflammation it is released from PMN with other lysosomal proteins [3, 4]. Measurement of I..ac may be useful because studies suggest that l.ae-<:oncentrations in blood might reflect the activation of the total blood granulocyte pool [5]. Here we describe an enzyme-(jnked immunosorbcnt assay (ELlSA) fo r human laclOferrin. Materials and methods LactofeTrin was isolatcd from human oolostrum by copper chelate chromatography, ion-exchange chromatography, and gel filtration. The purity of the preparatioos was assessed by SOS- and immuno-elcctTophoresis. Antibodies against punfied Lac were raised in sheep. Trace amounts of antibodies against human serum albumin wen: eliminated by absorption wiili nonnal human serum.IgG was isolated from rnonospecific anti-serum by precipitation with n-octanoic acid and further punfied 00 OEAE-ceJlulose. IgG was conjugatcd with calf intestinal alkaIine phosphatase (Boehringer Mannheim) by condensation with glutardialdehyde [6]. Polyslyrenc tubes were coated with IgG by incubation of tbe tubes with a solution containing 20 )1g IgG/ml in 0.01 molfl NH4 HC03 at Toom temperature o\'er night, washcd and lyophilized. For the assay serum, EDTA-plasma or eitrated pla~ma sampies were Toutinely dilutcd I/50 in phosphate buffer-saline, pH 7.4. 5(0)11 ofthediluted sampies were incubated in the IgG-<:03ted tubes for 2 h at 25°C. A standard eun 'e was derived oveT the range of 1 ng/ml 10 10 ng/ ml of human Lac as weU as a blank value. Lac-standards for Rl D (Behnng, Marburg) weTe ustd as extemal controls and diluted 1/10' before U5e. After removing the sampIes byaspira-tion and three washes with saline-polyoxyethylene(20}-sorbitan mono/aurate the tubes were reincubated another 2 h al 25°C with 5(0)11 ofconjugatein salinc-Tris/ HCI·bufl"er,pH 8.0. Afler aspiration of the conjugate the tubes were washcd 3 times. 500 )11 of 10 mmolfl 4-nilrophenylphosphsle in 1 mol/ l diethanol-amine/ HCI-buffer, pH 9.8, 0.5 mrnoljl MgCI2 were added and lhe lubes incubated at 25°C for 1 h. The enzymic reaction was stopped witb 5(0)11 of 2 molfl NaOH and the absorbancc was read al 405 nm. Clinical studies were perfonned with citrated plasma samplcs. Hesults and dlscussion Unear ca/ibralion curves were obtained in the range of 0 to 10 ng Lae/ml. Correlation eoefficients were bigher or equal to 0.995. Tbe Lae-<:oncentration of samplcs oould be calculated by linear regression. The detection limi t, defined as meao absorbance of a blank vaIue plus three times tbc standard devia-tion was about 0.25 ng tac/ml. Witrun-run imprccision (cocfficient ofvariation, ,, _ 10) was in the range of I.lto 6.6% with the standards and 2.310 5.3% with sampies (serum, EDTA· plasma, eitrated plasma) in the range of 65 to 2500g Lae/ml. Bctween-run imprecision (" _ 10) was from 2.8 to 7.9% witb the standards and 3.7 to 10.4% with the sampies. Within-run imprccisioo (n == 4) for one indoor control plasma and two ex-tcroal Lac-standards (see Materials snd methods) rangcd from 1.4 to 7.3%, the between-run imprecision (n = 10) was 12.5%. 10.3%, and 8.0% for these sampies. The nonnal rangt: of Lae-concentrations in serum, EOTA-plasma or citrated plasma of apparently healthy persons were (mean ± SO) found 10 be 202.4 ± 40.8 ng/ml (" = 10), 11 5.4 ± 35.4 ngJml (n = 35), and 136.6 ± 51.4 ngfml (n = 31), respeetively. Tbe bigher values for serum sampies oould be interpretcd as release of Lac during blood dotting. Similar differences bet\1"een serum and plasma were observed by others with RfA [7J. In dinical cases Lac-concentrations found excceded the normal ranges by a factor of 5 1010. Remarkably, preliminarydata of patientssuffering from multiple trauma andror severe infections showed a concomitant release of Lac from the specific granules and elastase from the azurophi lic oncs. The latter enzyme has been taken as a main indieator for the participation of polymorphonuclear pro teins in inflammatory reactions [8]. Tbe results suggest that in contrast 10 in-vitro stimulation of PMNs (9J bolb types of granulcs has been equally involved in phagocytosis and exlra-cellula r liberation of the lysosomal proteins in these underlying kinds of inOammation. Measured I..ac--concentrations exceeded that of the PMN-elastase-"l-proteinase inhibitor complex. On the other hand, measurement of PMN granule con-stituents in plasma sampies drawn in very short intervals (5-10min) before and after open heart surgery indicate that Lac is releascd more readily and to a greatcr degree Ihan elaslase in the first 2- 3 h following surgery. The clinical meaning of thc different in-vivo discharge of speeific granules compared to ll7.urophilic ones is currently undcr investigalion. Rererenct'5 1. Nemct K, Simonovits J (1985) Haematologia 18: 3- 12 2. Baggiolini M, DeDuve C, Masson PL, Heremans JF (1970) J Exp Med 131 :559-570 3. Leffell MS, Spil.7..nagel JK (J975) Infect Immunoll0 : 1241 -1249 4. Van Snick 1L, Masson PI... Heremans JF (1974) J Exp Med 140:1068 - 1084 5. Hansen NE, Malmquist J, Thorell J (1975) Acta Med Scand 198: 437-443 6. Avrameas S, Ternynck T (1969) Jmmunochemistry 6:53-66 7. Ol55On I, Olofsson T, Ohlsson K, Gust:avsson A(1979)Seand J HaemalOl22 :397-406 8. Duswald KR , Jochum M, Schramm W, Fritz H (1985) Surgery 98:892-899 9. Bentwood B1, Henson PM (1980)1 ImmunoI124 :855 - 862 

Quantification of human lactoferrin as an inflammation marker by an enzyme-linked immunosorbent assay (ELISA)

Universität München: Elektronischen Publikationen

Quantitation of human lactoferrin as an 
inflammation marker by an enzyme-linked 
immunosorbent assay (ELISA) 
W. Rautenberg 1, S. Neumann 1, G. Guozer l , H. Lang I , 
Marianne J ochum 1, and H. Fritz l 
I Biochemische Forschung E. Merck, Frankfurter Straße 250, 
0-6100 Oannstadt I , Fecleral Republic ofGermany 
1 Abteilung für Kliniscbe Chemie und Klinische Biocbemie, 
Chirurgische Klinik der Universität München, 
Nußbaumstraße 20, 0-8000 München 2, 
Federal Republic of Germany 
Quantifizierung , 'on Human Laclorerrin als 
Ent.tündungsparameter mittels Enzymlmrnunoassay 
vom Sand",·ich-Typ (ELISA) 
Human lactoferrin (Lac) is an iron·binding glycoprotein of 
Mt - 76,000 and is found especially in milk: and many other 
body fluids, such as tcars, saliva. nasal and bronchial secretions, 
urine, seminal fluid, and in pancreatic flu.id. Altbough ilie bio-
logical significance of Lac is still undcr discussion there is cvj-
dencc for its anribacterial activity in milk and blood. Olhcr 
biologieal roles of Lac include regulatory funetions during in-
testinal iron absorption. in myelopoiesis, in neutrophil aggrega-
tion and adherence and in inOammation [11. Lac appears lO be 
synlhesized mainly by polymorphonuelear leukocyles (PMN). 
11 15 localized intraccllularly in the seoondary or "spccific" 
granulcs of these ceHs [2]. The ce[[ular comcnt of Lac is around 
3 - 5 )1g/1 06 cells. Nonnally Lac occurs in plasma only in trace 
amounts. Ouring inflammation it is released from PMN with 
other lysosomal proteins [3, 4]. Measurement of I..ac may be 
useful because studies suggest that l.ae-<:oncentrations in blood 
might reflect the activation of the total blood granulocyte pool 
[5]. Here we describe an enzyme-(jnked immunosorbcnt assay 
(ELlSA) fo r human laclOferrin. 
Materials and methods 
LactofeTrin was isolatcd from human oolostrum by copper 
chelate chromatography, ion-exchange chromatography, and 
gel filtration. The purity of the preparatioos was assessed by 
SOS- and immuno-elcctTophoresis. Antibodies against punfied 
Lac were raised in sheep. Trace amounts of antibodies against 
human serum albumin wen: eliminated by absorption wiili 
nonnal human serum.IgG was isolated from rnonospecific anti-
serum by precipitation with n-octanoic acid and further punfied 
00 OEAE-ceJlulose. IgG was conjugatcd with calf intestinal 
alkaIine phosphatase (Boehringer Mannheim) by condensation 
with glutardialdehyde [6]. Polyslyrenc tubes were coated with 
IgG by incubation of tbe tubes with a solution containing 20 )1g 
IgG/ml in 0.01 molfl NH4 HC03 at Toom temperature o\'er 
night, washcd and lyophilized. For the assay serum, EDTA-
plasma or eitrated pla~ma sampies were Toutinely dilutcd I/50 
in phosphate buffer-saline, pH 7.4. 5(0)11 ofthediluted sampies 
were incubated in the IgG-<:03ted tubes for 2 h at 25°C. A 
standard eun 'e was derived oveT the range of 1 ng/ml 10 10 ng/ 
ml of human Lac as weU as a blank value. Lac-standards for 
Rl D (Behnng, Marburg) weTe ustd as extemal controls and 
diluted 1/10' before U5e. After removing the sampIes byaspira-
tion and three washes with saline-polyoxyethylene(20}-sorbitan 
mono/aurate the tubes were reincubated another 2 h al 25°C 
with 5(0)11 ofconjugatein salinc-Tris/ HCI·bufl"er,pH 8.0. Afler 
aspiration of the conjugate the tubes were washcd 3 times. 500 )11 
of 10 mmolfl 4-nilrophenylphosphsle in 1 mol/ l diethanol-
amine/ HCI-buffer, pH 9.8, 0.5 mrnoljl MgCI2 were added and 
lhe lubes incubated at 25°C for 1 h. The enzymic reaction was 
stopped witb 5(0)11 of 2 molfl NaOH and the absorbancc was 
read al 405 nm. Clinical studies were perfonned with citrated 
plasma samplcs. 
Hesults and dlscussion 
Unear ca/ibralion curves were obtained in the range of 0 to 
10 ng Lae/ml. Correlation eoefficients were bigher or equal to 
0.995. Tbe Lae-<:oncentration of samplcs oould be calculated by 
linear regression. The detection limi t, defined as meao 
absorbance of a blank vaIue plus three times tbc standard devia-
tion was about 0.25 ng tac/ml. Witrun-run imprccision 
(cocfficient ofvariation, ,, _ 10) was in the range of I.lto 6.6% 
with the standards and 2.310 5.3% with sampies (serum, EDTA· 
plasma, eitrated plasma) in the range of 65 to 2500g Lae/ml. 
Bctween-run imprecision (" _ 10) was from 2.8 to 7.9% witb 
the standards and 3.7 to 10.4% with the sampies. Within-run 
imprccisioo (n == 4) for one indoor control plasma and two ex-
tcroal Lac-standards (see Materials snd methods) rangcd from 
1.4 to 7.3%, the between-run imprecision (n = 10) was 12.5%. 
10.3%, and 8.0% for these sampies. The nonnal rangt: of Lae-
concentrations in serum, EOTA-plasma or citrated plasma of 
apparently healthy persons were (mean ± SO) found 10 be 
202.4 ± 40.8 ng/ml (" = 10), 11 5.4 ± 35.4 ngJml (n = 35), and 
136.6 ± 51.4 ngfml (n = 31), respeetively. Tbe bigher values for 
serum sampies oould be interpretcd as release of Lac during 
blood dotting. Similar differences bet\1"een serum and plasma 
were observed by others with RfA [7J. In dinical cases Lac-
concentrations found excceded the normal ranges by a factor of 
5 1010. Remarkably, preliminarydata of patientssuffering from 
multiple trauma andror severe infections showed a concomitant 
release of Lac from the specific granules and elastase from the 
azurophi lic oncs. The latter enzyme has been taken as a main 
indieator for the participation of polymorphonuclear pro teins 
in inflammatory reactions [8]. Tbe results suggest that in 
contrast 10 in-vitro stimulation of PMNs (9J bolb types of 
granulcs has been equally involved in phagocytosis and exlra-
cellula r liberation of the lysosomal proteins in these underlying 
kinds of inOammation. Measured I..ac--concentrations exceeded 
that of the PMN-elastase-"l-proteinase inhibitor complex. 
On the other hand, measurement of PMN granule con-
stituents in plasma sampies drawn in very short intervals (5-
10min) before and after open heart surgery indicate that Lac is 
releascd more readily and to a greatcr degree Ihan elaslase in 
the first 2- 3 h following surgery. The clinical meaning of thc 
different in-vivo discharge of speeific granules compared to 
ll7.urophilic ones is currently undcr investigalion. 
Rererenct'5 
1. Nemct K, Simonovits J (1985) Haematologia 18: 3- 12 
2. Baggiolini M, DeDuve C, Masson PL, Heremans JF (1970) 
J Exp Med 131 :559-570 
3. Leffell MS, Spil.7..nagel JK (J975) Infect Immunoll0 : 1241 -
1249 
4. Van Snick 1L, Masson PI... Heremans JF (1974) J Exp Med 
140:1068 - 1084 
5. Hansen NE, Malmquist J, Thorell J (1975) Acta Med Scand 
198: 437-443 
6. Avrameas S, Ternynck T (1969) Jmmunochemistry 6:53-
66 
7. Ol55On I, Olofsson T, Ohlsson K, Gust:avsson A(1979)Seand 
J HaemalOl22 :397-406 
8. Duswald KR , Jochum M, Schramm W, Fritz H (1985) 
Surgery 98:892-899 
9. Bentwood B1, Henson PM (1980)1 ImmunoI124 :855 - 862 


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Quantification of human lactoferrin as an inflammation marker by an enzyme-linked immunosorbent assay (ELISA)

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Universität München: Elektronischen Publikationen