Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
~3(~00S~
~EHRING~ERKE Aktiengesellschaft 86/~ 038~- Ma 583
Dr. Ha/3n/Li.
A lactoferrin-containing incubation medium for solid-
phase immunometric methods and its use
The invention relates to a lactoferrin-containing incuba-
tion ~edium for solid-phase immunometric assays and to
the use of th~s medium.
For solid-phase immunometric assays, for example ELISA,
the composition of the required incubation media t"buffer
solutions", "incubation milieus") must be such that non-
specific binding of concomitant substances in the sample
to the solid phase is prevented. Additives known for
this purpose are proteins such as albumin~ casein, mix~
tures of proteins such as animal sera, hydrolyzed gelatin
or its derivatives, as ~ell as surfactants.
Incubation media provided with such additives are unable
to prevent measurements being false, ~hich particularly
occurs ~hen samples have been heated. Heating is expe-
dient to reduce the infectiosity of the samples, ~hich
may derive from, for example, HIV (human immunodeficiency
virus~.
Hence the object was to find an incubation medium ~ith
which there is no occurrence, ~hen it is used, of false
values caused by heating of the sample.
:
It has been found, surprisingly, that an incubation medium
~hich contain~ lactoferrin achieves this object~
The invention relates to an incubation medium for solid-
phase immunometric assays containing lactoferrin~
The invention also relates to the use of lactoferrin in
an incubation medium ~hich is used for a solid-phase
immunometrlc assay.
~30~
-- 2
An incubation medium wi~hin the meaning of the invention
is the liquid phase,in ~hich the immunochemical reaction
takes place,of a solid-phase immunometric assay.
Lactof~rrin is added in a concentration o~ from O.OS to
20 g/l. A concentration of 0.15-0.25, in particular
0.2, g/l is preferred.
The lactoferrin can be added to incubation media or buffer
solutions kno~n per se for solid-phase immunometric
assays. One of these is, for example, the solution des-
cribed in Çerman Patent A-27 448 36 on page ~4, ~hich,
apart from buffer substance, contains 5 g/l bovine serum
albumin, 5 g/l polyoxyethylene (20) sorbitan monolaurate
~R)Tween 20) in phosphate-buffered physiological saline
solution (P9S). ~Another example which may be mentioned is
the solution described in German Patent A-31 15 115 on
page 10, a 0.2 mol/l NaH2P04/Na2HP04 buffer, pH 6.5, which
contains 2 g/l bovine serum albu~in and 2~0 ml/l normal
goat serum. A preferred buffer solution is used in
examPle 1.
A med;um of this type can advantageously contain a prote;n
kno~n per se, preferably casein, and/or a hydrolysis pro-
duct of gelatin, or hydrolyzed and chemically treated
gelatin.
The incubation medium of this invention can also advantageously
25 include a surfactant and/or a neutral salt.
The lactoferrin to be used according to the invention can
be obtiined from the 0ilk of a mammal by removing ehe
cream from the milk, adjusting the milk, fro0 ~hich the
cream has been removed, to a p~ of 3.5-4.5, removing the
protein precipitate ~hich has formed, and removing and
isolating the lactoferrin from the supernatant by chroma-
tography on an ion exchanger.
Lactoferrin is isolated from milk, preferably from cow's
.
13~0 [3S7
- 3 --
milk. In a preferred process for obtaining a lactoferrin
suitable for the purpose according to the ;nvention, the
fat is removed from the milk by, for exampLe, centrifuging
it. The upper of the t~o phases which form is removed
and discarded~ The lower phase ;s adjusted to pH 3.5-4.5
with an ac;d. This results in a precip;tate wh;ch is
sedjmented, for example by centrifugation, and is dis-
carded. The supernatant is adjusted to 3 conductivity of
6-7 mS/cm by dilution with ~ater. Then a cation exchanger,
for example CM-cellulose or SP-RSephadex, ~hich has been
swollen and equilibrated with a buffer of oH 4-5.5 and a
conductivity of, preferably, 6-7 mS/cm is introduced.
The suspension is filtered, and the residue ;s washed
several t;mes with the same buffer, and is then suspended
therein and packed into a column. It is eluted with a
gradient of, preferably, 0-1 mol/l NaCl at pH 5. The
fraction which is eluted in the range 500-650 mmol/l NaCl
as a homogeneous peak measured at 280 nm contains the
lactoferrin which is particularly suitable for the incu-
bation medium. On SDS-PAGE electrophoresis, this lacto-
ferrin appears as a band with a molecular ~eight of
80,000 ~ 3,000.
On chromatography on an anion exchanger, for example on
DEAE- or QAE-RSephadex, or on DEAE-cellulose, the super-
natant from the prec;p;tation at pH 3.5-4.5 which has been
descr;bed above ;s adjusted to pH 6.5-7.5~ and a conduc-
tiv;ty of 6-8 mStcm, and ;s appl;ed to 3 column packed
with the ;on exchanger which has previously been equ;l;-
brated ~ith a buffer of pH 6.5-7.5 and a conduct;v;ty of
6-8 mS/cm. The lactoferrin then passes through the column
or ;s eluted as the f;rst Peak on elution ~;th an NaCl
grad;ent.
The lactoferrin must be present on incubation of the sam-
ple or of the analyte w;th the reactants wh;ch are bound
to the sol;d phase. It can be added to the solut;on which
contains the analyte or to the solution or the buffer
~3~057
-- 4
~ith ~hich the analyte is diluted. However, it can also
be contacted in dissolved form with the reactant which
is bound to the solid phase and/or dried onto the solid
phase before this is contacted with the analyte.
s
Suitable reactants which are bound to solid phases are
the components of a bioaffinity binding system wh;ch are
able to bind an analyte of this typeO In ~he case of an
i~munDlogical binding system, these reactants are either
antigens or antibodies. Solid-phase immunometric assays
can be both competitive and two- side immunometric (sand-
wich) assays or even indirect assays. In the latter case
the analyte is an antibody, and the solid-phase reactant
is the corresponding antigen, and the amount of antibody
which is bound to the solid phase is determined by a
second, labeled antibody.
The assay system can be a single-step or multistep assay.
The lactoferrin prevents the b;nding of those constituents
of the sampLe which do not belong to the particular bio-
aftinity binding system. It thus averts false results in
the detection and determination of the analyte.
The examples which follow illustrate the invention and
demonstrate the appropriateness of lactoferrin as a com-
ponent of the incubation medium.
The examples are intended to show that Lactoferr;n can be
used in a variety of incubation media, introduced in
various amounts.
Example 1
.
1. Isolation of lactoferrin
Z0 l of cow's milk were centrifuged at 3400 x 9 for
30 m;n, and the milk fat ~as removed in the
~3~0~ii7
-- 5 --
supernatant. Small amounts of insoluble constituents
~ere removed as sediment and likewise discarded.
The pH of the milk from which the fat had been removed
was adjusted to pH 4.0 by addition of hydrochLoric
acid. The protein precipitate ~hich ~as produced by
this ~as removed by centrifugation at 3400 x 9
The supernatant, which had, after adj~ustment to pH 5.Q
with sodium hydroxide solution, a conductivity of
12 mS/cm at 20C, was diluted to 7 mS/c~ with water
and then mixed with 200 ml of the swollen ion exchanger
carboxymethylcellulose CM 32 supplied by What~ann, and
the mixture was stirred at room temperature for 4 h.
The ;on exchanger had previously been equilibrated
~ith 50 mmol/l sodium acetate buffer, pH 5Ø The ion
exchanger was then ren~oved by filtration on a suction
funnel and suspended ;n the sodium acetate buffer and
transferred into a chromatography column. The column
was then washed with 400 ml o~ the acetate buffer pre-
viously described. The proteins bound to the cation
exchanger were eluted with a solution o~ 1 mol/l sodium
chloride.
The eluate was adjusted to pH 5.0 ~ith 1-normal sodium
hydroxide solution and was converted by dialysis into
50 mmol/l sodium acetate, pH 5Ø The prote;n m;x-
ture was then rechromatographed on 200 ml of carboxy-
methylcellulose CM 32. The prote;ns ~ere eluted w;th
a linear gradient from 50 mmol/l sodium acetate, pH 5,
to 50 mmol/l sodium acetate, pH 5, and l mol/l sodium
chloride.
The 4th peak of the elution d;agram, whose solut;on
had a conduct;vity of 18-20 mS/cm, was collec~ed as
one fraction, concentrated by ultraf;ltration and sub-
jected to gel f;ltration on RSepharose AcA 34 wh;ch had
been egu;librated ~ith PBS containing O.S g/l sod;um
azide. The main component from the gel filtration,
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-- 6 --
which amounted to 1.2 9 of protein, contained the
lactoferrin.
2. Comparison of sample dilution buffer containing lacto-
ferrin with sample dilution buffer without addition
The EnzygnostR anti HSV ELISA kit of ~ehringwerke was
used for the solid-phase immunometric method. The
procedure described in the pack insert was followed for
carrying out the assay, and it is described briefly
below.
The samples ~ere diluted in the ratio 1:25, instead of
1:44 as recommended, with sample dilution butfer
(137 mmol/l NaCl, 7.247 mmol/l Na2HP04.2H20 and
2.7~0 mmol/l KH2P04 with the addition of 2.7 mmol/l
KCl, 0.4 mmol/l MgCl2, 3 mmol/l NaN3, 10 mltl fetal
calf serum and 40 ml/l (~)Tween 20) without or with the
addition of 0.2 g/l lactoferrin, and were incubated
with the antigens immobilized in the wells of a micro-
assay plate, the unbound ant;bodies were vashed out,
an enzyme was bound, using a conjugate solution, to
the ant;body/antigen complex ~hich had formed, the
excess conjugate solution was washed out, a chromogenic
substrate solution was used to develop a color via ~he
enzyme, and then the reaction was stopped ~ith a stop
solution. The samples which had thus been prepared
and had developed a color depending on the content of
the virus-specific antibodies were then evaLuated by
determination of their extinction tE) at 405 nm by
photometry.
One portion of each of 9 subjects' sera was heated at
56C for 30 min. The other portion remained un-
treated.
It was found that, in particular, in the case of heated
samples ;n dilution buffer without lactoferrin the
extinctions measured in the control wells were very
130~ 5~
high, but the extinctions in the antigen-coated wells
~ere also raised. This resulted ;n 5 false-negative
results. The control wells are treated ("coated")
with the preparation of non-virus-infected cells ~hich
are used for obtaining the viruses. When ~he 9 heated
samples were assayed using the diLution buffer con-
taining ~actoferrin, no false-negative value was found.
The unhea~ed samples ~ere tested using the dilution
buffer containing no lactoferrin. The results served
to check the results with the heated sera.
Exam
The effect of adding lactoferrin was tested in a single-
step sandwich assay. ~hen, in an assay of this type for
H3sAg, 7.5 g/l lactoferrin were added, all of 14 unheated
critical samples were correctly found to be ne~ative,
whereas all had been false-positive determinations with-
out addit;on of lactoferrin.
~0
Example 3
The effect of addition of lactoferrin was tested in a
three-step sandwich assay for detecting influenza A anti-
gen. Lactoferrin was used in the second and third stepsof this sol;d-phase immunochemical method.
When, in these two steps, first an antibody/biotin con-
jugate diluted for use and then a streptavidin/POD con-
jugate d;luted for use and conta;ning lactoferrin (~ore~han 0005 g/l) were incubated, it was poscible to reduce
the non-specific reactions, in terms of background and
influenza B foreign ant;gen, by more than 80~. An ELISA
result ~easured at 450 nm is given here as representative:
Non-specific binding ~ithout lactoferrin = 2.892 U.
Non-specific binding with lactoferrin = O.Z31 U.
