QUANTITATION OF LACTOFERRIN IN INFANT FORMULAS BY
ELECTROPHORESIS USING IR FLUORESCENCE IMAGING
Background of the Invention
Lactoferrin is an iron-binding glycoprotein which is a natural component of milk,
including human milk and bovine milk. Lactoferrin is believed to have antimicrobial,
anti-inflammatory and immunomodulating activity, and other benefits.
Lactoferrin may occur in infant formula either because the formula includes milk
components that contain lactoferrin or because lactoferrin has been added to the
formula. To ensure that the infant consuming the formula receives an appropriate
amount of lactoferrin, it is important to be able to accurately measure the amount of
lactoferrin in infant formula.
One challenge in measuring the lactoferrin content of infant formula is presented
by the heat treatment that the formula may undergo. Heat treatment causes proteins to
denature and aggregate; disulfide bonds may form between protein molecules, bonding
them together and making analysis difficult. For example, lactoferrin may bond to other
proteins and form aggregates that will not permit the lactoferrin to be detected by
standard protein analytical methods. As a result, analysis of the lactoferrin content of a
heat treated infant formula would be expected to yield a lower result than for a non-heat
treated infant formula, and not provide a result that reflects the total lactoferrin content.
One method for analyzing proteins so that both natured and denatured proteins
will be measured is to use sodium dodecylsulfate polyacrylamide gel electrophoresis
(SDS-PAGE) under reducing conditions. SDS-PAGE under non-reducing conditions
cannot measure lactoferrin that is covalently bonded to other proteins by disulfide bonds
to form aggregates. To break the disulfide bonds and disrupt the aggregates into
individual proteins, the SDS-PAGE must be conducted under reducing conditions. This
was demonstrated by G. Brisson, et al., Effect of Iron Saturation on the Recovery of
Lactoferrin in Rennet Whey Coming from Heat-Treated Skim Milk, J. Dairy Sci. 90,
2655-2664 (2007). However, Brisson, et al., did not demonstrate a highly precise
method for quantitatively measuring the amount of lactoferrin in the bands of the
electrophoresis gel.
Coomassie blue is commonly used to stain gels. This dye makes the proteins
visible so that the amount of protein may be measured semi-quantitatively by
densiometry. Coomassie blue also presents the opportunity to use a more precise
method of measurement than densitometry, because the dye fluoresces in the near
infrared part of the electromagnetic spectrum, and quantitative measurements may be
made using IR fluorescence detection, as demonstrated by S. Luo, et al., Quantitation
of protein on gels and blots by infrared fluorescence of Coomassie blue and Fast
Green , Anal. Biochem., 350, 233=238 (2006). The usefulness of this method for
detecting proteins on gels was also demonstrated by L. R. Harris, et al., Assessing
Detection Methods for Gel-Based Proteomic Analyses . J. Proteome Res., 6, 141 8-1425
(2007).
There is a need to develop a reliable and accurate method for the precise
quantitative measurement of total lactoferrin in an infant formula matrix
Summary of the Invention
The present invention provides a method of quantitatively measuring the total
lactoferrin content of an infant formula. In this method, standard solutions having
different known concentrations of lactoferrin are prepared and independently combined
with a reducing sample buffer. The resulting solutions are loaded onto the same
sodium dodecylsulfate polyacrylamide gel, heated for about 7-9 minutes at about 90-
100°C, and then subjected to electrophoresis using substantially the same power level
and time period for each solution. Following electrophoresis, the gel is stained with a
gel stain that fluoresces in the infrared, and then destained, leaving on the gel only the
stain that has bound to the lactoferrin in the standard solution. An infrared fluorescence
imaging system is then used to quantitatively measure, at a suitable wavelength, the
infrared fluorescence intensity of the stain for each standard solution to establish the
relationship between measured infrared fluorescence and total lactoferrin.
Essentially the same procedure is used to analyze an infant formula containing
an unknown amount of lactoferrin. The measured infrared fluorescence intensity of the
stain on the gel containing the infant formula sample is used to determine the amount of
lactoferrin in the infant formula sample by reference to the relationship between
fluorescence and lactoferrin amount that was established using the standard solutions.
Knowledge of the total amount of lactoferrin in the infant formula sample and the
quantity of infant formula analyzed allows calculation of the lactoferrin concentration in
the infant formula.
Detailed Description of the Invention
In the method of this invention, lactoferhn in an infant formula is measured by
using sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing
conditions to separate lactoferrin molecules from other compounds in the sample being
analyzed, and infrared fluorescence detection to obtain a quantitative result for the
lactoferrin content of the sample.
The term "infant formula" as used herein refers to a nutritional formulation (either
in the form of a liquid or in the form of a dry powder that may be reconstituted to form a
liquid infant formula upon addition of water) that provides complete nutrition for an infant
and may be used as a substitute for human milk in feeding an infant. Such formulae are
well-known in the art.
Typically, an infant formula in a ready-to-consume liquid form provides 60-70
Kcal/100 ml. Infant formula typically comprises, per 100 Kcal: 1.8-4.5 g protein; 3.3-6.0
g fat (lipids); 300-1 200 mg linoleic acid; 9-14 g carbohydrates selected from the group
consisting of lactose, sucrose, glucose, glucose syrup, starch, malto-dextrins and
maltose, and combinations thereof; and essential vitamins and minerals. Lactose may
be the pre-dominant carbohydrate in an infant formula. A liquid infant formula may
contain about 67 Kcal/1 00 ml. Infant formula may comprise about 1.8-3.3 g protein per
100 Kcal, for example, about 1.8-1 .1 g protein per 100 Kcal.
An infant formula may also comprise nucleotides selected from cytidine 5'-
monophosphate (CMP), uridine 5'-monophosphate (UMP), adenosine 5'-
monophosphate (AMP), guanosine 5'-monophosphate (GMP) and inosine 5'-
monophosphate (IMP), and mixtures thereof. Infant formula may also comprise lutein,
zeaxanthin, fructo-oligosaccharides, galacto-oligosaccharides, sialyl-lactose, and/or
fucosyl-lactose. Long chain polyunsaturated fatty acids, such as docosahexaenoic acid
(DHA) and arachidonic acid (AA) may be included in infant formula. Infant formula may
also include amino acids. Infant formula may also include other ingredients well-known
in the art.
One gel stain well-known in the art which is useful in the practice of this invention
is SimplyBlue™ SafeStain, a ready-to-use, proprietary Coomassie® G-250 stain that is
commercially available from Invitrogen Corporation (Carlsbad, California, USA). When
a gel containing protein (for example, lactoferrin) is treated with this gel stain, and the
excess stain is washed from the gel, only the stain that is bound to the protein remains
on the gel. Therefore the amount of stain remaining corresponds to the amount of
protein.
Coomassie blue stain fluoresces in the near infrared at wavelengths of about
670-800 nm. The 680-700 nm range is suitable for measuring the fluorescence of
Coomassie blue stained proteins. An infrared fluorescence scanner set to detect
fluorescence at these wavelengths may be used to quantitatively measure the amount
of Coomassie blue on a gel, and therefore to determine the amount of protein that binds
the Coomassie blue. In one embodiment, the fluorescence intensity is measured at
about 700nm. In another embodiment, the fluorescence intensity is measured at about
680nm.
In one embodiment, the invention is a method of quantitatively measuring the
total lactoferrin content of an infant formula by: preparing a solution of the infant formula
and combining it with a sample reducing agent to produce an infant formula sample
solution; heating the infant formula sample solution at about 90-1 00°C for about 7-9
minutes, for example at about 95 °C for about 8 minutes; loading the infant formula
sample solution onto a sodium dodecylsulfate polyacrylamide gel to obtain a loaded gel;
performing electrophoresis on the loaded gel; staining the gel with a Coomassie blue
stain and then destain the gel, leaving on the gel only the stain that has bound to the
lactoferrin; and, using an infrared fluorescence imaging system to quantitatively
measure the infrared fluorescence intensity at a suitable wavelength of the stain on the
gel. This intensity may be compared to the infrared fluorescence intensity at the same
wavelength of standard solutions in the same gel having known concentrations of
lactoferrin to determine the amount of lactoferrin in the infant formula. Those skilled in
the art will readily understand how to use such data to calculate the total amount of
lactoferrin, and the concentration of lactoferrin, in the infant formula sample. Those
skilled in the art will also be able to readily determine a suitable wavelength without
undue experimentation. For example, suitable wavelengths in the practice of this
invention include, but are not necessarily limited to, those within the ranges of 670-
800nm, 670-700nm, and 680-700nm, such as 680nm, 700nm, 780nm and 800nm.
In one embodiment of this invention, the infrared fluorescence intensity of
standard lactoferrin solutions is plotted versus lactoferrin content or concentration to
provide a standard curve. The infrared fluorescence intensity of an infant formula of
unknown lactoferrin concentration is compared to this curve to determine the lactoferrin
content or concentration of the unknown sample. In one embodiment, the curve is
substantially linear.
In infrared fluorescence spectroscopy, the material to be analyzed is irradiated
with infrared radiation at one wavelength (the excitation wavelength) and the
fluorescence at a longer wavelength (the fluorescence wavelength) is measured by the
instrument. For example, in the detection of lactoferrin stained with Coomassie blue the
material may be irradiated at about 550nm while fluorescence may be measured at, for
example, a wavelength in the range of about 680-800nm. The wavelength of excitation
is not limited to one particular wavelength, but may be any wavelength that produces
excitation and fluorescence; for example, the excitation wavelength may be in the range
of about 450-550nm. Any excitation wavelength that will produce the desired
fluorescence may be used, and those skilled in the art will readily be able to determine
suitable excitation wavelengths. The fluorescence wavelength also may vary, since the
material may fluoresce at more than one infrared wavelength. Those skilled in the art
will readily be able to determine the suitable wavelength(s) at which to detect
fluorescence for a particular material without undue experimentation.
In one embodiment of the invention, pre-cast sodium dodecylsulfate
polyacrylamide gels are used, for example NuPAGE Novex 4-1 2% Bis-Tris gels
commercially available from Invitrogen Corporation (Carlsbad, California, USA). Other
sodium dodecylsulfate polyacrylamide gels may be suitable in the practice of this
invention; those skilled in the art will readily be able to identify other suitable
electrophoresis gels useful in the practice of the present invention without undue
experimentation.
In one embodiment, prior to electrophoresis the sample to be analyzed is
combined with a reducing agent, such as NuPAGE sample reducing agent ( 10 X)
(Invitrogen Cat. No. NP0004) which contains dithiothreitol, or the like. In one
embodiment, prior to electrophoresis the sample to be analyzed is combined with a
buffer NuPAGE LDS sample buffer (4X) - Invitrogen Cat. No. NP0007), which is 40-70%
glycerol, or the like. In one embodiment, the reducing agent and buffer are combined
with each other prior to combining with the sample to be analyzed. Those skilled in the
art will readily be able to identify other suitable reducing agents and buffers useful in the
practice of the present invention without undue experimentation.
In an embodiment of this invention in which a powdered infant formula is to be
analyzed, the powder is dissolved in water to form a solution. In one embodiment, a
liquid infant formula is diluted with water to form a solution to be analyzed.
In one embodiment, the invention of this invention includes: preparing standard
solutions having different known concentrations of lactoferrin in the approximate range
of 0.01 -0.1 0 mg/ml and independently combining each standard solution with a sample
reducing agent and a buffer to produce standard sample solutions having lactoferrin
concentrations of approximately 0.005-0.075 mg/ml; preparing a solution of said infant
formula and combine it with a sample reducing agent and a buffer to produce an infant
formula sample solution; providing a sodium dodecylsulfate polyacrylamide gel for the
standard sample solutions and infant formula sample solution; heating the sample
solutions at about 95°C for about 8 minutes; and loading each such sample solution into
its respective unit or well of the gel; performing electrophoresis on the gel using
substantially the same electrophoresis power level and time period for each sample
solution; staining the gel with a Coomassie blue stain and then destaining, leaving on
the gel only the stain that has bound to the lactoferrin; using an infrared fluorescence
imaging system to quantitatively measure the infrared fluorescence of the stain on each
unit of gel at a wavelength that is in the range of 680-700nm; and, comparing the
measured infrared fluorescence of the stain on the unit of gel loaded with infant formula
sample solution to the infrared fluorescence of the stain on the units loaded with the
standard sample solutions to determine the lactoferrin concentration in the infant
formula.
In one embodiment, the infant formula sample(s) and the standard solutions are
all loaded onto the same gel, in separate wells, and all are analyzed at the same time.
Heat treatment of infant formula is standard practice to sterilize the formula, but it
results in denaturing and aggregation of proteins in the formula, such as lactoferrin. To
accurately measure the total amount of lactoferrin in a heat-treated infant formula
sample using SDS-PAGE and infrared fluorescence detection, it is necessary to break
up the aggregates by performing the SDS-PAGE under reducing conditions in which the
sample to be analyzed is heated sufficiently to break up the aggregates. It has been
discovered that heating for about 7-9 minutes at about 90-1 00°C is suitable for obtaining
an accurate analysis of total lactoferrin.
The following examples are presented to illustrate certain embodiments of the
present invention, but should not be construed as limiting the scope of this invention.
Example 1: Method for Analysis of Lactoferrin in Finished Product
A. Equipment
Odyssey Infrared Imaging System (LI-COR)
Micro centrifuge - VWR Galaxy Mini Spin - Cat. No. 37000-700 or equivalent.
Orbital shaker - Labline Maxi-Rotor or equivalent
Vortex Mixer - VWR Cat. No. 5881 6 or equivalent.
Electrophoresis system:
Novex Mini-cell Xcell Surelock - Invitrogen Cat. No. EI0001
B. Materials
Pre-cast gels: NuPAGE Novex 4-1 2 % Bis-Tris gel, 10 wells, 1.0 mm - Invitrogen Cat.
No. NPO321 Box
Micro centrifuge tubes - 1.5 ml (polypropylene) - VWR Cat. No.201 70 or equivalent
Simply Blue Safe Stain (1X) - Invitrogen Cat. No. LC6060
Lactoferrin standard - Sigma product # 9507
Water - Mill i water or deionized
NuPAGE MOPS SDS Running buffer (20X) - Invitrogen Cat. No. NP0001
Running buffer (1X) - mix 50 ml_ of NuPAGE MOPS SDS Running buffer (20X)
and 950 ml_ of deionized water.
NuPAGE LDS sample buffer (4X) - Invitrogen Cat. No. NP0007.
NuPAGE sample reducing agent ( 10 X) - Invitrogen Cat. No. NP0004.
Reducing sample buffer ( 1X) - 1000 uL of NuPAGE LDS sample buffer with 400 uL of
NuPAGE sample reducing agent ( 10 X).
C. Procedure
Stock Standard : Weigh about 24 mg of lactoferrin standard (about 87% pure),
transfer to a 10 ml volumetric flask, add 8 ml water, sonicate for about 15 minutes to
dissolve the LF and add water to volume; mix by vortex mixer. This lactoferrin stock
solution is ~ 2 mg/ml.
Working Standard : Make 1: 0 dilution (add 100 ul of lactoferrin stock standard to
900 ul water in a micro centrifuge tube, mix by vortex). The final concentration of this
lactoferrin working standard stock is ~ 0.2 mg/ml.
Calibration Standards :
Calibration Standard 1: add 500.0 ul of lactoferrin working standard to 800.0 ul of water
in a micro centrifuge tube and mix.
Calibration Standard 2 : add 250.0 ul of lactoferrin working standard to 1050.0 ul of
water in a micro centrifuge tube and mix.
Calibration Standard 3 : add 125.0 ul of lactoferrin working standard to 1175.0 ul of
water in a micro centrifuge tube and mix.
Aliquot 65 ul of each calibration standard into a micro centrifuge tube with a screw cap.
Store this calibration standard stock at -20 °C.
Finished Product : Weigh about 20 mg of sample in a 5 ml volumetric flask, add
about 4 ml water, sonicate for about 15 minutes to dissolve the sample, add water to
volume, mix by vortex. Aliquot 65.0 uL sample each into 2 micro centrifuge tubes.
Prepare reduced standard and samples :
Add 35.0 ul of reducing sample buffer to each calibration standards and sample (65 ul).
Mix by vortex. Heat the micro centrifuge tubes at 95°C for 8 minutes. Let the tubes cool
down for 1 minute. Spin down tubes in a Micro centrifuge for a few seconds.
Running SDS -PAGE : Rinse pre-cast gel with water. Insert the pre-cast gel into
the Xcell Mini-cell apparatus. Fill the Xcell Mini-cell apparatus with running buffer (1X).
Using gel loading pipette tips, load 20.0 uL of lactoferrin standards and samples into
each well; all samples are duplicated. Cover Xcell Mini-cell apparatus and perform
electrophoresis at 200V for 65 minutes., then place the gel in a staining dish. Wash the
gel with water. Discard water. Replace it with approx. 20 mL of SimplyBlue™ SafeStain
reagent. Shake for 1 hour. Discard staining reagent and replace it with water. Destain
by leaving it over night (or 3 hours) with shaking at medium setting.
Gel scanning and Data Analysis : The gels are scanned with the Odyssey
Infrared Imaging System. The lactoferrin calibration standards is used to make a
standard curve (Integrated Intensity v. Concentration). The lactoferrin concentrations of
the samples are determined by measuring their IR integrated intensity, and using the
standard curve to determine the lactoferrin concentration which correlates with the
measured intensity.
Example 2 : Validation of Method of Measuring Lactoferrin in Infant Formula
A bovine lactoferrin reference standard from Sigma (product # L9507, Lot #
097K3779, purity: 87.42% based on COA) was used to test the method.
Linearity was determined by using 4 different amounts of bovine lactoferrin
reference standards over a range of approximately 1 ug to 0.1 25 ug on a the same day.
A least-square regression equation was constructed using the standard amount versus
the instrument response. The correlation coefficient was 0.998, and the method was
linear over the entire range.
Repeatability was determined by measuring the lactoferrin content 6 independent
samples of the same product on the same day. Each sample was measured twice on
separate gels. The results are shown in Table 1, below.
Table 1: Data for Repeatability
Gel 1 Gel 2 AVG STDEV CV
g/1 00g g/1 00 g/1 00 g/1 00g %
g g
Sample 1 1.089 1.104 1.10 0.01 0.97
Sample 2 1.105 1.103 1.10 0.00 0.1 3
Sample 3 1.054 1.057 1.06 0.00 0.22
Sample 4 1.047 1.071 1.06 0.02 1.63
Sample 5 0.984 1.029 1.01 0.03 3.1 2
Sample 6 0.926 0.937 0.93 0.01 0.84
AVG 1.03 1.05
STDEV 0.07 0.06
CV % 6.53 5.94
Intermediate precision was determined by measuring 6 independent samples of
the same product on three different days by two analysts. The results are shown in
Tables 2 and 3, below.
Table 2 : Data for Precision (Analyst 1)
Day 1 Day 2 Day 3 3 Days
g/1 00g g/1 00g g/1 00g g/1 00g
Sample 1 1.096 1.071 0.9975
Sample 2 1.104 1.020 0.9733
Sample 3 1.056 1.053 0.8831
Sample 4 1.059 1.021 0.9552
Sample 5 1.006 0.988 0.9583
Sample 6 0.931 0.925 0.9382
AVG 1.04 1.01 0.95 1.00
STDEV 0.06 0.05 0.04 0.06
CV % 6.1 9 5.1 2 4.08 6.30
Table 3 : Data for Precision (Analyst 2)
Day 1 Day 2 Day 3 3 Days
g/1 00g g/1 00g g/1 00g g/1 00g
Sample 1 0.982 1.189 1.01 8
Sample 2 0.970 1.099 1.004
Sample 3 0.945 1.186 0.846
Sample 4 1.036 1.21 0 1.106
Sample 5 0.91 0 1.092 1.078
Sample 6 0.902 1.042 1.058
AVG 0.96 1.14 1.02 1.04
STDEV 0.05 0.07 0.09 0.1 0
CV % 5.20 5.95 9.1 0 9.85
Accuracy was determined by spiking commercially available Wyeth S26 Gold
Infant Formula with 1.334 wt%, 1.068 wt% and 0.795 wt % of the reference standard, in
duplicate. Both spiked samples were tested separately by two different analysts.
Results are shown in Tables 4 and 5, below.
System suitability was determined by analyzing 6 loadings of the same sample.
The CV% ranged from 2.277% to 5.320%
Ruggedness was evaluated by having three analysts analyze 6 samples that
contained concentrations of lactoferrin unknown to the analysts, with the concentrations
ranging from 1 g/L to 2g/L lactoferrin. The lactoferrin determinations by the three
analysts ranged from 85-1 19% of the actual amounts of lactoferrin in the samples.
These results indicated that the method has good linearity, repeatability,
ruggedness, system suitability, precision and accuracy.
Many variations of the present invention not illustrated herein will occur to those
skilled in the art. The present invention is not limited to the embodiments illustrated and
described herein, but encompasses all the subject matter within the scope of the
appended claims.
Claims
1. A method of quantitatively measuring the total lactoferrin content of an infant
formula comprising the following steps:
a) prepare a solution of said infant formula and combine it with a sample
reducing agent to produce an infant formula sample solution;
b) load the infant formula sample solution onto a sodium dodecylsulfate
polyacrylamide gel to obtain a loaded gel;
c) heating the infant formula sample solution at about 90-1 00°C for about 7-9
minutes;
d) perform electrophoresis on the loaded gel;
e) following electrophoresis, stain the gel with a Coomassie blue stain and then
destain the gel, leaving on the gel only the stain that has bound to the lactoferrin
and,
f) after staining and destaining the gel, quantitatively measure the infrared
fluorescence intensity of the stain on the gel at a suitable infrared wavelength.
2 . The method of claim 1 further comprising correlating the measured infrared
fluorescence of the stain on the gel to an amount of lactoferrin using data derived from
reference standards and then calculating the lactoferrin concentration of the infant
formula by dividing the amount of lactoferrin so determined by the amount of infant
formula loaded onto the gel.
3 . The method of claim 2 wherein the data derived from reference standards is
obtained by:
a) preparing solutions having a range known lactoferrin concentrations;
b) combining these solutions with a sample reducing agent to produce standard
sample solutions;
c) loading each standard sample solution onto its respective unit of a sodium
dodecylsulfate polyacrylamide gel to obtain a loaded gel;
d) heating the standard sample solutions at about 90-1 00°C for about 7-9
minutes;
e) perform electrophoresis on the loaded gel using the procedure used for the
infant formula;
f) following electrophoresis, stain the gels with a Coomassie blue stain and then
destain, leaving on the gel only the stain that has bound to the lactoferrin; and,
g) after staining and destaining the gel, quantitatively measure the infrared
fluorescence of the stain on each standard solution on the gel at the same
fluorescence wavelength as used for the infant formula loaded gel.
4 . The method of any of claims 1-3 wherein the infrared fluorescence intensity is
measured at a wavelength of about 680-700nm.
5 . The method of claim 3 or claim 4 wherein the standard sample solutions and the
infant formula sample solution further comprise a buffer which is combined with the
standard solutions and with the solution of infant formula before, after or simultaneously
with the sample reducing agent.
6 . The method of claim 5 wherein the buffer is mixed with the sample reducing
agent prior to combining the buffer and sample reducing agent with the standard
solutions and the solution of infant formula.
7 . The method of any of claims 1-6 wherein the infant formula is in the form of a
powder and the solution of infant formula is prepared by dissolving the powder in water.
8 . A method of quantitatively measuring the total lactoferrin content of an infant
formula comprising the following steps:
a) prepare standard solutions having different known concentrations of lactoferrin
in the approximate range of 0.01 -0.1 0 mg/ml and independently combine each
standard solution with a sample reducing agent and a buffer to produce standard
sample solutions having lactoferrin concentrations of approximately 0.005-0.075
mg/ml;
b) prepare a solution of said infant formula and combine it with a sample
reducing agent and a buffer to produce an infant formula sample solution;
c) provide a sodium dodecylsulfate polyacrylamide gel for each of the standard
sample solutions and infant formula sample solution and load each such sample
solution into its respective unit of the gel to obtain a loaded gel;
d) heating the infant formula sample solution and the standard sample solutions
at about 95°C for about 8 minutes;
e) perform electrophoresis on the loaded gel using substantially the same
electrophoresis power level and time period for each sample solution on the gel;
f) following electrophoresis, stain the gel with a Coonnassie blue stain and then
destain, leaving on the gel only the stain that has bound to the lactoferrin;
g) after staining and destaining the gel, quantitatively measure the infrared
fluorescence of the stain on each unit of gel at a wavelength that is in the range
of 680-700nm with an infrared fluorescence imaging system; and,
h) compare the measured infrared fluorescence of the stain on the unit of gel
loaded with infant formula sample solution to the infrared fluorescence of the
stain on the units loaded with the standard sample solutions to determine the
lactoferrin concentration in the infant formula.