U.S. patent application number 14/482339 was filed with the patent office on 2015-04-02 for method for determining the concentration of the adipocytic form of the fatty acid binding protein (a-fabp, fabp4, p2).
This patent application is currently assigned to BIOVENDOR LABORATORY MEDICINE, INC.. The applicant listed for this patent is BIOVENDOR LABORATORY MEDICINE, INC.. Invention is credited to VIKTOR RUZICKA.
Application Number | 20150093769 14/482339 |
Document ID | / |
Family ID | 38048068 |
Filed Date | 2015-04-02 |
United States Patent
Application |
20150093769 |
Kind Code |
A1 |
RUZICKA; VIKTOR |
April 2, 2015 |
METHOD FOR DETERMINING THE CONCENTRATION OF THE ADIPOCYTIC FORM OF
THE FATTY ACID BINDING PROTEIN (A-FABP, FABP4, P2)
Abstract
Products and methods for the research, diagnosis, risk
assessment, course monitoring, treatment and prophylaxis of various
metabolic disorders and their early forms, concomitant diseases and
secondary diseases are provided. Metabolic diseases include, for
example, metabolic syndrome, non-insulin-dependent diabetes, (type
II diabetes), insulin resistance, obesity (adiposis), in addition
to diseases that are associated with disorders of the fatty acid
metabolism. Methods may include analysis of the concentration of
the adipocytic form of the fatty acid binding protein (A-FABP, FABP
4, P2) in various bodily fluids.
Inventors: |
RUZICKA; VIKTOR; (BRNO,
CZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
BIOVENDOR LABORATORY MEDICINE, INC. |
BRNO |
|
CZ |
|
|
Assignee: |
BIOVENDOR LABORATORY MEDICINE,
INC.
BRNO
CZ
|
Family ID: |
38048068 |
Appl. No.: |
14/482339 |
Filed: |
September 10, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11988822 |
Mar 3, 2009 |
8846413 |
|
|
PCT/IB2006/002383 |
Jul 21, 2006 |
|
|
|
14482339 |
|
|
|
|
Current U.S.
Class: |
435/7.95 ;
435/287.2; 435/7.92; 436/501 |
Current CPC
Class: |
A61P 5/50 20180101; A61P
43/00 20180101; G01N 2800/044 20130101; A61P 3/10 20180101; A61P
3/00 20180101; Y10S 436/811 20130101; G01N 2800/042 20130101; A61P
3/04 20180101; G01N 33/92 20130101 |
Class at
Publication: |
435/7.95 ;
435/7.92; 436/501; 435/287.2 |
International
Class: |
G01N 33/92 20060101
G01N033/92 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 21, 2005 |
DE |
10 2005 034 788.6 |
Claims
1. A test kit for the diagnosis of metabolic syndrome, of its
preliminary stages, early forms, concomitant and secondary
diseases, type II diabetes, insulin resistance, obesity or related
disorders of the metabolism, wherein the concentration of the
adipocytic form of the fatty acid binding protein (A-FABP) in
bodily fluids is determined and is used as an indicator for the
fatty acid transport capacity, comprising: a suitable carrier
material on which substances with affinity to A-FABP or parts
thereof have been immobilised.
2. The test kit of claim 1, wherein the immobilised substances with
affinity to A-FABP or parts thereof are manifested as
antibodies.
3. The test kit of claim 1, wherein the carrier material has at
least a partly planar surface.
4. The test kit of claim 1, wherein the carrier material is
encompassed by an ELISA plate.
5. The test kit of claim 1, wherein the test kit contains carrier
material, substances and solutions suited for the implementation of
an ELISA or a comparable dye assay.
6. The test kit of claim 5, wherein the test kit contains
anti-A-FABP antibodies, secondary detection antibodies, carrier
materials, washing, incubation and detection solutions.
7. The test kit of claim 5, wherein the test kit contains at least
one calibration solution containing recombinant A-FABP.
8. The test kit of claim 6, wherein the test kit contains at least
one calibration solution containing recombinant A-FABP.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/988,822, filed Jan. 15, 2008 which is the United States
National Phase under 35 U.S.C. .sctn.371 of PCT International
Application No. PCT/IB2006/002383, filed on Jul. 21, 2006, and
claiming priority to German national application no. 10 2005 034
788.6, filed on Jul. 21, 2005.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The invention relates to a method for determining the
concentration of the adipocytic form of the fatty acid binding
protein (A-FABP, FABP4, P2) for diagnostics and research of the
metabolic syndrome, of non-insulin-dependent diabetes (type II
diabetes), insulin resistance, obesity
(obesitas/adipositas/fatness) and related disorders of the
metabolism. It further relates to a test kit for implementation of
this method.
[0004] Areas of application are medicine and, in particular,
medicinal diagnostics.
[0005] 2. Background of the Related Art
[0006] Metabolic syndrome, also known as Syndrome X, insulin
resistance syndrome or multiple metabolic syndrome, represents a
disorder of metabolism of lipids, carbohydrates, proteins, minerals
etc. of the organism, which can be caused by hereditary factors
and/or conditions of life. The metabolic syndrome includes: insulin
resistance, dyslipidaemia, arterial hypertonia and adipositas.
[0007] Insulin resistance is defined as an insensitivity against
the body's own insulin. Not only the patients with type II
diabetes, but also about 20% of the practically healthy patients
who are not overweight suffer from insulin resistance. The causes
of insulin resistance have not yet been unambiguously clarified.
The characteristics of insulin resistance also include exhaustion
of beta cells, their desensitisation and apoptosis caused by
hyperinsulinaemia with gluco- and lipotoxicity.
[0008] Dyslipidaemia is a disorder of the fat metabolism in form of
hypercholesteraemia, hypertriglyceridaemia, hyperlipidaemia
(increased blood fat values as a generic term). The latter is
characterised by high triglyceride and low HDL cholesterol values,
small, dense LDL particles and a high content of free fatty acids.
The spectrum of affects ranges from "customary" (polygenic)
hypercholerstinaemia (about 10% of the population) to rare,
genetically induced lipid metabolism disorders. Dyslipidaemias are
risk factors for atherosclerosis, in particular for coronary heart
disease. Over and above this, the free fatty acids contribute to
maintaining the state of insulin resistance.
[0009] Cardiovascular diseases and in particular coronary heart
disease (CHD) are the essential complications of metabolic syndrome
as regards prognosis and thus the main cause of death for this
group of patients.
BRIEF SUMMARY OF THE INVENTION
[0010] The objective of the invention is early recognition and/or
monitoring of the metabolic syndrome, of type II diabetes, insulin
resistance, obesity and/or related disorders of the metabolism. The
invention is based on the task of developing methods and test kits
with which it is possible to determine suitable parameters for risk
assessment and monitoring of the course in diagnosis and therapy of
metabolic syndrome, type I diabetes, insulin resistance, obesity I
and/or related disorders of the metabolism.
[0011] The invention is based on the central idea that the
concentration of A-FABP in serum, plasma, urine or other
extra-cellular bodily fluids or in the fatty tissue is determined
and used for diagnosis or research of the metabolic syndrome, its
concomitant diseases, early forms and secondary diseases connected
with a disturbance of the fat metabolism.
[0012] A very central role in the origination, development and
course of the metabolic syndrome and type II diabetes is played by
the fat cells and fat metabolism. A cytoplasmatic protein of the
fat cells is the adipocytic form of the fatty acid binding protein
(abbreviated to A-FABP, FABP4 or P2), which transports longer fatty
acids, prostanoids, retinoic acid and similar, non-water-soluble
molecules in the cytoplasma of the fat cells. The concentration of
A-FABP was compared in the serum of healthy persons and type II
diabetics, the body mass index being taken into account as an
indication for the share of fatty tissue in the body mass of the
persons examined. Although A-FABP is an intra-cellular protein of
the fat cells, which hardly ought to appear in the circulation,
A-FABP levels in the nanogram range (ng/ml) are found in the serum
of normally healthy persons.
[0013] As the A-FABP levels in the serum are about twice as high as
those of the adipocytic hormone leptin (leptin concentrations.+-.8
ng/ml; A-FABP is .+-.16-20 ng/ml), it is obvious that A-FABP is
actively secreted into the circulation. Further, it cannot be ruled
out that a further (signal) effect independent of its
intra-cellular function can be ascribed to A-FABP. However, the
establishment that measurement of the concentration of A-FABP in
bodily fluids is suitable for establishment and monitoring of the
metabolic syndrome was completely unexpected.
[0014] Thus, the invention is based on the completely surprising
knowledge that a significantly increased content of F-ABP in
extra-cellular bodily fluids is connected with the metabolic
syndrome, its early forms, concomitant diseases and secondary
diseases, non-insulin-dependent diabetes (type II diabetes),
insulin resistance, obesity (obesitas/adipositas/fatness) and
related disorders of the metabolism.
BRIEF DESCRIPTION OF THE FIGURES
[0015] FIG. 1: Determination of A-FABP in the serum of 486
volunteers (100 standard-weight, healthy volunteers, 100 patients
with dyslipidaemia without obesity and without further features of
the metabolic syndrome, 86 type II diabetics, 100 obese and 100
patients with metabolic syndrome.
[0016] In the groups of volunteers in question, the A-FABP
concentration ranges shown were found (values on the Y axis are
ng/ml).
[0017] FIG. 2: Correction of the values from FIG. 1 to the Body
Mass Index (A-FABP divided by BMI=A_BMI) resulted in the
distributions as shown.
[0018] FIG. 3: A-FABP values divided by the values of the QUICKI
Index
[0019] FIG. 4: Correlation between measured concentrations of
A-FABP and insulin of volunteers.
[0020] FIG. 5: Correlation between measured concentrations of
A-FABP and glucose of volunteers.
[0021] FIG. 6: Correlation between measured concentrations of
A-FABP and QUICKI Index.
[0022] FIG. 7: Correlation between measured concentrations of
A-FABP and triglycerides
[0023] FIG. 8: Correlation of the concentration of A-FABP and
E-FABP with total cholesterol from serum (examination of samples
from 48 women).
[0024] FIG. 9: Correlation of the concentration of A-FABP and
E-FABP with HDL cholesterol from serum (examination of samples from
48 women).
[0025] FIG. 10: Correlation of the concentration of A-FABP and
E-FABP with LDL cholesterol from serum (examination of samples from
48 women).
[0026] FIG. 11: Electrophoretic separation by SDS PAGE of cell
lysates and visualisation of the protein bands received by
Coomassie dyeing.
Description of the tracks [0027] 1. T.sub.0, i.e. at the moment of
addition of IPTG [0028] 2. T.sub.2, i.e. 2 hours after addition of
IPTG [0029] 3. T.sub.4, i.e. after 4 hours at the end of the
expression [0030] 4. Supernatant after ultrasound treatment of the
bacteria [0031] 5. Inclusion corpuscles
[0032] FIG. 12: Result of the affinity chromatography of
anti-A-FABP antibodies from rabbits from a column with bound
A-FABP.
[0033] FIG. 13: Result of the affinity chromatography of
anti-A-FABP antibodies from goats from a column with bound
A-FABP.
[0034] FIG. 14: The cleaned antibodies were separated in 12%
SDS-PAGE and dyed with Coomassie Blue (left--goat,
right--rabbit).
[0035] FIG. 15: Implementation of an ELISA to determine the
concentration of F-ABP from bodily fluid.
[0036] FIG. 16: Typical calibration curve.
[0037] FIG. 17: Distribution of values for A-FABP in serum.
[0038] FIG. 18: Influence of age on the A-FABP values.
[0039] FIG. 19: Establishment of the standard range for A-FABP
values in a group of 66 women.
[0040] FIG. 20: Two-dimensional electrophoresis of he cell culture
supernatant of an adipocytic culture.
[0041] FIG. 21: Circulating levels of A-FABP in non-obese
(BMI<25) and obese (BMI>25) individuals.
[0042] FIG. 22: Serum levels of A-FABP positively correlated with
various indexes of obesity (BMI, WHR, WC and fat percentage)
[0043] FIG. 23: Correlations of serum A-FABP with insulin
sensitivity (HOMA), Fasting insulin, fasting and 2 h FP glucose
[0044] FIG. 24: Positive correlation of A-FABP with LDL, TG and
FFA
[0045] FIG. 25: Avandia.RTM. (rogiclitazone) treatment increases
A-FABP in T2DM patients (n=28)
DETAILED DESCRIPTION OF THE INVENTION
[0046] The invention relates to a method in which the concentration
of the F-ABP in bodily fluids, in particular from serum (fatty acid
transport capacity), is made use of for diagnosis and/or research
of the metabolic syndrome and related metabolic diseases. What was
also completely unexpected was the establishment that the
concentration of A-FABP in bodily fluids, in particular serum,
significantly correlates with the concentration of other parameters
of metabolic syndrome, type II diabetes, insulin resistance,
obesity and related disorders of the metabolism. For diagnosis and
monitoring of the metabolic syndrome and the aforementioned
disorders of the metabolism, the concentration of F-ABP in bodily
fluids and/or diluted solutions of them is determined.
[0047] For the implementation of the invention, immunometric
systems, in particular in combination with an optical and/or
biosensory evaluation, are particularly suited, as they allow a
simple determination of concentration of A-FABP in or from bodily
fluids. In this context, various systems, with the help of which
the concentration of A-FABP can be determined, for example by
parallel or subsequent measurement of various concentrations of the
bodily fluid to be determined, can be used, especially if a
normalisation/calibration with defined or known A-FABP
concentrations is done alongside this, by titration or by the use
of various defined concentration of capture molecules, which are
bound to a solid phase/carrier material and have affinity to A-FABP
or structures derived therefrom.
[0048] In this context, methods which entail carrier materials on
the surface of which substances suited for determination of the
concentration of A-FABP in or from bodily fluids have been
immobilised are favourable. These can in particular be molecules or
substances composed thereof, preferably proteins, which have an
affinity to F-ABP, for example anti-F-ABP-antibodies. What is also
possible is the use of immobilised substances which have affinity
to part fragments of A-FABP, in particular peptide chains, or
peptides composed of part fragments.
[0049] As a carrier material, all possible materials or coated
materials on the surfaces of which molecules can be immobilised are
suited, in particular nitrocellulose or PVDF or other materials for
bio-physical immobilisation, e.g. in wells of ELISA plates. But an
immobilisation via electrostatic interaction or immobilisation by
chemical compounding, for example by use of so-called
(cross-)linkers, is also possible. For example, gold or coated gold
surfaces are also suited for immobilisation, in particular for
biosensory measurements, for example of surface plasmon resonance
or electrochemical variables. A benefit for the implementation of
the method according to the invention is in particular carrier
materials with surfaces which have at least a partly planar
surface, for example glass or plastic chips or ELISA plates, as
they permit a particularly simple implementation of the method
according to the invention, for example with the help of scanners
or ELISA readers. Alongside planar surfaces, on which locally
addressed areas, for example spots, have been formed with
immobilised capture molecules, in particular with anti-FABP
antibodies, for example on surfaces of cellulose or chips, the base
surfaces of the wells of ELISA plates are in particular suited for
the determination of the A-FBP concentration from bodily
fluids.
[0050] As an immunometric system, the so-called Enzyme Linked
Immunosorbent Assay (ELISA) or comparable systems using dye-marked
molecules are particularly sensible, as a result of which a
particularly high sensitivity of the system for determination of
the A-FABP concentration is achieved. In this context, a carrier
material on which one or more substance(s), e.g. antibodies, with
affinity to A-FABP or derived molecules has/have been immobilised
is put into contact with bodily fluid or solutions containing
bodily fluid, with the result that the A-FABP or the derived
form/course, if applicable in the solution with bodily fluid, can
associate with the substances in the vicinity of the surface of the
carrier material. For this purpose, the bodily fluid can have been
dissolved in an incubation solution, e.g. blocking buffer, which
leads, for example, to an improvement of the signal/sound ratio as
a result of reduction of unspecific interactions. As proof of the
associated A-FABP or derived molecules on the surface, a dissolved
substance, preferably anti-A-FABP antibodies, is put onto the
carrier material, mainly following a washing step, in which context
the dissolved detection substance likewise has affinity to A-FABP
and binds to the associated A-FABP in the vicinity of the carrier
material.
[0051] The detection substance/antibodies bound on the A-FABP or
the molecules derived therefrom can be detected in various ways. It
is possible that the detection substance or anti-A-FABP antibodies
has/have already been provided with a marking, for example with a
dye, in particular fluorescent dye, or with an enzyme which can
convert a substrate, for example from a detection solution, with
optical detection, e.g. horseradish peroxidase or alkaline
phosphatase. The detection substance, in particular antibodies, can
however also have been provided with a molecule, e.g. biotin, which
for its part also has affinity to another marked substance, e.g.
streptavidin. If the detection substance/anti-A-FABP antibody
itself has not been marked, marked secondary antibodies are in
particular suited, these being added, mainly after a washing step,
and making the detection substance/anti-A-FABP antibody visible on
the surface of the carrier material or making the subsequent
optical evaluation possible.
[0052] For the diagnosis and research of metabolic syndrome, its
early forms, concomitant and secondary diseases, type II diabetes,
insulin resistance, obesity or related disorders of the metabolism,
a further benefit which is suitable is the fact that the A-FABP
concentration measured in accordance with the invention is placed
in a reference with other characteristics, e.g. age or gender, the
body mass index or with components of the metabolism which are
characteristic for the metabolic syndrome, in particular insulin
(for example Quicki values), glucose, triglyceride, adiponectin,
leptin, total cholesterol, HDL cholesterol and/or LDL cholesterol.
As a result of the combination of A-FABP with at least one further
parameter, a prognosis of other parameters of the metabolic
syndrome is also beneficially possible.
[0053] As a result of the implementation of the invention, a
deduction to characteristics of the metabolic syndrome is possible
in a very simple way, also enabling a particularly simple and
favourably priced diagnosis or monitoring of the metabolic
syndrome, of type II diabetes, insulin resistance, obesity
(obesitas/adipositas/fatness) and related disorders of the
metabolism. For example, no removal of tissue samples is necessary,
as deduction directly to the pathogenic processes in the tissue or
in the cells, as the case may be, is possible. A further particular
benefit of the invention is the fact that early stages of the
metabolic syndrome are detectable. In this way, it becomes
possible, for the first time, to initiate countermeasures for
patients before the occurrence of the pathogenic phenomena
connected with the metabolic syndrome, for example by prescription
of a diet or sport or by administration of a suitable
medication.
[0054] As a result of the invention, the use of A-FABP as a
substance similar to a hormone for influencing the fat, sugar and
energy metabolism is made possible (cf. FIG. 20). If a positive
effect of A-FABP is seen, it or its fragments or its mimetics are
applicable as medication. If the effect is negative, A-FABP
blockers are of medicinal benefit.
[0055] FIG. 20 shows a two-dimensional electrophoresis of the cell
culture supernatant of an adipocytic culture, in which a very large
and distinct spot of the A-FABP can be seen (the identity of the
protein is determined by means of N-terminal sequencing with a mass
spectrometer). The fact that no beta-tubulin (an intra-cellular
structure protein) could be detected in the supernatant with the
Western Blot is an indication for the fact that the A-FABP does NOT
enter the medium by the decay of the fat cells, but really by
secretion.
[0056] Thus, the invention provides a completely new possibility of
risk assessment and course monitoring in the diagnostics and
therapy of metabolic syndrome and type II diabetes.
[0057] The invention is to be explained in more detail below with
the help of examples of application:
EMBODIMENT 1
[0058] In 486 volunteers, A-FABP in the serum was determined. These
were 100 healthy volunteers of standard weight, 100 patients with
dyslipidaemia without obesity and without other features of
metabolic syndrome, 86 type II diabetics, 100 obese and 100
patients with metabolic syndrome. Alongside A-FABP, the following
parameters were also determined: body mass index (BMI), glucose,
insulin, quantitative insulin sensitivity check index (QUICKI),
adiponectin and E-FABP (epidermal fatty acid binding protein).
[0059] In the individual groups of volunteers, A-FABP concentration
ranges were found as portrayed in FIG. 1.
[0060] A correction to the body mass index (A-FABP divided by BMI)
resulted in distributions according to FIG. 2.
[0061] After the division of the A-FABP values by the values of the
QUICKI index, the distribution shown in FIG. 3 resulted.
[0062] From the measured data, it was seen as a complete surprise
and unambiguously that the A-FABP levels discriminate very
effectively between healthy standard volunteers and patients with
metabolic syndrome, this being independent of the body mass
index.
[0063] In addition, the concentrations of glucose, insulin,
quantitative insulin sensitivity check index (QUICKI) and
triglycerides were measured and placed into correlation with the
measured values of A-FABP (FIGS. 4-7).
[0064] It was clearly recognisable that the measured values of
glucose, insulin, QUICKI and triglycerides, i.e. characterising
parameters for the metabolic syndrome, are in a direct correlation
with the concentration of the measured A-FABP.
[0065] If the values from FIG. 1 and FIG. 2 are additionally used,
it becomes clear that the measured values of the A-FABP from bodily
fluids are in a direct connection with the metabolic syndrome and
the related metabolic diseases.
Embodiment 2
[0066] On 48 women whose serum samples were available, amongst whom
there could also be women with metabolic syndrome, the
concentrations of A-FABP, E-FABP, total cholesterol, HDL
cholesterol and LDL cholesterol were determined. Application of the
values of A-FABP and E-FABP against each of the values of total
cholesterol (FIG. 8), HDL cholesterol (FIG. 9) and LDL cholesterol
(FIG. 10) clearly showed that A-FABP closely correlates with the
most important parameters of the metabolic syndrome, of type II
diabetes, insulin resistance, obesity (obesitas/adipositas/fatness)
and related disorders of the metabolism.
Embodiment 3
[0067] Production of the recombinant human A-FABP:
TABLE-US-00001 cDNA sequence: (SEQ ID NO: 1)
ATGTGCGATGCGTTTGTGGGCACCTGGAAACTGGTTAGCAGCGAAAACTTCGATGATTACAT
GAAAGAAGTGGGCGTTGGTTTTGCGACCCGCAAAGTTGCGGGTATGGCGAAACCGAACATGA
TTATCAGCGTGAACGGCGATGTGATTACCATCAAAAGCGAAAGCACCTTCAAAAACACCGAA
ATCAGCTTTATCCTGGGCCAGGAATTTGATGAAGTGACCGCGGATGATCGTAAAGTGAAAAG
CACCATCACCCTGGATGGTGGTGTTCTGGTGCATGTGCAGAAATGGGATGGCAAAAGCACCA
CCATCAAACGCAAACGCGAAGATGATAAACTGGTGGTGGAATGCGTGATGAAAGGTGTTACC
AGCACCCGTGTTTATGAACGTGCG Amino acid sequence: (SEQ ID NO: 2)
MCDAFVGTWKLVSSENFDDYMKEVGVGFATRKVAGMAKPNMIISVNGDVITIKSESTFKNTE
ISFILGQEFDEVTADDRKVKSTITLDGGVLVHVQKWDGKSTTIKRKREDDKLVVECVNKGVT
STRVYERA
[0068] The expressed protein contained no additional amino acids or
tags.
[0069] Protein size: 132 amino acids; molar mass: approx. 14,720
Da
[0070] Expression stem: E. coli: BL21 DE3(pRSET-FABP4)-1
[0071] Expression Method: [0072] SOB-Medium (4.5 I) was inoculated
with an overnight culture of E. coli BL21 DE3(pRSET-FABP4)-1.
[0073] Cultivation was on a shaker at 37.degree. C. [0074] Inductor
IPTG was added after 3.5 hours of the cultivation at OD=0.69.
[0075] The expression was ended after 4 hours. The optical density
reached OD=1.52. [0076] The bacteria were centrifuged off and
concentrated to a theoretical OD=100 in 68.4 ml PBS buffer. [0077]
Soluble A-FABP was obtained by ultrasound treatment of the
bacteria.
[0078] Expression analysis of A-FABP (FIG. 11).
[0079] As the result of the protein separation, A-FABP was
detectable within tracks 2-5 as distinct protein bands, whereas
control track 1 did not show any corresponding bands.
Obtaining Antibodies and Processing
[0080] Against A-FABP, polyclonal antibodies were produced and
affinity-cleaned.
[0081] 1. Immunisation of the Animals
[0082] 1.1 Goat
[0083] Goat no. 589 was immunised according to the following
scheme:
TABLE-US-00002 1.sup.st day 8x intradermal 1000 .mu.g antigen +
CFA, 1:1 21.sup.st day 2x subcutaneous 1000 .mu.g antigen + ICFA,
1:1 28.sup.th day 2x subcutaneous 500 .mu.g antigen + ICFA, 1:1
35.sup.th day 2x subcutaneous 500 .mu.g antigen + ICFA, 1:1
45.sup.th day blood taken 160 ml of blood 105.sup.th day 2x
subcutaneous 500 .mu.g antigen + ICFA, 1:1 112.sup.th day 2x
subcutaneous 500 .mu.g antigen + ICFA, 1:1 122.sup.nd day blood
taken 200 ml of blood 182.sup.nd day 2x subcutaneous 500 .mu.g
antigen + ICFA, 1:1 189.sup.th day 2x subcutaneous 500 .mu.g
antigen + ICFA, 1:1 199.sup.th day blood taken 200 ml of blood
CFA--complete Freund adjuvant IFCA--incomplete Freund adjuvant
[0084] 1.2 Rabbits
[0085] Rabbits no. 68 and 69 were immunised according to the
following scheme:
TABLE-US-00003 1.sup.st day 8x intradermal 200 .mu.g antigen + CFA,
1:1 21.sup.st day 2x subcutaneous 200 .mu.g antigen + ICFA, 1:1
28.sup.th day 2x subcutaneous 100 .mu.g antigen + ICFA, 1:1
35.sup.th day 2x subcutaneous 100 .mu.g antigen + ICFA, 1:1
45.sup.th day blood taken 50 ml of blood 105.sup.th day 2x
subcutaneous 100 .mu.g antigen + ICFA, 1:1 112.sup.th day 2x
subcutaneous 100 .mu.g antigen + ICFA, 1:1 122.sup.nd day blood
taken 50 ml of blood 182.sup.nd day 2x subcutaneous 100 .mu.g
antigen + ICFA, 1:1 189.sup.th day 2x subcutaneous 100 .mu.g
antigen + ICFA, 1:1 199.sup.th day blood taken 50 ml of blood
CFA--complete Freund adjuvant IFCA--incomplete Freund adjuvant
[0086] 2. Obtaining of Serum
[0087] Blood (from both goat and also rabbit) was centrifuged for
20 minutes at 4.degree. C. and at 2,400 G, the serum obtained
stored at -20.degree. C.
[0088] 3. Affinity Cleaning
[0089] 3.1 Production of the Affinity Column
[0090] Poros AL (Applied Biosystems), 0.45 g, was provided with 1
mg of A-FABP in accordance with the manufacturer's
instructions.
[0091] 3.2 Antibody Cleaning
[0092] Antibodies were bound to the column in 0.1M PBS, pH 7.4 and
eluted with 0.1 PBS, 13 mM HCl, 0.15 mM NaCl.
[0093] The results of the affinity chromatograms can be seen in
FIG. 12 (cleaning of antibodies from rabbits) and in FIG. 13
(cleaning of antibodies from goats).
[0094] 3.3 Testing the Antibodies for Purity and Titre
Determination
[0095] 3.3.1 The cleaned antibodies were separated in 12% SDS-PAGE
and dyed with Coomassie Blue (FIG. 14).
[0096] 3.3.2 Titre Determination with Indirect ELISA
[0097] A microtitre plate (Nunc) was coated with 25 mg/well
A-FABP.
[0098] Affinity-cleaned antibodies were pipetted in an initial
concentration of 1 mg/ml and diluted 1:3 in series.
[0099] Titre was defined as an antibody dilution which has an
absorbance of less than 1.5:
[0100] Goat:
[0101] Titre: 270,000.-, quantity 4.4 mg
[0102] Rabbit
[0103] Titre: larger than 90,000, quantity: 4.8 mg
[0104] ELISA Development:
[0105] ELISA Plate:
[0106] For coating of the microtitre plate, the affinity-cleaned
goat antibody (4 .mu.g/ml) was used in hydrogen carbonate buffer
(0.1 M). After an overnight coating at 4.0.degree. C., the plates
were washed with PBS and blocked for 30 minutes at room temperature
with 0.5% BSA in TPS, 4% saccharose.
[0107] Conjugate:
[0108] Affinity-cleaned rabbit antibody was conjugated with
Biotin-LC-LC-NHS-sulfo, Pierce, cat. no. 21338, in accordance with
the manufacturer's instructions and used in a concentration of 33
.mu.g/ml.
[0109] Streptavidin-HRP conjugate was purchased from the firm of
Roche (catalogue number 1 089 153).
[0110] Calibrator:
[0111] Recombinant human A-FABP was lyophilised and used as a
master calibrator in an initial concentration of 500 ng/ml.
[0112] The actual ELISA test was held as shown in FIG. 15.
[0113] Finally, the following characteristics of the tests were
set: [0114] a) typical calibration curve for human A-FABP: with the
ELISA held, a sensitivity of the concentration measurement of
A-FABP of less than 100 pg/ml resulted (FIG. 16). [0115] b)
Recovery/dilution (see table 1)
TABLE-US-00004 [0115] TABLE 1 Monitored Expected Recovery Sample
Dilution (ng/ml) (ng/ml) O/E (%) 1 -- 36.8 -- -- 1:2 19.6 18.4
106.5 1:4 9.9 9.2 107.6 1:8 4.9 4.6 106.5 2 -- 28.1 -- -- 1:2 14.1
14.1 100.0 1:4 7.8 7.0 111.0 1:8 3.9 3.5 111.0
[0116] c) Recovery/Addition (see Table 2)
TABLE-US-00005 [0116] TABLE 2 Monitored Expected Recovery Sample
(ng/ml) (ng/ml) O/E (%) 1 8.9 -- -- 16.5 18.9 87.3 24.1 28.9 83.4
55.5 58.9 94.2 2 6.8 -- -- 15.4 16.8 91.7 24.2 26.8 90.3 53.1 36.8
93150
[0117] d) Precision intra-assay (see Table 3)
TABLE-US-00006 [0117] TABLE 3 Mean SD Sample (ng/ml) (ng/ml) CV (%)
1 13.9 0.92 6.6 2 27.3 1.08 3.9
[0118] e) Precision intra-assay (see Table 4)
TABLE-US-00007 [0118] TABLE 4 Mean SD Sample (ng/ml) (ng/ml) CV (%)
1 12.5 0.32 2.6 2 31.1 1.58 5.1
[0119] f) A value distribution for A-FABP for healthy adults as
portrayed in FIG. 17 resulted. [0120] g) The influence of age on
the A-FABP values was established in accordance with FIG. 18.
[0121] h) The influence of gender on the A-FABP values was
established (see Table 5).
TABLE-US-00008 [0121] TABLE 5 Total HDL- LDL- Triglyc- cholesterol
chol. chol. eride AFABP Age mmol/l mmol/l mmol/l mmol/l ng/ml Mean
45.5 5.21 1.46 3.35 1.13 21.18 (women): Mean 43.7 5.08 1.06 3.70
1.61 21.44 (men):
[0122] i) In a group of 66 women, the standard area was established
or set (see also FIG. 18): [0123] Minimum: 7.7 ng/ml [0124]
Maximum: 45.1 ng/ml [0125] Mean: 19.58 ng/ml [0126] Standard
deviation: 8.16 [0127] Normal range: (x.+-.2 s): 19.8.+-.16.32
ng/ml
Sequence CWU 1
1
21396DNAHomo sapiensmisc_featureA-FABP cDNA 1atgtgcgatg cgtttgtggg
cacctggaaa ctggttagca gcgaaaactt cgatgattac 60atgaaagaag tgggcgttgg
ttttgcgacc cgcaaagttg cgggtatggc gaaaccgaac 120atgattatca
gcgtgaacgg cgatgtgatt accatcaaaa gcgaaagcac cttcaaaaac
180accgaaatca gctttatcct gggccaggaa tttgatgaag tgaccgcgga
tgatcgtaaa 240gtgaaaagca ccatcaccct ggatggtggt gttctggtgc
atgtgcagaa atgggatggc 300aaaagcacca ccatcaaacg caaacgcgaa
gatgataaac tggtggtgga atgcgtgatg 360aaaggtgtta ccagcacccg
tgtttatgaa cgtgcg 3962132PRTHomo sapiensMISC_FEATUREA-FABP Protein
2Met Cys Asp Ala Phe Val Gly Thr Trp Lys Leu Val Ser Ser Glu Asn 1
5 10 15 Phe Asp Asp Tyr Met Lys Glu Val Gly Val Gly Phe Ala Thr Arg
Lys 20 25 30 Val Ala Gly Met Ala Lys Pro Asn Met Ile Ile Ser Val
Asn Gly Asp 35 40 45 Val Ile Thr Ile Lys Ser Glu Ser Thr Phe Lys
Asn Thr Glu Ile Ser 50 55 60 Phe Ile Leu Gly Gln Glu Phe Asp Glu
Val Thr Ala Asp Asp Arg Lys 65 70 75 80 Val Lys Ser Thr Ile Thr Leu
Asp Gly Gly Val Leu Val His Val Gln 85 90 95 Lys Trp Asp Gly Lys
Ser Thr Thr Ile Lys Arg Lys Arg Glu Asp Asp 100 105 110 Lys Leu Val
Val Glu Cys Val Met Lys Gly Val Thr Ser Thr Arg Val 115 120 125 Tyr
Glu Arg Ala 130
* * * * *