Note: Descriptions are shown in the official language in which they were submitted.
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~EHRINGWERKE AKTIENGESELLSCHAFT 86/~ 036 - Ma 603
Dr. Ha/~n
A method for the determination of anti-HlV, means there-
S fore and their use in this method
The invention relates to a method for the imrunochemical
determination of HIV-sPecific antibodies, which employs
the competition principle, as well as to means suitable
for this method and their use. The method is suitable
for the highly sensitive and specific detection and det-
ermination of HIV-specific antibodies of the IgG and IgM
classes in humans.
A virus (retrovirus) or a closely related group of v;rus-
es, which have recently been designated as human immune
deficiency virus (HIV) but are also referred to as T-lym-
photropic virus type III (HTLV III) or lymphadenopathy-
associated ~irus (LAY) is regarded as being the causative
; 20 a~ent of the recently recognized infectious disease AIDS
(Acquired I~mune Deficiency Syndrome), which shows a
high mortality rate.
The virus can be transmitted by human blood and contamin-
ated blood products and has been isolated from cerebro-
- spinal fluid, seminal fluid, lacrimal fluid, and saliva.
The serological determination of HIV-specific antibodies
is carried out to clarify whether an individual has had
contact with the virus and is to be regarded as a potent-
ial carrier of the infectious agent~ An essential signif-
icance of the ant;body determination lies in the use as a
diagnostic tool for AIDS (together with other investiga-
tions and the case history) as well as in the lowering of
~ 35 the risk of transmission of the causatiue agent of the
j~ disease via transfusion, organ transplantation, blood
products or semen by excluding material donated by
~ antibody-positive subjects. Moreover, the HIV antibody
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determination ;s of importance in members of so-called
risk groups (homosexuals, drug addicts, hemophiliacs,
children of infected mothers) as well as in epidemio-
logical investigations.
The detection of anti-HIV using the Western blot tech-
nique as is described for example in US Patent 4,520,113
is regarded as being the most sensitive detection method
for HIV-specific antibodies at present. In this method,
concentrated and inactivated HIV protein material is sep-
arated according to molecular weights by means of elect-
rophoresis and then transferred to nitrocellulose paper,
preferably electrophoretically. As a modification of US
Patent 4,520,113, where an RIA is used, an antigen immob-
ilized in such a way can be used for an indirect ELISAwithout loss of sensitivity, and in this the Jse of spar-
ingly soluble dye systems in the enzyme reaction makes
it possible to visualize the bands at which an immune
reaction has taken place. Stained virus-specific bands
can then be differentiated from non-virus antigen-specific
and cross- or unspecific-reacting contaminants after cali-
bration with suitable control material, and therefore a
discrimination between genuine- and false-positive reac-
tions can be accomplished.
~ 25
; Furthermore a method is described which is a competitive
; ELISA and is said to fulfil the criteria o~ the Western
blot technique with regard to specificity and sensitiv-
ity (Arzte-Zeitung/No. 1,25./26.10.8S, p.2û).
In this method undiluted samples are used. It can be
used for the simultaneous detection of HIV-specific an~i-
bodies of the IgG and IgM classes. The method can be
carried out using a Wellcome test kit, named We~lcozyme
3~5 Anti-HTLV III. Microassay plates onto which purified HIV
antibodies are bound, and onto the latter, in turn, HIV
antigens are immunochemically immobilized, are used for
the solid phase. The samples are incubated in the wells
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together with horseradish peroxidase-labeled anti-HIV. In
this, the antibodies against HIV from the sample compete
with the labeled anti-HIV for the HIV binding sites on the
solid phase. The amount of labeled antibody bound in the
wells is inversely proportional to the anti-HlV concen-
tration in the sample.
Very frequently class II human leukocyte antigens tHLA Il,
particularly DR4), which originate from the human lymph-
ocyte cell lines usually used for HIV culture, and canlead to false-positive results particularly in polytrans-
fused patients (anti-HLA DR4), are held responsible for
false-positive results in the other presently used tests.
This type of unspecific reactions is excluded in the com-
petitive test kit mentioned for the detection of HIV-
specific antibodies (Walgate R., ~eardsley T~, (1986)
Nature 320, 96) by the use of the human cell line CCRF-
CEM to culture the virus, since these cells carry no HLA
of class II.
If, in the competitive ELISA described, use was made of
HIV which was cultured in the H9 cell line which is
known for better yields, it was found that extremely
large amounts of virus starting material are necessary
to purify the trapping antibodies for immobilization on
the solicd phase, on the one hand, and to achieve the
saturation necessary for maximum sensitivity of the trap-
ping antibodies immobilized on the synthetic material
with antiyen, on the other hand, and that furthermore
the immunoadsorptive purification of human serum only
proceeds unsatisfactorily with regard to an improvement
of the specificity when HIV antigenic material cultured
on H9 and thereby contaminated with nuclear, mitochondrial
and leukocytic antigens is used. In addition, onty very
low yields of specific human antibodies were obtained,
which makes use as a screening test impossible on econ-
omic grounds.
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It was found that a competitive method for the detection
and for the determination of antibodies against HIV,
both of the IgM and the IgG classes, is possible on use
of a solid phase onto which HIV antigens, which may also
be contaminated by antigens originating from the host
cell, may be bound directly, i.e. without antibody-
mediated binding, when labeled antibodies against HIV
are used, which react neither with antigens or the host
cell nor with any antigens from human tissue.
The invention relates to an immunochemical competitive
method for the detection and for the determination of
antibodies against HIV using a solid phase consisting of
a carrier and, bound thereto, envelope and core proteins
of HIV and labeled antibodies, directed against envelope
and core proteins of HIV, in which the antibodies comp-
ete for the binding sites on the soLid phase, wherein
the proteins are irreversibly bound directly or via a
non-immunochemically binding spacer to the carrier.
The labeled antibodies used in the method are of a type
not reacting with antigens of an HIV-free host cell or a
medium for the host cell, or with human, HIV-free tissue.
The invention also relates to an assay kit for the detec-
tion and for the determination of antibodies against HIV
in an immunochemical competitive method comprising a
carrier to which envelope and core proteins of HIV are
bound irreversibly either directly or through a non-
;mmunochemically binding spacer, as well as labeledantibod;es against envelope and core proteins and, where
appropriate, means for the detection of the labeling.
It is advantageous that the sensitivity of the method
according to the invention corresponds to the sensitivity
of the Western blot technique since even samples with
low HIV antibody titers lead to a significant inhibition
of the bind;ng of the labeled antibodies which a.lows
13~5;4~.1
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visual evaluation of the color development when, for
example, an enzyme marker is used, and makes possible a
discrimination of positive and negative samples with ex-
tremely high selectivity, that even early seroconversion,
caused by HlY-specific IgM antibodies, can be detected
reliably, that no interference and thus false-positive
results occur through human leukocyte antigens (HLA),
nuclear antigens or mitochondrial ant;gens, so that a
trial, using conventionaL control antigens from the host
cell, as to ~hether a positive result is specific is su-
perfLuous, and that in sera, citrated, heparinized and
EDTA-plasma and also in recalcified plasma of human ori-
gin reliable determination of HIY-specific antibodies
is guaranteed and inactivation of the samples for 30
minutes at 55C (HIV inactivation) and even for 10 hours
at 60C (hepatitis ~ virus inactivation) does not lead to
false-positive results.
Antigenic preparations of HIV suitable for the solid phase can
be prepared by first obtaining HIV-positive permanent T cells
a~ described in Canadian Patent 1,266,243 and obtaining an ~IV
preparation as de~cribed in U.S. Patent 4,520,113.
In th;s, the HIV is first propagated after transmission
to the human T cell line H9 by co-cultivation ~ith lym-
phocytes of an AIDS patient and then separated from thehost cell culture supernatant by ultracentrifugation.
After removal of cell debris and fat the virus material
;s purif;ed by sucrose density gradient centrifugation
and finally inactivated by addition of Tr;ton~ and heat
treatment.
Su;table carrier materials for the solid phase include
plastics such as polystyrene, polyvinyl chloride, nylon
:,
and other synthetic polymers, natural polymers ~uch as
cellulose~ as well as derivatized natural polymers such
as cellulose acetate and nitrocellulose as well as glass,
particularly as glass fibers.
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The carriers can be in the form of spheres, rods, tubes,
and microassay plates. Sheet-like structures such as
paper strips, platelets and membranes are also suitable.
The surface of the carriers can be permeable as well as
impermeable to aqueous solutions.
Spheres, tubes, paper strips and membranes are preferred
carriers. Microassay plates are particularly preferred
carriers~
The solid phase is prepared by irreversibly binding an
antigen preparation of HIV to the carrier.
Irreversible binding in the sense of the invention is
present, for example, in tne case of
1) adsorptive binding, which is not split by immuno-
chemical agents, such as high-affinity antibodies or
by the agents, such as labeled antibodies, dilution
and buffer solutions, used in the method,
2) bioaffinity binding mediated by a non-immunochemically
binding spacer~ in which the spacer may consist of
biotin and avidin or of other pairs of receptors and
ligands,
3) direct covalent binding,
4) covalent binding brought about through a chemically
bifunctional spacer
Covalent binding is preferred when water-permeable car-
riers are used, and adsorptive binding is preferred when
water-permeable or ~ater-impermeable carriers are used.
Direct adsorptive binding of HIV antigen preparations to
gamma ray-treated polystyrene as a carrier is particular-
ly preferred.
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Antibodies intended for labeling are selected on the
basis of the results from the Western blot analysis of
sera from AIDS patients.
5 In order to minimize the risk of false-negative diagno-
sis, in a method for the detection of pre-AIDS and AIDS
the possibility of detection of at least two antibody
specificities, namely those for a core protein and an
envelope protein, must be given in accordance with the
present state of knowledge.
Suitable combinations of specificities are, for example,
those which recognize
1. the core protein designated p24 and the envelope
protein designated gp41 or
2. p24 and the envelope protein designated as gp120.
The detection of further antibody specificities, namely
those for the core proteins designated p18, p30, and p55
and the HIV polymerases designated as p53 and p65 means
an additional safety measure in the detection of anti-HIV-
positive samples.
In order to exclude a false-positive diagnosis the anti-
bodies intended for labeling may not have any specificit-
ies which detect constitutents of human tissue such as,
for example, leukocyte antigens, nuclear and mytochond-
rial proteins.
Antibodies of AIDS patients can be used for labeling pro-
vided they fulfil the previously described criteria as
ascertained in the Western blot technique.
Furthermore, polyclonal antiboclies obtained from sera
of animals immunized with HI~ can be used provided these,
after absorption of the specificities which recognize
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constituents of the host cell and of the culture medium,
fulfil the above criteria after testing in the Western blot.
Monoclonal antibodies are also suitable. In this case,
at least two must be used in one of the above combinat-
ions of specificities, i.e. for example in the combin-
ation anti-p24 and anti-gp41.
Radioactive isotopes, fluorescent and chemiluminescent
dyes as ~elL as enzymes, whose detection is possible by
chromogenic, luminogenic or fluorogenic substrate systems,
can be used as markers.
Labeling is performed by methods which are described as
the state of the art for the above markers.
Where the antibodies are labeled with peroxidase the
periodate technique of Nakane et al., 1974, J. Histochem.
Cytochem. 22, 1084-1090, can be used or a method accord-
ing to Ishikawa et al.~ 1983, J. Immunoassay 4, 209-327,
;n which the partners are linked with a heterobifunctional
reagent.
The method according to the invention can be performed,
for example, by, into the wells of a microassay plate
which contains bound a previously described HIV antigen
preparation,
a3 simultaneously adding the sample and a solut;on of
the labeled antibody, optionally adding the sample
first and a solution of the labeled antibody after a
certain time, removing the solution after a certain
incubation time and washing the wells with a buffer
and then measuring the labeling in the wells
or
b) first adding a solution containing the labeled anti-
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body, removing the solution after a certa;n incubat-
ion ti-e and washing the wells with a buffer, opt-
ionally drying the microassay plate, then adding the
diluted sample and incubating for a certain time,
then removing the sample from the wells and measur-
ing the labeling in the sample.
The method according to the invention can also be per-
formed in the case the use of a diagnostic element which
contains the solid phase and some or all the necessary
reagents ;n dry form.
The following example represents an embodiment of the
invention, without, however, limiting it thereto.
Example
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1. Binding of HIV antigen onto microassay plates
An HIV antigen preparation prepared according to US
Patent 4,5Z0,113~ pur;fied by sucrose density gradi-
ent centrifugation and heat-inactivated was predilu-
ted to 50 ~g/ml protein w;th 100 mmol/l sod;um bicar-
bonate of pH 9.6. From this dilution a 2-fold dilution
series was set up with 10 dilutions in the same buf-
fer, i.e. a series with the concentrations 50, 25,
1Z.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.2 and 0.1 ~g/ml
was obtained. Each 100 ~l portion of each dilution was
placed in 16 wells of type ~ microassay plates from Nunc,
Roskilde, Denmark. The assay plates filled with the
dilutions were left at Z0C for 18 hours, the sol-
ut;ons in the wells were then aspirated and the wells
were washed 3-4 times with 200 ~l of a solution of
10 g/l of bovine serum albumin in phosphate-buffered
physiological saline, pH 7.4 (PEIS) by filling and
aspiration, and the assay plates were then dried
over silica gel at 20C.
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2. Preparation of a peroxidase-labeled antibody against
HIV
Serus from an AIDS patient which was selected by the
Western blot technique and contained antibody spec-
ificities for the core proteins p18, p24, p30 and
p55, for the envelope proteins gp41 and gp120 as
well as for the HIV polymerases p53 and p65 was frac-
tionated by chromatography on DEAE cellulose. For
this, the serum was dialyzed against a buffer of 30
mmol/l Na2HP04/NaH2P04, pH 7.0, and added onto a
column filled with DEAE cellulose DE 32 (Whatman)
and equilibrated with the same buffer. The IgG fra-
ction obtained in the forerun was dialyzed against
PBS and adjusted to 4 mg/ml protein.
A further test with the Western blot technique show-
ed that all the above antibody specificities were
recovered in the IgG fraction.
The IgG fraction was then reacted with N-gamma-malei-
midobutyloxysuccinimide (GM~S), obtained from æehring
Diagnostics, as described by Tanamori et al., 1983
in J. Immunol. Meth. 62, 123-131. 2-Iminothiolanhyd-
rochloride (Sigma, catalog no. I 6256) was reacted
with horseradish peroxidase (POD), obtained from
Boehringer Mannheim, catalog no. 413470, as described
by King et al. 1978 in ~iochem. 17, 1499-1506. An
IgG-POD conjugate was prepared from the GM~S-IgG
conjugate and the iminothiolane-POD conjugate, as
described by Tanamori.
The solution of the IgG-POD conjugate obtained had a
protein content of 440 ~g/ml. The ratio of POD to
IgG was determined to be 2.5. The solution was then
diluted to 500 ng/ml IgG-POD with a solution of 50
ml/l of fetal calf serum, 5 g/l polyoxyethylene-(20)-
sorbitan monolaurate (Tween 20R) in PBS and was
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designated as ant;-HIV-POD.
3. Obtaining a TM~ substrate preparation
A substrate system or a substrate preparation con-
taining hydrogen peroxide and tetramethylbenzidine
(TM~), which was prepared from two stock solutions,
was used for the detection of anti-HIV-POD.
Stock solution 1: TMB dihydrochloride was dissolved
w;th stirring at a concentration of 5 g/l, i.e. 16
mmol/l, in twice-distilled water and adjusted to pH
1.5 with 5 N hydrochloric acid. Penicillin G was
added to this solution with stirring in a final con-
centration of 200 mg/l, i.e. 0.56 mmol/l.
Stock solution 2: 1.4 ml glacial acetic acid, 1.5 ml
1 N NaOH and 250 mg, i.e. 3 mmol H22 as a urea-
hydrogen peroxide adduct were added to 900 ml of
twice-distilled water. After dissolution was com-
plete it was made up to 1 l with twice-distilled
water.
TM~ substrate preparation: one part by volume of
stock solution 1 and 10 parts by volume of stock
solution ~ were mixed with one another.
4. Optimization of the method
3û 16 sera, which were confirmed as being HIV negative
by the ~estern blot technique, were diluted 1:5 with
anti-HIV-POD (1 part by volume serum plus 4 parts by
volume anti-HlV-POD).
125 ~l of the dilution was added to each of ~e wells
of the microassay plates treated with HIV antigen
preparation and incubated for 1 h at 27C. The
dilutions were aspirated and the wells washed four
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times with a solution of 1 g/l Tween 2 ~ in PBS by
filling and aspiration. 100 ~l of the TMB substrate
preparation was then added to each well and incubated
for 30 min at 20 to 22C. The incubation was ended
by addition of 100 ~l portions of 1 N sulfuric acid.
The extinction of the solutions was measured against
PBS at 450 nm.
The solutions from the wells which had been loaded
with 3.12 ~g/ml HIV antigen showed an E4so between
1.6~ and 1.74. The solutions from the wells loaded
with 1.56 ~g/ml HIV antigen showed an E4so between
1.38 and 1.45.
Wells of microassay plates were then treated with an
HIV antigen preparation of exclusively 2.5 ~g/ml in
the previously described manner. The evaluation of
the 16 sera in the wells treated ~ith 2.5 ~g/ml HIV
antigen preparation gave a mean E4so of 1.56 + 0.05
w;th the same test procedure.
Since in the determination of anti-HIV a limiting
value fixing of E greater than 0.8 for anti-HIV-neg-
ative and of E less than 0.8 for anti-HIV-positive
samples is regarded as being favorable (the limiting
value is defined as half the mean extinction of sam-
ples confirmed to be negative), a concentration of
2.5 ~g/ml HIV antigen preparation was selected for
the preparation of microassay plates for the deter-
mination of anti-HIV (anti-HIY assay plates) in the
treatment of the wells which is also designated as
loading.
In all the cdeterminat;ons of anti-HIV described in
the ~ollowin~ text, the reagents used in the hitherto
described preliminary tests, namely the anti-HIV-POD,
the TM~ substrate preparation and the solution of
1 g/l Tween 20 in PBS designated as washing buffer
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below~ were furthermore used.
5. Determination of human antibodies against HIV
a) 25 ~l serum or plasma and 100 ~l anti-HIV-POD
were added to the wells of anti-HIV assay plates
and incubated for 1 h at 37C~ The content of
the wells was removed by aspiration and the wells
were washed four times with washing buffer. 100 ~l
TM~ substrate preparation was added to each well,
incubated for 30 min at 20-22C and the incu-
bation ended by the addition of 100 ~l of 1 N
sulfuric acid. E450 of the colored solution
was measured against a blank PEIS.
Samples which produced an E4so of less than 0.30
were graded as anti-HIV positive, those whose
E4so was in the range of 0.31 to 0.80 as anti-
HIV borderline, and those which produced an E450
above 0.81 as anti-HIV-negative.
Of 420 samples which were anti-HIV-positive in
the Western blot technique, 419 samples were like-
wise found to be positive.
On 1a96 plasma or serum samples of healthy sub-
jects or of subjects showing other pathological
conditions, the results shown in Table 1 were
obtained.
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The 5 samples from subjects from risk groups
which initially gave a positive or borderline
reaction were aditionally investigated with the
Western blot technique. Of these, only the 2 sam-
ples were positive which according to the method of
the invention were repeatedly found to be positive.
The findings thus show that the method according
to the invention is in good agreement with the
Western blot technique.
b) Samples of 50 ~L whole blood were diluted with 50
~l distilled water containing 20 g/l Triton X-100R
and 0.2 mol/l trisodium citrate, and SO ~l of
this lysate was added to wells of anti-HIV assay
plates. After an incubation of 1 h at 37C 100 ~l
of anti-HIV-POD was added and then incubated again
for 1 h at 37C. The content of the wells was
removed by aspiration and the wells washed five
times with washing buffer. 100 ~l TMB substrate
preparation was added to each well, incubated for
30 min at 20-25C and the reaction ended by
addition of 100 ~l 1 N sulfuric acid. The ex-
; tinction of the solutions was measured at 450 nm
Z5 against a blank of PBS ~E4so).
The evaLuation of the samples is as described
under 5 a). Of 51 and 5 blood samples which, as
serum, were positive and weakly positive, res-
pectively, in the Western blot technique, all the
samples were likewise found to be positive.
: :
Of 226 blood samples which reacted as serum anti-
HIV-negative, all the samples were likewise found
to be negative. This shows that the method accord-
ing to the invention is in good agreement with
the Western blot technique regarded as a reference
method.
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c) Samples of 50 ~l serum or plasma or of 50 ~l
lysate of whole blood obtained as described ;n 5 b)
were added to the wells of the microassay plates
with 100 ~l of a solution of 5 ~g/ml IgG-POD, pre-
pared by dilution of the ~t40 ~g/ml IgG-POD con-
jugate described in ~xample 2, to 5 ~g/ml with
the already described solution containing fetal
calf serum and Tween 2 ~ in P~S and incubated
for 10 min at 45C.
The wells were then washed 5 times with washing
buffer. 100 ~l TM~ substrate preparation was
then added to each well and incubated for 5 min
at 20-22C. The incubation was ended by addit-
ion of 1 N normal sulfuric acid. The evaluation
was as described in 5 a)~
Of 51 and 5 samples which, as serum, were positive
and weakly positive, respectively, in the Western
blot technique, all the s3mples were likewise
found to be positive.
Of 225 samples which gave an anti-HIY-negative
reaction, all the samples were likewise found to
be negative.
Thus it is evident that the variant, according to
the invention, of a rapid method is in good agree-
ment with the Western blot technique regarded as
being a reference method, with regard to sensit-
;vity and specificity.
d) 100 ~l of anti-HIV-POD was added to the wells of
anti-HIV assay plates and incubated for 1 h at
37C~ the anti-HIV-POD wash aspirated and the
wells were washed four times with washing buffer.
The anti-HIV assay plates treated in this way were
dried over silica gel at 20-22C.
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25 ~l sample and 100 ~l of a solution of 10 g/l
bovine serum alburnin in 50 mmol/l Tris, adjusted
to pH 7.4 with HCl, were then added to the wells.
After incubation at 37C for 1 h the content of
the wells was removed and added tc, wells of an
unloaded assay plate, mixed there with 100 ~l
TM8 substrate preparation, the mixture was left
for 30 min at 20-22C and 100 ~l 1 N sulfuric acid
was added. E4so was then measured against PBS.
The mean E4so values from triple determination
per sample are given in Table 2 for samples which
were graded according to the method described
under a~ as anti-HIV-negative, anti-HIV border-
line and anti-HIV-positive.
TA~LE 2
E450
Anti-HIV-negative
samples 1 0.140 0.148 0.136
2 0.218 0.206 0.205
3 0.267 0.279 0.270
4 0.275 0.274 0.272
Anti-HIV borderline
samples 5 0.609 0.454 0.457
6 0.308 0.316 0.351
Anti-HlV-positive
samples 7 0.769 0.674 0.773
8 0.891 0.865 0.820
9 1.202 0.953 0.863
- 10 1.678 2~020 2.202
Table 2 shows that in contrast with embodiments a) ~o c)
the extinction of anti-HIV-negative samples is low and
of anti-HIV-positive samples is high here. The border-
line value for E4so for anti-HIV-negative samples is also
to be set at 0.30 here.
