Note: Descriptions are shown in the official language in which they were submitted.
CA 02216189 1997-11-12
Specification:
This invention related to the one-step colloidal gold two-site sandwich immllnoassay test
kit for the determination of substances such as proteins, antibodies, hollllolles or antigens
(analyte). One-step two-site sandwich immunc.~say is con~i~te~ of three major parts,
those are: the colloidal gold-monoclonal or/and polyclonal antibody conjugate, a test
region coated with monoclonal or/and polyclonal antibodies and a control region coated
with monoclonal or/and polyclonal antibodies. The second and the third part are usually
a piece of reaction membrane such as nitrocellulose melllbl~le. During the test, the
sample is added on to a region co~ inillg colloidal gold antibody co~jug~te The target
sul.slal ce, if any, present in the sample will bind with the precoated antibody, which is
conj~lg~ed with colloidal gold. The whole complex will move up to the reaction region
by capillary action. The sul,s~ce-colloidal gold complex will then bind with the pre-
coated antibody on the test region. Because of the color of the colloidal gold conjug~te a
CA 02216189 1997-11-12
.
distinct red line will be seen on this region. If the analyte is not present in the sample the
colloidal gold complex will not react with the test region, hence no red line is formed.
The third region(control region) will react with the antibody portion of the colloidal gold
complex regardless the presence of the analyte, so that, a distinct red color band will
appear to prove the test is valid. A typical appe~ ~nce of a diagnostic test is shown in Fig
1 andFig2.
2 3
1. Control region ( pre-coated with antibody against the antibody portion of the colloidal gold conjugate)
2. Test region ( pre-coated with antibody against the analyte)
3. Colloidal Gold conjugate region ( pre-coated with colloidal gold-antibody
conjugate, which the antibody is against the analyte)
Fig 1. Side view of a one-step diagnostic test
Control
~egion
Test
region
Negative Positive
Fig.2 Top view of a bpical one-step diagnostic test
(Positive and negative results)
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For most clinical important substances eg. hormones or protein markers, an estim~tion of
concentration is required for precise clinical inte~ elaLion. In many cases, if the patient
is conr~ ed with the presence of the tested analyte, if the concentration is required, the
sample is usually re-collect for either EIA or RIA.
Even though the color intensity of the test bands are related to the concentration of the
target substance in the sample, the current One-step colloidal gold two-site sandwich
immunoassay is, however, inaccurate for semi-qu~ live purpose because the high dose
hook effect limitation.
High dose hook effect is a phenomenon which the color intensity of the test banddecreasing as the concentration of the analyte increasing. Therefore, it is not possible to
~listin~ h between low concentration and very high concentration of the analyte
presence in the sample. The high dose hook effect makes the one step diagnostic test not
suitable for semi-qu~ live purpose.
I have found out that this limitation can be overcome by providing an extra 'semi-
qu~ntit;~tive~ band and modify the control band. Such bands allow user to estim~te the
concentration of the analyte without affected by the high dose hook effect.
On the 'semi-qu~ntit~tive' band, monoclonal antibody against mouse immunoglobin is
coated. And on the 'control band', monoclonal antibody against colloidal gold particles
is coated. During the test the color intensity of the control band will not affected by the
concentration of the analyte, i.e. the color intensity of the control band will always the
same to serve as a reference color. When the semi-qu~ntit~tive band is darker than the
control band, it means that the color intensity of the test band is directly proportional to
the conce~ ion of the analyte. When the semi-qu~ntit~tive band is lighter that or equal
to the control band, it means that the color intensity of the test band is inversely
plupolLional to the concentration ofthe analyte.
A color ~Illpalison chart is included with the test kit, which can be used for direct
comparison to obtain the inrol ma~ion on the range of the analyte.
Between each band, there is a visible marker line print on, this is for the purpose of
tin~ h the function of each band.
Figure 3 which illustrate the appearance and principle of the invention.
2 3 4
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1. Semi-qu~ntit~tive band (pre-coated with monoclonal/polyclonal antibody
against the antibody portion of the colloidal gold conjugate)
2. Control band (pre-coated with monoclonaVpolyclonal antibody against the
colloidal gold conjugate)
3. Test region (pre-coated with monoclonal/polyclonal antibody against the
analyte)
Fig3. Side view of the reaction part of the semi-quantitative one stepimmunoassay test.
Semi~ t;l ~;ve
region
--~,~ ~ I~ Control
regio
Test
region
A B. C.
Fig 4. Top view of the reaction part of the semi-quantitative test kit
(three cases)
A. When the semi-qu~ a~;ve band at the semi-q~l~ntit~tive region is darker than the
control band, it means the that the concentration of the analyte in sample is directly
proportional to the color intensity of the test band at the test region.
B. When the color intensity of the semi-q~ ;ve band is the same as the control
band, this is the point where further higher concentration of the analyte will change
the test region to a lesser color intensity.
C. When the color intensity of the semi-q~l~ntitative band is lighter than the control
band, it means that the lighter the color of the test band it is, the higher theconcentration of the analyte.
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Examples:
The following examples illustrate this invention without, however, limiting the same
thereto.
EXAMPLE 1
TEST KIT FOR HORMONES
Human chorionic gonadotropin (hCG)is a glycoprotein hormone secreted by the
developing placenta shortly a~er fertilization. hCG can be detected in serum and urine of
pregnancy women as early as 6 to 15 days after conception. Most of the pregnancy test is
by means of testing the presence of hCG.
By using the concept of this invention, the one step pregnancy test kit can be used for
semi-qu~ ive purpose. There are three sites on the nitrocellulose membrane whereimmllnoreaction occur. 1. The test region, 2. The semi-qll~ntit~tive region, and 3. The
control region. During the test, i.e. immerse the test strip into the urine and serum, the
beta sub-unit of hCG in patient's urine or serum, if any, will react with colloidal gold-
anti-hCG conjugate. The complex will move upward to the test region by capillaryaction. The alpha sub-unit of hCG will then bind to the anti-alpha hCG anitbody which is
coated on the test region. This is the so called sandwich immuno-reaction and hCG, in
this case, acts as a bridge between the two antibodies. For positive result, a color band
will appear on test region because of the colloidal gold. Absence of the color band
suggests a negative result. To serve as a control and control, a color band at the control
region will always appear regardless of the presence of hCG in the test specimen.
The one-step semi-qll~ntit~tive pregnancy test kit is shown in Fig. 5. The tip of the test
kit (part 5 as shown below) is immen~ed into sample during the testing. The sample will
move to a piece of nitrocellulose membrane which colllaills part 2,3,and 4, which
corresponding to semi-q~l~ntit~tive region, control region and the test region respectively.
2 3 4 5
D DD,~ / ~
1. Absorbent mat
2. Antibody coated (anti-mouse IgG) semi-qll~ntit~tive region
3. Antibody coated (anti-colloidal gold) control region
4. Antibody coated (anti-hCG) test region
5. Colloidal gold-antibody conjugate region
Fig: 5 One-step Semi-quantitative Pregnancy test
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A color reference card will be accompany with the test kit for semi-quantify of hCG, as
showed in Fig.6.
C ~ ~__
c~ L ~ _ ~ ,=
T ~ _
OmIU/ml lOmIU/ml SOmIU/m1 200mIU~ml 5IU/ml 50IU/ml 200IU/ml
Concentration of hCG in sample
Fig.6 Semi-quantitative Color Reference Card
EXAM[PLE 2.
TEST FOR TWO HORMONES:
The described test strip can able to detect both hCG and Luteini7:in~ Hormone (LH) in
one test strip. Fig.7 demonstrate the appearance of one step hCG/LH combination test
strip.
Control band
HCG band
LH band
Fig. 7 Illustration of a hCG and LH combination one step test strip
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On a single device both anti-hCG and anti-LH are coated on two di~ele,.L region which
separated by visible marker lines.
