“United States Patent
[19]
[11]
Morris et a1.
3,905,767
[451 Sept. 16, 1975
[54] PROCESS FOR QUALITATIVE ANALYSIS
3,614,231
10/1971
OR QUANTITATION OF ANTIGENS OR
3,730,842
5/1973
Wyatt.
ANTIBODIES
3,734,622
5/1973
Adler . . . . . . . 1 .
3,754,867
8/1973
Guentherh.
[751 Inventors: David Alexander Nathaniel Morris,
Elkhart; Melvin Dee Smith,
Shaw ............................. .. 250/574 X
356/103 X
. . . . . ..
356/103
23/253 TP
3,770,380
11/1973
Smith . . . . . . . . .
3,776,817
12/1973
Pfordten .................... .. 195/1035 R
. . . . . ..
23/253 TP
. Mishawaka, both of Ind.
[731 Assignee: Miles Laboratories, Inc., Elkhart,
Ind.
Jan. 30, 1974
[22] Filed:
[21] Appl. No.: 437,732
Primary Examiner-Morris O. Wolk
Assistant Examiner-Sidney Marantz
Attorney, Agent, or Firm—George R. Caruso
[5 7]
ABSTRACT
A process for rapid qualitative analysis or quantitation
of antigens or antibodies with an antigen-antibody re
[52]
US. Cl. .... .. 23/230 B; 23/253 R; 195/1035 R;
[51]
[58]
Int; Cl.2 ................. .. G01N 21/22; GOlN 33/16
250/574; 356/103; 424/12
Field of Search ...... .. 23/253 TP, 230 B, 253 R;
424/12; 356/103; 250/574; 195/103.5 R
[56]
References Cited
UNITED STATES PATENTS
2,666,355
l/1954
Trurnit ............................ .. 424/12 X
action in which a precipitate is formed, which process
comprises contacting a surface of a gel having a ?rst
reagent incorporated in at least a surface part thereof
with a second reagent which is reactible with said ?rst
reagent to form a precipitate which remains in a sur
face portion of the gel, projecting a light beam
through the area of contact and measuring the extent
to which the light beam is scattered by the precipitate.
14 Claims, 4 Drawing Figures
3,905,767
1
2
PROCESS FOR QUALITATIVE ANALYSIS OR
QUANTITATION OF ANTIGENS OR ANTIBODIES
cred a major drawback in the use thereof. For single or
double diffusion at least six hours and usually days are
required to obtain an end point. With immunoe~
lectrophoresis, at least 18 hours are usually. required
before a determination may bemade. With such long
time requirements for the generation of data, it can be
seen that these techniques are not compatible with
BACKGROUND OF THE INVENTION
1. Field of The Invention
This invention relates to a process for rapid qualita
tive analysis or quantitation of antigens or antibodies
modern diagnostic practices which require almost‘ im
with an antigen-antibody reaction in which a precipi
tate is formed. More speci?cally this invention con
mediate results.
cerns an immunochemical process in which an antigen l0
It is also known that with these techniques it is criti
‘ antibody precipitate is formed and remains in a surface
cal that the size and shape of the openings in the gel
layer be correct and that the reagents be properly
portion of a gel and in which one of the antigen or ‘the
antibody is rapidly qualitatively analyzed or measured
quantitatively while the precipitate‘ so remains.
2. Description Of The Prior Art
placed therein. 7
15
I
SUMMARY OF THE INVENTION
The process of this invention for rapid qualitative
analysis or quantitation of antigens or antibodies with
an antigen-antibody reaction in which a precipitate is
In recent years the formation of a precipitate by a re
action of an antigen with an antibody in a suitable gel
has become an essential tool in biochemical analysis.
Although such reactions were generally observed in the
double diffusion and immunoelectrophoresis. Each cat
formed includes contacting a surface of a gel having a
?rst reagent incorporated in at least a surface part
thereof with a second reagent which is reactible with
said ?rst reagent to form a precipitate. A light beam is
projected through the area of contact away from the
egory will be discussed only brie?y herein, since it is
surface of the gel and the light scattered by the precipi
late l800’s and used as clinical tools soon thereafter,
recent re?nements in procedures have developed three
distinct categories thereof, namely, single diffusion,
recognized that they are all well known to those skilled 25 tate is qualitatively analyzed or quantitatively mea
in this art and complete descriptions thereof may be
sured with suitable measuring means.
with the new and novel process of this invention an
found in references such as Work, TS. and Work, E.,
immunochemical assay is completed in a much shorter
Laboratory Techniques in Biochemistry and Molecular
time than was possible with prior art techniques. Fur
Biology, Vol. 1, 1972, North-Holland Publishing Co.,
ther, the preparation of the gel in which the reaction
Amsterdam.
occurs is simpli?ed as compared to the preparation of
For single diffusion a layer of a gel is prepared con“
prior art gels for immunochemical assays.
taining either an antigen or a speci?c antibody there
for. Since an antibody is normally incorporated in the
gel, our discussion will proceed accordingly. An open
ing is-formed in the gel and a quantity of antigen placed
therein. A ring of precipitate forms about the opening
and migrates radially away therefrom. After a certain
The novel process of this invention will be more eas
ily understood by reference to the accompanying draw
ings in which:
.
FIG. 1 is a semi-schematic view of an apparatus used
fixed time the area or diameter of the ring is measured.
This measurement provides data for determining the
antigen concentration and, if the antigen is unknown,
its identity.
BRIEF DESCRIPTION OF THE DRAWINGS
35
.
' Double diffusion may be carried out in. a glass tube
in which an antigen and an antibody are introduced
into compartments on opposite sides of a common gel
in practicing the invention;
FIG. 2 is a cross-sectional view taken along the line
2—2 of FIG. 1 of a gel and a supporting substrate mem
ber. therefor used in the present invention;
FIG. 3 is a fragmentary semi-schematic view of an al
ternative apparatus used in practicing this invention;
,
compartment. As the antigen and antibody diffuse 45 and
FIG. 4 is a graph which is usedrin the practice of this
through the gel a precipitate forms at a place of equiva
invention to determine unknown IgM (immunoglobu
lence thereby providing data for determining the un
lin-M) concentrations, which graph consists of a family
known concentration and/or material. Another double
of curves for different concentrations of goat anti
diffusion technique employs a layer of common gel
50
human IgM in a gel, the abscissa being calibrated in
which is prepared with openings or wells into which an
human IgM concentrations and the ordinate calibrated
tigen and antibody are selectively placed. A line or arc
in microamperes.
forms in the gel at the points of equivalence of the anti
‘ gen-antibody reaction thereby providing data for deter
DESCRIPTION OF THE PREFERRED
mining the unknown concentration or identifying the 55
EMBODIMENTS
unknown material.
An apparatus is shown in the accompanying FIGS. 1
The third technique, immunoelectrophoresis, in
and 2 which is suitable for practicing the invention. It
volves the movement of molecules in an electric ?eld.
is believed that the process disclosed herein willgbe
In this technique, an electric potential is applied across
more easily understood if such apparatus by which it
a layer of gel to cause a movement through the gel of
may be practiced is ?rst described. It is to be recog
an unknown material placed in an opening in the gel.
A material capable of forming a precipitate with the ‘ nized that the novel process of this invention is not lim
ited in any way by the apparatus illustrated and dis—
unknown is then introduced into another opening in
cussed herein.
.
, the gel. The resulting precipitate pattern provides data
for determining the unknown concentration or mate
rial.
,
'
‘
Although these techniques provide accurate results,
the time required to generate these results is consid
.
Referring to FIGS. 1 and 2, a substrate member 12
is shown which includes a top plane surface 13 and a
re?ective bottom plane surface 15. A gel layer 17 is in
laminar relation with member 12, being disposed on
3,905,767
3
4
and in intimate contact with'surface 13. Gel layer 17
has an exposed .top surface 21. Substrate 12 and gel‘ 17
?ected light beam 28 is scattered thereby and‘ a seg
ment of‘ this scatteredlight is transmitted by light col
lecting tube 32 to phototube 33 which generates an
electrical current in proportion thereto. This electrical
form a unitarydevice l8.
'
1
In the practice of this invention device 18 is placed
on a suitable support (not shown) in operative position
current is sensed by current measuring means 34 and
converted to a suitable readout form of microamperes
which is displayed on dial 35. If there is no precipitate
at the contact area to scatter re?ected light beam 28,
less light strikes light collecting tube 32 and a corre
with respect to alight source 25 and a light sensing -
means ‘31. Light source 25 projects a light beam 26 in
a direction to strike the re?ective surface 15 at an
oblique angle and be reflected away therefrom through
Light sensing means 31 consists of a light'collecting
tube 32, a phototube 33 optically aligned therewith and
spondingly low ?gure is displayed by the dial.
It will be recognized that substantially the same pro
cedural steps are‘ practiced with the alternative appara
tus shown in FIG. 3. Therefore, such step by step pro
a current measuring means 34. Light sensing means 31
cedure will not be set forth herein. .
is mounted so that light collecting tube 32 is spaced
from surface 21 and is optically aligned with intersec
tion' point 27, being clear of light beam 28. Current
The graph of FIG. 4' shows typical- relationships be
tween goat anti-human IgM (WI-IO nomenclature)
concentrations in the gel of 0.995 mg/ml, 0.68 mg/ml
measuring means 34 has a calibrated dial 35.
and 0.405 mg/ml and observed opacity, as expressed in
microamperes, for various known concentrations of
an intersection point 27'with top surface 21 as are
flected light beam 28 and as scattered light beams 29.
1 Referring now to FIG. 3 in which an alternative appa
10
ratus is represented, a transparent substrate member 51
is‘shown which includes a top plane surface 53. A gel
human IgM in the reactant material as determined with
layer 55ris in laminar relation with member 51, being
constant slope was observed for human IgM concentra
tions up to about 0.40 mg/ml. In this working range, un
disposed on and in intimate contact with surface 53.
the process of this invention. A working range of large
Gel layer '55 has an exposed top surface 56. Substrate
known antigen concentrations are readily identifiable‘
51 and gel 55 form a substantially transparent unitary 25 from observed opacities. It is also to be noted from
device 57 which is similar in general appearance to uni
FIG. 4 that with properly selected or adjusted antigenv
tary device 18.
concentrations the working range is substantially inde-,
In the practice of this invention which this alternative
pendent of the antibody concentration in the gel. How
apparatus, device 57 is placed on a suitable transparent i ever, it has been found that the antibody concentration
support (not shown) in operative position with respect 30 may be reduced such that the working range is also re
to a light source 61. Light source 61 projects a light
duced. Working ranges of 'large constant slope similar
beam 62 through substrate 51, gel 55 and an intersec
to that shown in FIG. 4 were also observed for other an
tion point 65 with top surface 56 which continues away
tigen-antibody reactions.
'
therefrom as a reduced light beam 66 and scattered
~In practicing the process of this invention for the
light beams 67. Av light collecting tube 68', which is a 35 qualitative analysis and quantitation of haptens, an an
part of a- light sensing means (not shown) correspond
tiserum to a hapten conjugate, such as antiserum to es
ing to light sensing means 31, is spaced from surface 56
triol conjugated to bovinev serum albumin, is dispersed
and is optically ‘aligned with intersection point 65,
in the gel as the ?rst reagent.'The‘ surface of the geltis
being clear of light beam 66.
e
then contacted with the hapten. After a reaction per
In practicing the process of this invention a ?rst rea
iod, preferably at least about 5 minutes, the surface is
gent, which is either an antigen or an antibody, is pref
?ooded with the second reagent reactible with the antierably uniformly incorporated or dispersed in a suitable
serum to form a precipitate. When‘the hapten is pres
liqui?ed gel. For the sake of this discussion the ?rst rea
ent the areas contacted thereby are substantially free of
gent will be an antibody. However, it is to be under
precipitate and the amount of scattered light is accord
stood that the ?rst reagent may equally well be an anti 45 ingly reduced. Therefore, the quantity of hapten may
‘gen. The method by which this reagent is dispersed is
be determined by the scattered light.
not critical. Accordingly, laboratory techniques which
If desirable, the hapten and the second reagent may
are commonly used to obtain such dispersions may be
be brought into contact with the surface of the gel at
practiced,.such as, agitating a mixture of the liqui?ed
a common time. The resulting precipitate and scattered
gel and antibody by hand or with a suitable mixer.
light is reduced proportional to the hapten present and
" vThe liqui?ed gel, with the antibody dispersed therein,
the quantity thereof may be determined by such reduc
is deposited or cast on a suitable substratejm'ember fol
lowing known procedures and allowed to set up as a
Antigens identi?able with the process ‘of this inven-'
?rm gel. In this manner the unitary device 18 shown in
tion include antigenic proteins, materials having anti
55
FIGS. 1 and 2 is formed.
genie properties and bacteria. Antibodies identi?able
A drop of reactant material, which may include a sec
with the process of this invention include proteins
ond reagent reactible with the ?rst reagent to form a
which are formed when animals are immunized with
precipitate, in this case an antigen, is then placed on
antigenic proteins, materials having antigenic proper
they exposed surface 21 of the gel. If the reactant mate
ties or bacteria. Such antigens and antibodies more spe
tion.
rial contains the antigen reactible with the antibody, a
partially opaque area- (the precipitate) forms within
‘
=
'
ci?cally include, but are not limited thereto, immuno
globulins, such as lgG, IgA and IgM; haptens; hor
about- 10 to 15 minutes in a surface portion of gel 17
mones, such as HCG (human chorionic gondotropin);
under the area where the reactant material contacts the
hapten conjugates, such as dinitrophenyl conjugated to
gel. The unitary device is then placed in the operative
65 bovine serum albumin, estriol conjugated to bovine
position shown in FIG. 1 so that the partially opaque
area is optically aligned with light‘ collecting tube 32. If
a precipitate formed at the area of contact, the re
serum albumin, or estriol conjugated to thyroglobulin;
antigens of E. Coli; and antibodies or antiserums there
for.
3,905,767
6
Since techniques‘ of preparing and collecting suitable
antigens and antibodies are'gwell known and the partic
ated by suchlight source may or may not be in the visi
ble light range according to the requirements of the
light sensing means used in practicing the process of
ular technique practiced is not critical to this invention,
the preparation and collection‘ thereof will not be dis
cussed herein.
‘
g
.
this invention. Such light sources may include the fol
lowing, each of which has been found acceptable in
_
It is recognized that at times it is dif?cult to de?nitely
categorize a reagent solelyfas an antigen or solely as an
antibody. \Since this invention is concerned with a pro
cess in whicha precipitate is formed rather than with
the particular categorization of reagents, the name ap
practicing this invention, NORMAIODE H3 lamp
(12v. 55 watt), GEl630 lamp (6.5V. 2.75 amp.) and
METROLOGIC He/Ne laser, Model 310.
In the preferred apparatus of this invention the light
beam is projected onto a re?ective surface. An angle of
incidence between about 40° and 60° is preferred when
the light beam is projected in this manner. With a trans
plied to a reagent is not considered to be critical. In this
application an endeavor has been made to use consis
tent nomenclature in accordance with current prac
tices of the related art.
parent substrate the light source is situated on the side
of the substrate opposite the gel and the light beam pro
jected through the substrate and, gel. When projected
The gel may be prepared from one of the many gel
forming materials which are acceptable for use in im
through the gel an angle of incidence of between about
40° and 60° may be used for the light beam. Of course,
other angles may be used so long as the re?ected light
beam and the light beam do not interfere with the de
munodiffusion. Such gel forming materials include, for
example, agar, cellulose acetate, starch gel, gelatin,
polyacrylamide gel, or combinations thereof. This list
is not considered exhaustive in view of the large num 20 tection of scattered light beams by the light sensing
ber of gel forming materials which will be recognized
means.
.
by one skilled in the art as suitable for use in this inven
The light sensing means which measures the scat
tion.
With agar, a liqui?ed gel is prepared with a concen
tered light is bene?cially selected from apparatus
known for this capability. Such apparatus can include
tration of between about 0.5-l% by weight of agar. 25 a phototube which senses the scattered light and gener
Lower concentrations may be used, but they lack a de
ates an electrical output and a current measuring
sirable degree of ?rmness. Higher concentrations may
means which converts the output of the phototube to
also be used, but they form gel layers with a corre
a suitable readout. Of course, the scattered light may
be observed visually, although quantitation is then
sponding increase in background scattering of light.
The concentration of the ?rst reagent dispersed ‘in the 30 somewhat subjective, and the desired information re
corded based upon such observation.
liqui?ed gel is determined by referring to a graph for
In practicing the process of this invention the volume
that particular reagent corresponding to FIG. 4. Ad
of reactant material contacting the surface of the gel is
vantageously the concentration of the ?rst reagent is
not critical. This process is particularly bene?cial since
selected such that a reasonable range of concentrations
for the second reagent fall under the constant slope 35 only a drop of reactant material including the second
reagent is required although larger quantities may be
portion of the curve. As an example, when the second
used. The small quantity of reactant material required
reagent is human IgM, a goat anti-human IgM concen
to obtain signi?cant results is advantageous in that
tration of between about 0.4 mg/ml and 0.8 mg/ml is
many evaluations may be performed with a single gel
preferred. Although higher concentrations may be
substrate.
Should the concentration of the second reagent in
the reactant material exceed the highest working range
concentration therefor, the reactant material can be
used, such are not considered bene?cial in view of the
slight increase in the constant slope portion of the
curve ‘as opposed to the increased reagent requirement.
The liqui?ed gel is then cast on the substrate and al
lowed to become ?rm, thereby‘forming the unitary de
vice previously discussed. In this combination, the agar
diluted so as to obtain a suitable concentration. In a
45 usual clinical environment the expected normal immu
thickness may be between about 0.01 and 2 cm or more
noglobulin ranges in reactant material collected as
and is preferably between about 0.01 and 0.15 cm.
Unitary devices used in the process of this invention
samples are expected to exceed the working ranges of
when prepared in this manner are stable after 1 year of
storage in a high humidity environment at between 4”
and 10°C. Accordingly, large quantities of unitary de
vices may be prepared at one time with a resulting sub
the process of this invention. Accordingly, it is benefi
cial to dilute each sample a predetermined ?xed
50
amount which is expected to bring- it within the working
ranges. In the following Table 1 working ranges along
with the expected normal clinical ranges and predeter
mined dilutions to obtain a concentration within the
stantial savings in personnel and in greater consistency
working ranges are set forth for human IgG, human
from device to device.
55 IgM and human IgA.
For the process of this invention, the substrate must
be capable of suitably supporting the gel in a stable
TABLE 1
' physical and chemical condition. Preferably, this sub
strate will be partially transparent so that light is at least
partially transferable therethrough. For example, the
substrate may be selected from a glass slide, a glass mir
ror, a plastic slide or the like. A substrate which is re
IgG
lgM
IgA
60 Working Range, mg/lOO ml
2-30
2-35
2-30
Normal Clinical Range,
600-1500
50-200
50-250
mg/lOO ml
Dilution Ratio
l/60
l/6
l/l0
?ective on the ‘surface on which the gel is cast, although
Dilution Normal Range,
l0-25
8-33
5-25
not transparent, may also be used.
mg/ 100 ml
The light source may, be selected from the many that 65
are readily available and known tobe capable of pro
viding a beam; or ray of light of controlled dimension
This process is signi?cantly different from prior art
and substantially constant intensity. The beam gener
techniques in that the second reagent is substantially
3,905,767
7
8
totally depleted in the formation of the precipitate
‘For this Example the light source was a NORMA]
ODE H3 lamp (l2v., 55 watt). The‘ scattered light was
which unexpectedly forms and remains in the surface
of the gel.
detected withv an'RCA 931-A phototube and the cur
rent from the converted signal measured with a Kieth
'
In the practice of this invention the ?rst reagent is in
corporated in the gel in a concentration which is in ex
ley 610C Electrometer.
'
'
For each qualitative analysis and quantitation the fol
cess of its point of equivalence in the working range of
lowing procedure was followed.
the second reagent. Conversely the reactant material is
Six drops of antigen solution, each of a volume of 5
diluted such that the concentration of the second rea
gent therein is less than its point of equivalence in the
microliters, were placed on the gel surface within a 5
working range of the ?rst reagent.
The precipitate is visible within about 10 to 15 min
utes following application of the second reagent to the
minute interval. Opacity was observed within 15 min
utes for the solutions with lgA and measurements
thereof made at 30 minutes. The observed measure-l
gel. Reproducible measurements are possible after only
ments are set forth in Table 2.
I
'
about thirty minutes ‘ and the measurements remain
substantially constant for at least 60 minutes or more.
TABLE 2
Of course, these times may be reduced or increased by
Anti lgA Con
raising or lowering the temperature of the gel and its
surroundings. Accordingly, with the process of this in
centration,
vention measurements are achieved in shorter periods
of time and with smaller amounts of samples including 20
the second reagent than was previously possible.
The amount of light scattering resulting from the uni
tary device or background scattering is readily deter
mined by positioning a portion of the device which is
25
free of the reactant material, preferably a portion adja
cent to a test area on which the reactant material has
lgA Concentra-
Opacity,
mg/ml
tion, mg/lOOml
Microamperes
0.44
0.44
0.44
0.44
0.44
0.44
6.1
10.2
20.4
20.1
39.4
52.5
22
38
80
125
170
188
The observed measurements show that as the antigen
concentration increased the opacity increased, i.e.
been placed, in line with the light collecting tube. The
more precipitate was formed, and more light was scat
resulting measurement may be subtracted from the
tered which resulted in a proportional increase in the
measurement of the test area to obtain a corrected 30
value. In a similar manner a device may be standard
microamperes measured. Accordingly, the amount of
scattered light is dependent upon antigen concentra
ized with solutions of known reagent concentrations.
tion and therefore this process provides a basis for
quantitative analysis of lgA.
The following examples demonstrate the process of
this invention as practiced with different gels, reactant
EXAMPLE 2
materials, concentrations, etc. These examples are not 35
The
process
of
this
invention was used for the rapid
considered as exhaustive of this invention, and it is to
qualitative analysis and quantitation of human IgG ac
be recognized that one skilled in this art can readily
cording to the following procedure.
identify equivalents that may be practiced within the
Gel-substrates were prepared in accordance with the
scope of the process of this invention.
40
EXAMPLE 1
procedure of Example 1 with the exception that goat
anti-human IgG (antiserum obtained from the ' Re
search Division of Miles Laboratories, Inc.) at a con
The process of this invention was used for the rapid
centration of 1.61 mg/ml (15% antiserum) was used in
stead of lgA antiserum and IgG was substituted for IgA.
qualitative analysis and quantitation of human lgA ac
cording to the following procedure.
A 1 percent liqui?ed agar solution was prepared by 45 The observed results are'set forth in Table 3.
TABLE 3
dissolving’ 1 gm. of agar (Noble agar from Difco‘Labo
ratories) and 0.1 gm of sodium azide in 99 ml. of bu?l
ered (Michaelis’ barbital sodium-acetate buffer pH 7)
saline solution and heating the mixture, with stirring, to
just below boiling.
50
'
Solutions containing goat anti-human lgA were pre
pared by adding lgA antiserum (obtained from Miles
Research Products Division) with an assay value of 2.0
mg/ml of anti lgA, to the 1% liqui?ed agar at 50°C.
Ami lgG,
lgG,
Opacity,
mg/ml
mg/ 100 ml
Microamperes
1.61
1.61
1.61
1.61
1.61
1.61
5.2
10.6
15.9
20.6
20.6
23.1
70
120
205
250
235
290
Buffer solution was then added to the mixture to yield 55
a ?nal concentration of 0.44 mg/ml-anti lgA ( 15% anti
The observed opacity measurements show that the
serum) and 0.7% agar. Volumes of 1.5 ml. of this solu
tion were then cast on individual glass mirrors which
were 0.19 cm. thick and 2 inches on a side. The gel was '
between 0.06 cm and 0.08 cm in thickness. The mirrors 1
were kept in an H2O saturated environment thereafter
to retard evaporation and allowed to cool to room tem
perature.
'
I
Antigen solutions were prepared by dilution of lgA
standard sera (obtained from Hyland Division of Trav
enol Laboratories). The sera were'diluted :to desired
concentrations with Michaelis‘ buffer.
opacity, i.e. light scattered, is dependent upon antigen
concentration and therefore this invention provides a
60
basis for quantitative analysis of IgG.
EXAMPLE 3
The process of this invention was used for the rapid
qualitative analysis and quantitation of human lgM ac
_ cording to the following procedure.
l?elésubstratesl were prepared in accordance with the
procedure of Example 1 with the exception that goat
anti-human 'lzgMw( ntise‘rum, obtained from the Re
3,905,767
9
search Division of Miles Laboratories Inc.) at a‘ con
centration of 0.54 mg/ml (20% antiserum) was used in
stead of goat anti-human IgA and IgM was substituted
Amount
for IgA. The observed results are set forth in Table 4.
Solution A
5
TABLE 4
Anti lgM,
mg/ml
lgM,
Opacity.
mg/ I00 ml
Microamperes
0.54
0.54
4.0
5.6
15
20
0.54
'
0.54
0.54
0.54
l0.0
45
I70
25.2
32.0
60
95
100
0.2 ml '
Solution B
0.4 ml
Anti lgA serum
2.3 mg/ml
Solution C
0.2 ml
0.8 ml
10 The resulting mixture was cast on a transparent plastic
plate substrate. The unitary device was maintained at
_40°C for 30 minutes at which time the gel was ?rm.
This gel was about 0.07 cm in thickness.
For this example, the lightisource was placed under
the unitary device such that the light beam passed only
once through the substrate and gel.
The unitary devices prepared in this manner devel
oped gradations of opacity when challenged with lgA
The observed measurements show that the ‘opacity is
substantially the same as were observed in Example 4.
dependent upon antigen concentration and therefore
this process provides a basis for quantitative analysis of 20
EXAMPLE 6
lgM.
The process of the invention was used for the rapid
qualitative analysis of a hapten by an inhibition effect
EXAMPLE 4
The process of this invention was used with a gel
formed from gelatin for ‘the rapid qualitative analysis
and quantitation of IgA according to the following pro
cedure.
25
a 0.7% agar (Noble) containing 20% goat DNP
antisera- (obtained from Miles-Yeda) on 2 inches X 2
’
inches mirrored slides. (DNP=Dinitrophenyl)
A gel solution was prepared containing 10% gelatin
Each unitary device was allowed to cool to room
(from Difco Laboratories) in a pH 7 buffered solution
(sodium barbital, sodium acetate and HCI in 0.85% sa
line). To l .2 ml portions of this solution 0.3 ml portions
of anti lgA serum (l-Iyland) were added. Individual por
tions of the thus formed 20% antiserum solution (0.46
mg/ml antiserum) in an 8% gelatin solution were cast 35
on glass mirrors which were 2 inches on a side and the
devices placed in a room maintained at 4°C. for at least
60 minutes to permit gelling. The gel was between 0.06
cm and 0.08 cm in thickness.
In separate positions on the gels, 5 pl drops of IgA so
lutions at concentrations of 31 .2 mg/I‘OO ml, 15.8
mg/ 100 ml and 7.2 mg/ I00 ml were placed. Gradations
of opacity were observed which corresponded to the
increase in IgA concentration.
according to the following procedure.
Unitary devices were prepared by depositing 2 ml of
45
EXAMPLE 5
In this example the gel was formed with polyacryl
amide. The following three solutions were ?rst pre
temperature (about 23°C.) in a water saturated envi
ronment for 30 minutes. The gel was between 0.06 cm
and 0.08 cm in thickness. Five microliter drops of
e-DNP-Lysine, a hapten, (obtained from Nutritional
Biochemicals Corporation), at concentrations from
0.005 to 0.02 mg/ml were spotted on the gels and al
lowed to react for 5 minutes. The entire surface of each
gel was then ?ooded with 0.5 ml of a 0.3 mg/ml solu
tion of DNP-BSA conjugate (BSA=Bovine serum albu
min) and allowed to react for 5 minutes. The unitary
device was rinsed gently with normal saline (0.85%)
and placed in a constant humidity chamber. After 45
minutes of incubation, a general area of precipitation
had developed with clear or semiclear areas related to
the locations of the hapten.
The difference in opacity between the general pre
cipitation background and the hapten locations were
then measured. Microampere differences ranging from
28:4 to 96:13 were obtained as set forth in Table 4
pared.
TABLE 4
50
'
Solution A
I N HCl
48
ml
Tn's( hydroxymethyl )aminomethane
36.6
g
N ,N ,N'.N'-tetramethylethylene
‘
0.23 ‘
i
Hapten. mg/ml
Amount
55
ml
A Opacity.
Microamperes
0.005
28:14
0.007
0.01
0.02
46tl l
66112
96:13
diamine
Water to
Solution B
Acrylamidc‘
100.
ml
Amount
.
N,N’-Methylenebisacrylamide
28.0
(Eastman 8383)
Water to
Solution C ‘
Ammonium persulfatc
Water to
The observed results demonstrated the use of this in
g
0.735 g
60
‘
I00.
ml
ability thereof to inhibit the normal development of a
Amount
0. l4
100.‘
surface precipitate area.
g
‘
vention to‘measure the concentration of small hapten
molecules (non-precipitating antigen molecules) by the
ml
'
EXAMPLE 7
A gel solution was prepared by mixing the following
procedure of Example 6 was used in this exam
ple with the exception that rabbit anti-estriol was used
materials:
instead of DNP-antisera, estriol was used as the hapten
‘
3,905,767
‘
11
_
instead of e-DNP-Lysine, and estriol-thyroglobulin was
used as the conjugate instead of DNP-BSA.
The resulting inhibited precipitation areas were sub
stantially the same as those observed in Example 6.
EXAMPLE 8
The process of this invention was used for the quali
tative analysis of HCG according to the following pro
1-2
process comprising
contacting a surface of a gel having, a ?rst reagent
which includes an antigen or an antibody incorpo
rated in at least a surface part thereof with a second
reagent which includes an antigen or an antibody
and which is reactive with said ?rst reagent to form
a precipitate which remains in a surface portion of
said gel,
.
'
cedure.
projecting a light beam through saidsurface portion
Lyophilized antiserum to l-ICG (obtained from Nutri
tionalBiochemicals Corporation, Cleveland, Ohio) was
measuring the extent to which said light beam is scat
diluted according to manufacturer’s instructions.
so
of said gel,
'
tered by said precipitate, and
lution was prepared consisting of the antiserum, liqui
?ed 2% agar and phosphate-buffered physiological sa
correlating the extent to which said light beam is
line so that ?nal ‘concentrations of 50% antiserum and
0.7% agar were obtained. The solution was deposited
upon a mirror and allowed to gel.
2. The process of claim 1 in which said ?rst reagent
scattered to analytical data sought. '
is an antigen and said second reagent is an antibody.
3. The process of claim 1 in which said ?rst reagent
'One hour after deposition upon the gel of 5 ul of a
is an antibody and said second reagent is an antigen.
solution containing 1 unit of l-lCG/ml in buffered sa
4. The process of claim 1 in which said light beam'is
7 line, a spot of precipitate was observed in the area of 20 projected through said surface portion in a direction
contact. This result indicated that the process of this
away from said gel surface.
'
'
'
’ invention provides a rapid and early visual indication of
5. The process of claim 1 in which said light beam is
pregnancy.
projected at an angle oblique to said surface of said gel.
EXAMPLE 9 _
The process of this invention was used to determine
‘ whether or not a speci?c bacterial species was present
in a reactant material according to the following proce
dure.
Unitary devices were prepared by depositing a 0.7%
agar solution containing 20% E. Coli Poly A antiserum
on mirrors.
' E. Coli Ol 1 1:84 was. incubated for 18 hours in 10 ml.
6. The process of claim 1 including as additional
25 steps projecting said light beam through a second sur
face portion of said gel which is free of said second rea
gent, and measuring the extent to which said light beam
is scattered by said second surface portion, whereby
the background scattering of a unitary device compris
ing a substrate and said gel is measured.
I
7:. The process of claim 1 including as an additional
step prior to contacting said surface with said second
reagent,_ contacting a surface area of said gel with a’
of Bill nutrient broth and the cells harvested by centrif
ugation. The cells were twice washed with saline solu 35 third reagent reactible with said first reagent to inhibit
subsequent formation by said ?rst reagent of a precipi
tion, centrifuged and the supernatant discarded. The
tate
in a surface portion of said gel adjacent said sur
recovered cells, estimated to be 1010 in number, were
face area when said surface area is contacted with said
mixed with 50 ,ul of a saturated aqueous solution of eth
ylenediaminetetraacetic acid at pH 7.0 and incubated
at 55°C. for_3O minutes. Then 50 pl of lysozyme solu
tion (16 mg/ml in water) was added to the cell mixture
and incubation continued an additional 30 minutes
with occassional stirring. After cooling, cell debris was
I removed by centrifugation. The clear supernatant was
second reagent, whereby said third reagent may be
qualitatively analyzed or quantitated according to the
measured scattering of said light beam.
‘
-
8. The process of claim 1 including as an additional
step prior to contacting said surface with said second
reagent, mixing with said second reagent a third rea
maintained at 100°C. for 30 minutes to precipitate lyso 45 gent reactible with said ?rst reagent to inhibit the sub
sequent formation thereby of a precipitate with said
zyme, which was removed by centrifugation.
The clear supernatant was diluted lzlO, 1:100 and
1:1000 respectively with an aqueous solution buffered
to a pH of 7 with‘ phosphate buffer. Five /.|.l aliquots of
each dilution were deposited on separated areas of the
surface of the gel and incubated for 1 hour at about
i . 23°C. in a high humidity chamber. Spots of precipitate
of decreasingv opacity were observed in the areas of
second reagent, whereby said third reagent may be
qualitatively analyzed or quantitated according to the
measured scattering of said light beam.
9. The process of claim 1 in which said gel is'sup-A
ported by and is in intimate contact with a substrate
member.
’
'
A
I
10. The process of claim 9 in which said substrate
contact for the 1:10 and 1:100 dilutions respectively.
Y ‘1 l. Theisprocess
transparent.
of claim
’
10
- in which
r
said
> light beam
No spots were visually observed for the area contacted 55 member
is ?rst projected through said transparent substrate
by the 1:1000 dilution.
member and then through said surface portion of said
These observations established that the process of
gel.
‘
this invention is effective in detecting bacteria.
12. The process of claim 9 in which said substrate '
It has thus been shown with the above examples that
this invention provides a new and novel process which 60 member is a mirror which re?ects towardvsaid gel. _
13. The process of claim 12 in which the re?ective
results in more'rapid qualitative analysis or quantita-j
surface of said mirror is adjacent said gel.
tion of antigens or antibodies.
14. The process of claim 12 in which the re?ective
-What is claimed is:
surface of said mirror is the surface thereof opposite
1. A process for rapid qualitative analysis and/or
quantitation of antigens or antibodies with an antigen 65 the surface thereof supporting said gel.
*
*
*
*
*
antibody reaction in which a precipitate is formed, the