U.S. patent number 3,645,687 [Application Number 05/000,642] was granted by the patent office on 1972-02-29 for immunodiffusion plate apparatus.
Invention is credited to Samuel T. Nerenberg.
United States Patent |
3,645,687 |
Nerenberg |
February 29, 1972 |
IMMUNODIFFUSION PLATE APPARATUS
Abstract
A device for the quantitative determination of protein by radial
diffusion thereof through a coating of antibody on a sheet formed
of a paper-thin liquid-permeable chemically inert layer (e.g.,
cellulose acetate) on a resilient liquid-impermeable backing (e.g.,
flexible plastic). The device includes a wall and marginal
framework therearound, having a slot for slidably receiving a
perforate or solid plate into a fixed position above the wall. The
device further includes means for slidably receiving said sheet
between the wall and either of the plates in position. Blood serum
may be applied via a capillary tube through the openings in the
perforated plate onto a sheet in predetermined placement. In one
embodiment, the device contains identical back-to-back units as
above described. In a second embodiment, one side of such
back-to-back units contains parallel members mounted on one wall
and cooperating therewith to form parallel slots adapted to receive
a number of sheets.
Inventors: |
Nerenberg; Samuel T.
(Burlingame, CA) |
Family
ID: |
21692396 |
Appl.
No.: |
05/000,642 |
Filed: |
January 5, 1970 |
Current U.S.
Class: |
436/514; 422/430;
D24/225; 422/69; 436/530; 436/807; 435/287.2; 435/287.7 |
Current CPC
Class: |
G01N
33/558 (20130101); Y10S 436/807 (20130101) |
Current International
Class: |
G01N
33/558 (20060101); C12b 001/00 (); G01n
033/16 () |
Field of
Search: |
;23/230,253,23B
;195/139LE |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Wolk; Morris O.
Assistant Examiner: Reese; R. M.
Claims
We claim:
1. In a method for the quantitative determination of protein, the
steps of layering antibody specific to said protein onto a
paper-thin cellulose acetate coating on a liquid impermeable
resilient backing, placing said backed layered coating into a
frame, sliding a protective plate into a groove in said frame,
sealing said coating from the air, incubating said sealed coating
for a sufficient time to cause the antiserum to be uniformly
absorbed into the coating, removing said protective plate from said
groove, sliding into said groove a perforate plate having a
plurality of openings in spaced-apart relation, applying an unknown
protein antigen through said template to said coating, reacting
said antibody and antigen on said coating to form precipitin rings,
and quantitating the area of said rings.
2. A device for the quantitative determination of protein by radial
diffusion comprising open-topped container means, a sheet disposed
in said container means, said sheet being formed of a cellulose
acetate layer on a resilient impermeable backing, said layer being
saturated with antibody specifically reactive with said protein, a
perforate template adapted to fit over said container means, said
template having holes in spaced-apart relation for applying protein
sample to said layer in a predetermined pattern.
3. In a device for the quantitative determination of protein by
radial diffusion thereof through a coating of antibody specifically
reactive with the protein, a sheet formed of a paper-thin
liquid-permeable chemically inert layer on a resilient
liquid-impermeable backing, wall means, means forming a marginal
framework on each edge of said wall means extending outwardly
therefrom, said framework means having slot means adapted to
slidably receive a plate, holding means disposed at lateral edges
of said wall means at a level between said wall means and said slot
means, said sheet being inserted in said holding means.
4. A device as in claim 3 wherein said coating is formed of a
cellulose acetate and said backing is comprised of a pliable
plastic.
5. A device as in claim 3 together with a perforate plate having a
plurality of openings in spaced apart relationship and slidably
received in said slot means whereby liquid sample may be applied
via a tube through said perforations onto said sheet in
predetermined placement.
6. A device as in claim 5 wherein said sheet is disposed between
said wall means and said perforate plate.
7. A device as in claim 3 including a solid protective plate
slideably received in said slot means, said marginal framework
means, wall means and protective plate cooperating to form an
air-sealed compartment for said sheet.
8. A device as in claim 7 wherein said sheet is disposed between
said wall means and said protective plate within said air-sealed
compartment.
Description
In a method for using the above device for the quantitation of
protein, the antibody layered sheets are placed into their holders
in the device, the protective plate is slid into its groove of the
device to seal the coating from the air during incubation period,
the protective plate is removed and replaced with the perforate
plate, blood serum is applied via a capillary tube through the
perforations onto the coating, the serum and antibody reacts to
form precipitin rings and the area of said rings (representing the
quantity of antigen in the blood serum) is quantitated.
In another embodiment of the device, the above antibody layered
sheet is placed at the bottom of an open-topped container, a lid is
placed on the container for sealing during incubation, the lid is
replaced with a perforate template, serum is applied through the
perforations, and the resulting precipitin rings are
quantitated.
BACKGROUND OF THE INVENTION
Immunological procedures for the quantitation of specific protein
antigens have been widely used for both clinical and research
analyses. Such procedures are based on the measurement of the
degree of precipitation in the reaction between such protein
antigens and their specifically reactive antibody. Perhaps the most
widespread usage of this reaction is in quantitative single radial
immunodiffusion. The first such technique, performed in agar gels,
was described in Mancini et al., "A Single Radial Diffusion Method
for the Immunological Quantitation of Proteins," 11 Prot. Biol.
Fluids 370 (1964). According to that technique, antibody was
incorporated in uniform concentration in an agar gel with a
thickness of about 1 mm. formed by means of plastic frames. After
solidification of the gel into a plate, a series of spaced-apart
wells were cut therein and filled with precise volumes of antigen
solution determined by means of a calibrated micropipette. The
immunodiffusion reaction between antigen and antibody was allowed
to proceed in the gel in the form of a precipitin ring during
incubation for about 14 days in an oil bath. The bath functioned to
prevent drying. A photographic enlarger was used to project the
thus-formed circular shaped precipitin line images onto
photographic paper. These images were then cut out, weighed, and
plotted on semilog paper for quantitation. The accuracy of
quantitation was found to be on the order of .+-. 5 percent. The
major drawback of the Mancini technique is that the 14-day
diffusion period is impractical for clinical use since laboratory
tests normally are required within a 12-24 hour period to gauge the
need for immediate therapy.
In view of the pressing need for a more rapid assay procedure for
the clinical determination of serum immunoglobulins, Hyland
Laboratories, of Los Angeles, California, developed a product
called an "Immunoplate" for quantitating the three common serum
immunoglobulins: .gamma. A Globulin (IgA), .gamma. M Globulin
(IgM), and .gamma. G Globulin (IgG). These plates embody the
principles of Mancini but require only overnight incubation to
yield complete ring precipitin reactions. The ring diameters are
plotted on a linear portion of semilogarithmic paper with the
values of the reference sera on the coordinate logarithmic axis to
obtain a standard curve. Values for the unknown samples were taken
from the standard curve. In contrast to the Mancini technique which
employs precisely measured volumes of antigen solution in the
sample wells, the Hyland technique is less accurate recommending
measurement by the mere filling of wells to the gel surface. This
technique is presently being used extensively.
In 1965, an analytical study of the Hyland Immunoplates was
published by Fahey, J. L., et al., in "Quantitative Determination
of Serum Immunoglobulins in Antibody Agar Plates," 94 J. Immunol.
84 (1965). The probable error determined by Fahey et al. was about
.+-. 10 percent compared to the .+-.5 percent reported by Mancini
in his more refined and prolonged procedure. In particular Fahey,
et al. calculated the standard deviations for the three
immunoglobulins to be: IgA .+-. 70 mg./100 ml., IgG .+-. 220
mg./100 ml., and IgM .+-. 35 mg./100 ml. Furthermore, experience in
the clinical laboratory with Hyland Immunoplates, monitored by a
quality-control procedure, indicate that the probable error is much
larger than .+-. 10 percent under actual hospital conditions,
approximating .+-. 20-25 percent when a number of technologists are
responsible for carrying out the procedure on a routine basis.
Although fairly good straight-line relationships are obtained
between precipitin ring diameters and logarithmic concentration of
antigen, the slopes of the curves are very steep so that even small
variations in reading precipitin ring diameters lead to significant
differences in final results. A further source of error is
attributable to divergence of the ring precipitin diameters from
true circles. Although results could be improved somewhat by
multiple readings of a single precipitin ring taken in different
directions, this would prolong the procedure significantly when
numerous samples are taken. In summary, some of the major drawbacks
to the use of the Hyland Immunoplates include its inaccuracy;
refrigerated storage temperatures; relatively large volumes of
undiluted serum sample (on the order of 0.25 ml.) is necessary to
determine the level of all five immunoglobulins; and inaccuracies
in the system.
In order to overcome certain of the aforementioned disadvantages,
C. Vergani et al. developed a technique which was published in a
paper entitled "Quantitative Determination of Serum Immunoglobulins
by Single Radial Immunodiffusion on Cellulose Acetate," 4
Immunochemistry 233 (1967). According to this technique, strips of
unbacked cellulose acetate membranes are utilized as the carrier
medium for specific antiserum instead of the agar gel in
Immunoplates. In the Vergani method, the cellulose acetate strips
are stretched between clamps to form a bridge in a moist boxlike
chamber wherein diluted antiserum is sprayed onto the strips. Since
the antiserum soaks though the strips, they must be suspended to
avoid contamination of surfaces therebelow. Following spraying, the
strips are left at room temperature in the moist chamber sealed via
a lid. The test sample is then applied to the strip by means of a
micropipette. In order to avoid drying, the strips are then placed
into a mineral oil bath where they remain throughout the period of
diffusion. After diffusion and the accompanying precipitin ring
formation the strips are removed from the oil, immersed in a
detergent solution to wash off traces of oil, and washed under tap
water. The standard deviation for the results of this procedure was
stated by Vergani et al. to be comparable to that of the
aforementioned Fahey et al. study (.+-.10 percent). The obvious
advantages of this procedure as compared to agar immunodiffusion
include: uniform thickness of the cellulose acetate supporting
medium which yields a sharp outline of precipitin rings;
significantly greater serum economy (e.g., a 4.times.28 cm. strip,
accommodating 50 determinations, consumes only 1.2 ml. of diluted
antiserum); and a 2-day procedure compared to 14 days in the
original Mancini technique.
In spite of the aforementioned advantages, the Vergani technique
has not achieved widespread use for a number of reasons. Perhaps
the most important is that unbacked cellulose acetate membranes are
quite brittle and delicate which renders them extremely difficult
to handle without breaking. Furthermore, the strips lack rigidity
and so must be stretched to apply the sample accurately In
addition, the strips must be suspended on a bridge in a rather
cumbersome apparatus to avoid contamination of surfaces therebelow
since fluid will travel through the strips. Also, there is a
certain loss of precision resulting from incomplete equilibration
of the suspended strips and from the requirement for direct
measurement by calipers rather than use of photographic enlargement
prior to performing a reading. Finally, Vergani et al. instructs
that to use the strips, the antigen (e.g., blood serum containing
immunoglobulins) must be diluted prior to application, a
time-consuming step which multiplies in a procedure designed to
screen the blood serum of a large number of patients entering a
hospital.
SUMMARY OF THE INVENTION AND OBJECTS
This invention relates to certain holding devices for use in a
single radial immunodiffusion technique on a paper-thin, liquid
permeable, inert layer (e.g., cellulose acetate) on a resilient
liquid-impermeable backing (e.g., flexible plastic).
In one embodiment of the invention, a holder is formed of a wall
with a marginal framework on each side thereof extending outward
therefrom. The framework is provided with plate slots on the
lateral sides of said wall adopted to receive either a perforate
template or a solid protective plate. Sheet slots are also provided
internally of the plate slots for receiving an immunodiffusion
sheet formed of a cellulose acetate layer on a flexible plastic
backing. Multiple elongated slots in parallel, spaced-apart
relationship may be mounted on either surface of the wall for
slidably receiving a plurality of the aforementioned
immunodiffusion sheets. Thus, a variety of sheet sizes may be
utilized in the holder in accordance with the number of samples
selected to be run on a single sheet (described hereinafter)
resulting in a conservation of antiserum.
According to one procedural embodiment of the invention, antiserum
is spread evenly onto the above immunodiffusion sheet which is then
inserted, acetate layer up, via the sheet slots into the
aforementioned holder. A protective plate is then slid into the
plate slots above the sheet to form a compartment which protects
the sheet from drying. The antiserum is then allowed to diffuse
evenly in an "equilibration stage," after which the protective
plate is removed from the holder and replaced with the
aforementioned template During a "serum application stage,"
measured amounts of antigen sample (e.g., blood serum) are applied
by means of micropipettes through the openings in the template. In
a "diffusion or incubation stage," the sheet is removed from the
holder and placed under mineral oil for about 18 hours. The
precipitin ring-containing sheet is then washed and stained and the
ring images enlarged onto photographic paper for viewing. During
this procedure, the backed film is extremely easy to handle, much
like photographic film.
In another embodiment of the technique and apparatus of the
invention, quantitation can be performed on the above
immunodiffusion sheet in a base dishlike device such as a
conventional flat-bottomed Petri dish. Thus, the sheet is cut to
fit snugly into the dish. The cut sheet is coated with antiserum
and placed at the bottom of the base dish, a protective cover lid
is placed thereover to protect the sheet from drying, and the
antiserum is allowed to equilibrate. Then the lid is removed,
replaced with a template lid having a series of radially placed
holes, and serum is applied. Thereafter, mineral oil is poured in
the dish for coverage during incubation during which the
immunodiffusion reaction is completed without drying out of the
antiserum. The remainder of the steps performed on the sheet are as
described above.
It is an object of the invention to provide an improved device and
technique for quantitating proteins by radial diffusion through
specifically reactive antibodies which overcomes the disadvantages
of the prior art.
It is a further object of the present invention to provide a device
of the above type which eliminates the need for the cumbersome
equipment of the prior art and which conserves serum and
antiserum.
It is a further objection of the invention to provide a device of
the above type which decreases the time of quantitation of protein
in blood serum whereby a large number of patients may be screened
simultaneously.
Additional objects and features of the invention will appear from
the following description in which the preferred embodiments are
set forth in detail in conjunction with the accompanying
drawings.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a top view of one embodiment of a holder according to the
invention.
FIG. 2 is a cross-sectional view of the device in FIG. 2 taken
along the line 2--2.
FIG. 3 is a cross-sectional view of the device in FIG. 2 taken
along line 3--3.
FIG. 4 is an enlarged portion of the device in FIG. 3 taken in the
area 4--4. FIG. 5 is a top sectional view of FIG. 4 taken along the
line 5--5.
FIG. 6 is a top view of another embodiment of a holder according to
the invention.
FIG. 7 is a cross-sectional view of the device in FIG. 6 taken
along the line 7--7.
FIG. 8 is a top view of a dishlike container embodiment according
to the invention.
FIG. 9 is a cross-sectional view of the device in FIG. 8 taken
along the line 9--9.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Referring to the drawings, rectangular immunodiffusion sheet 11
includes a paper-thin diffusion layer 12 integral with a resilient
backing 13. Layer 12 is formed of any coatable material that is
chemically inert and readily permeable to liquid, such as cellulose
acetate. Backing 13 is formed of any suitable material that is both
flexible and impermeable to liquid such as plastic. I have found
that a particular effective material for use as sheet 11 is a
cellulose acetate layer on a backing of polyethylene terephthalate
plastic (trademark Mylar). This integral sheet is sold under the
name "Titan III-X-100" by Helena Laboratories, Allen Park,
Michigan.
Referring to FIGS. 1-4, holder 14 includes facing upper and lower
walls 16 and 17 bordered on all sides by a marginal framework 18.
Framework 18 includes an upper portion 19 which cooperates with
wall 16 to form an upper container 20 and a lower portion 21
cooperating with wall 17 to form a lower container 22. Since
containers 20 and 21 have identical construction and function, the
description herein of container 20 applies equally to container 21
with like part numbers assigned to each container. Holding means
for sheet 11 are provided at opposing edges of wall 17 and
comprising in this instance bent portions at lateral edges 23 and
24 forming lateral grooves 26 and 27 respectively. The forward edge
of wall 17 is also bent over forming a similar groove 29. Grooves
26 and 27 serve to slidably receive sheet 11 at the rear edge 30 of
wall 16 while groove 29 functions to stop the sliding sheet in a
reproducible fixed position relative to holder 14. A recess 31 is
provided at edge 30 to facilitate removal of sheet 11 from holder
14.
Framework 18 includes an integral frame member 32 with inwardly
extending ridges 33 on all four sides which separates wall 16 from
wall 17. Member 32 includes lateral sections 34 and 36, front
section 37 and rear section 38. The inner surfaces of sections 34
and 36 contain slots 39 and 40 respectively and lips 35 which
overlap and, thus, retain wall 16 in a fixed position. A flat
coverplate 41 is attached to section 38, suitably by bolting to
upwardly projecting ridges on the lateral sides of section 28 to
form an opening 45 therebetween communicating with slots 39 and 40.
Hinged member 42 and the outer edge of section are suitably
interconnected by means of a flexible fabriclike tape 43 with
adhesive backing forming the pivotal line of the hinge. A slot 44
is provided in member 42 as a stop for a plate sliding through
slots 39 and 40 and opening 45. Such a plate in a stopped position
would form an essentially sealed container.
Holder 14 may be formed of any rigid material such as wood, plastic
or metal. It will be apparent that the above holder construction
may be varied without altering the function thereof. For example,
the entire holder could be formed of one integral structure as of a
plastic material.
Template 46 is of a suitable rectangular shape and size for sliding
through opening 45 and along slots 39 and 40 and into slot 44.
Template 46 is provided with spaced-apart fluid application holes
47 through which serum may be fed in a predetermined placement onto
layer 12. By way of example, 42 holes, 1 mm. in diameter, may be
drilled through the template. A handle 48 may be attached to
template 46 to facilitate gripping on sliding into and out of
holder 14. Template may be formed of any rigid or semirigid
material, such as plastic. For visual monitoring of applied sample,
as described hereinafter, the material is preferably transparent.
It is understood that although template 46 is illustrated in
position in upper container 20, that interchangeably it could be
placed into lower container 22 for applying serum to a sheet 11
placed therein.
Protective plate 49 is of approximately the same size as template
46 and may be provided with a similar handle 50 The plate material
may be rigid or semirigid. Plate 49 slides into the same slots as
template 46 to cooperate with either container 20 or 22 to form
chambers shielded from substantial contact with surrounding air.
Plate 48 is illustrated in FIGS. 1-4 to be in a reserve
nonfunctional position. Plate 49 would be functional if a second
sheet 11 were placed in container 22. Alternatively, if template 46
is slid out and replaced with plate 49, container 20 would be
sealed thereby.
Referring to FIGS. 6 and 7, an alternative holder 14a is
illustrated. Only the components in holder 14a that vary from those
in holder 14 will be described in detail or assigned different part
numbers. Lower container 22a has the same structure and function as
that of container 22. On the other hand, upper container 20a
functions to slidably receive three different size sheets 11a of
the same material as sheet 11 but transverse to sheet 11. To
accomplish this, three elongated members 51, 52 and 53 having
overhanging portions on both sides thereof are mounted in place of
wall 16. The overhanging portions form slots 54 which, along with
grooves 26 and 27, form channels for three sheets 11a. In contrast
to stationary section 36, section 36a is hinged in the same manner
as member 42 to provide ready access to sheets 11a. Since these
sheets 11a are received from the side, there is no reason to hinge
member 42a and so it is stationary (in contrast to hinged member
42). The only functional distinction between holders 14 and 14a is
the ability to utilize variable sheet sizes in the latter, thus
conserving antiserum for runs involving a small number of
samples.
The foregoing description relates to a two-chambered holder with a
convenient slot for reserve storage of the template or protective
plate when not in use. It is to be understood that a
single-chambered device (e.g., formed of the upper or lower half of
the holder 14 or 14a) when used for quantitating proteins on a
sheet similar to sheet 11 is considered to be within the scope of
my invention.
In one embodiment of the technique of the present invention
performed either on holder 14 or 14a, large sheets of cellulose
acetate backed with Mylar plastic (commercially available as Titan
III-X-100 from Helena Laboratories, Allen Park, Michigan) were cut
into rectangular segments to fit slots 20 of holder 11. The upper
left-hand corner may be notched or cut off for orientation
purposes.
In a saturation step, monospecific antibody is spread across the
cellulose acetate by suitable means such as a plastic disposable
razor with the blade removable. The antibody is reactive with a
specific protein to produce a precipitin. For purposes of this
description, commercial antisera monospecifically reactive with the
three immunoglobulins, IgG, IgA, and IgM of blood serum are used as
the antibody for the quantitative determination of the same
immunoglobulin. The antisera are preferably diluted with saline
solution prior to application to the cellulose acetate. The optimum
dilution (i.e., the one producing the heaviest precipitin rings)
must be determined for each commercial antiserum batch by trial and
error since there are certain variations from batch to batch. By
way of example, it has been found that using antisera obtained from
Hyland Laboratories, Los Angeles, California, a dilution with a 0.9
percent saline solution is effective in the following ratios of
antiserum to diluent; IgA 1:10, IgG 1:20, and IgM 1:10. The diluted
antiserum is then spread uniformly onto an immunodiffusion sheet as
with a plastic razor with its blade removed. As a quantitative
example, 1.5 ml. would be sufficient quantity for a 4.times. 5 inch
sheet.
In an equilibration step, the coated sheet is slid into grooves 26
and 27 of holder 14 by pivoting member 42 out of the way.
Protective plate is then slid into its guiding slots thereover into
the slot of member 42 in a seated position. Thus, antiserum is
prevented from drying during equilibration or uniform saturation
throughout the cellulose acetate layer. Equilibration takes
approximately 10-15 minutes at room temperature or about 5-10
minutes at 37.degree. C.
The reason that equilibration may be performed in efficient holder
14 is that backing 13 of sheet 11 is impermeable to liquid and so
there is no soaking therethrough onto wall 16. This is a marked
contrast to the unbacked cellulose acetate membrane of Vergani
which necessitates the use of a cumbersome apparatus and technique
for quantitation.
Following equilibration, the protective plate is removed from the
holder and the surfaces of layer 12 may be inspected by an oblique
light. If an irregular layer of superficial fluid is seen on the
surface, that is allowed to evaporate by leaving the sheet exposed
to air for approximately 3 to 5 minutes. If the layer of fluid is
unusually heavy, it may be spread more uniformly as with the
plastic razor and allowed to evaporate. Drying of focal areas
should be avoided to prevent distortion of the ultimate precipitin
rings. Excess drying is recognizable by the appearance of
occasional opaque, white spots on a dull grey background. If drying
occurs, the oblique spots may be moistened with a drop of antiserum
and uniformly spread with the plastic razor.
In preparation for a serum application step, a transparent template
46 is inserted into holder 14 via grooves 26 and 27. The template
prevents rapid drying during application of the blood serum sample.
Concentrated reference samples of each immunoglobulin are diluted
for application to form three precipitins for construction of
standard curves in the anticipated range of sample values as
explained hereinafter. Referring to FIG. 5, in decreasing order of
concentration points 56 a-c represent applied reference sera and
the corresponding ultimately stained precipitin rings are
represented at 57a-c. In like manner, points 58a-c represent
applied unknown sera and precipitin rings 59a-c represent
corresponding stained precipitin rings. Sera, both reference and
unknown samples, may be labeled with bromphenol blue dye by mixing
a few grains of dye powder with each sample for easier viewing of
the precipitin.
Sample is applied through the template holes suitably by means of a
tube which accurately measures small quantities of fluid. A
preferred applicator is a microcapillary tube holder fitted with
disposable 0.5 .mu. 1 self-filling capillary tube such as the
Drummond type sold by Helena Laboratories, Allen Park, Michigan.
For the most accurate control of samples, it has been found that
mouth pressure exerted through narrow gauge tubing is more
effective in forcing the sample from the capillary tubes than is
the use of a compressible rubber bulb as supplied with the Drummond
capillary tube holder. Sample in the microcapillary tubes is
applied through template holes directly onto the cellulose acetate
surfaces. During application of the dye-colored samples, total
emptying of each capillary tube was determined by monitoring or
viewing through the transparent plastic template. Each sample forms
a concentric blue-colored spot on the surface of the cellulose
acetate sheet.
For incubation, the template is slid out of the holder, member 42
is pivoted outwardly, and sheet 11 is removed from the holder and
placed under the mineral oil. The sheet is then incubated overnight
(e.g., about 18 hours) suitably at the following temperatures:
about 25.degree. C. for IgA, about 4.degree.-10.degree. C. for IgG,
and about 37.degree. C. for IgM. The purpose of the mineral oil is
to shield the sheet from drying out during incubation. It is noted
that if holder 14 could be completely air sealed with the
protective plate, incubation could be performed directly in the
holder.
Following incubation, the sheet is removed from the mineral oil and
suspended as from a clamp for a few minutes to allow residual oil
to drip free of the sheet. In order to remove any remaining oil,
the sheet may then be washed in a detergent-saline solution for
approximately 5 minutes. In a second wash the sheet may be retained
in a 0.9 percent saline solution for approximately 30 minutes to
remove unreacted proteins. The saline solution can be removed from
these sheets by rinsing in tap water. To render the precipitin
rings clearly visible, they may be stained as with Thiazine Red in
a 1 percent acetic acid solution for approximately 30 minutes
followed by rinsing as with 1 percent acetic acid solution. After
allowing the sheets to dry, the stained precipitin rings
represented at 57 and 59 are readily visible.
To quantitate the precipitin, it is preferred to project the dried
unstained cellulose acetate sheets through a standard photographic
enlarger for final viewing on photographic paper. In one system,
the images projected onto sensitized paper such as "Ectamatic SC"
paper produced by Eastman Kodak Co. and then passed through an
Ectamatic Processor which provides enlarged images on a fixed
photographic paper in a semidried state in less than a minute. The
photographic paper may then be removed from the dark room for
analysis. Diameters of each precipitin ring may be compared in
several directions with a compass to confirm the formation of true
circles. If so, the magnitude of the ring diameter may be measured.
On those infrequent occasions when the ring precipitin do not form
true circles, the area enclosed by the rings may be cut out along
with the reference areas for weighing. A standard curve may be
prepared by plotting the ring diameters and/or the weights of the
paper areas circumscribed by the precipitin rings 57 of the
reference specimens on the linear axis of two-cycle semilog graph
paper against their known protein values plotted on the logarithmic
axis. Unknown samples may then be read directly from the standard
curve.
A statistical analysis of commercial standard reference sera for
the immunoglobulins IgA, IgG, and IgM, was performed by applying
blood samples to antisera-coated, backed cellulose acetate sheets
according to the aforementioned technique and using reference sera
as above-described for points of the standard curve. The results of
this analysis are summarized in Table I.
---------------------------------------------------------------------------
TABLE I
Protein Listed value of control Determined value serum in mg./100
ml. in mg./100 ml.
__________________________________________________________________________
IgA 260 262 .+-. 14.4 IgG 450 450 .+-. 24.5 IgM 115 113 .+-. 9.9
__________________________________________________________________________
referring to Table I, it is apparent from the above standard
deviations that a significant improvement in precision of
quantitation is achieved by the present device and technique in
comparison to that of the aforementioned agar gel technique as
studied by Fahey et al. with standard deviations of IgA - .+-. 70
mg./100 ml., IgG-220 mg./100 ml., and IgM-35 mg./100 ml. The
present invention has the same superiority of precision over the
Vergani unbacked cellulose acetate technique since the latter is
comparable in accuracy to that of Fahey.
An important factor in the improved accuracy of the technique of
the present invention is the ability of the plastic-backed
cellulose acetate sheet to be handled as a regular photographic
film. Thus, projection and enlargement of the stained precipitin
rings onto the photographic paper enables the enlarged
photographically developed precipitin images to be more accurately
measured than precipitin rings viewed through a microscope. It is
noted that even a slight error in direct measurement of a ring
diameter would introduce a relatively large error in the final
result due to its logarithmic value. Furthermore, recording of
precipitin rings on photographic paper may be performed almost
instantaneously with great precision compared to the tedious and
relatively inaccurate technique of tracing the images in a darkroom
recommended by Mancini et al. In addition, the time utilized for
processing the photographic paper (less than a minute) is
exceedingly more rapid than the tracing of individual images of
many samples. Finally, increased accuracy and speed of an analysis
are provided by the use of the compass to detect "distorted"
precipitin rings. It is only those infrequently occuring distorted
rings which require the time consuming process of weighing the
closed areas for quantitation.
Referring to FIGS. 8 and 9, another embodiment is illustrated of a
device which may utilize the aforementioned backed cellulose
acetate sheet in a technique analogous to that described above.
This dishlike device includes a container dish 60, an overlaying
replaceable perforate template lid 61, and a solid protective lid
(not shown) interchangeable with template 61. Dish 60 is suitably a
flat-bottomed, open-topped rigid or semirigid dish such as
disposable plastic, commercially available Petri dish. The size of
dish 60 may vary in accordance with the number of samples to be
applied in a single run. To conserve antiserum, an 85 mm. diameter
dish 60 may be employed along with template lid 61, with 16 holes
to accommodate a like number of samples as described hereinafter.
Template 61 (and the protective lid) may be of a flat-bottomed
dishlike shape similar to dish 60 but with a slightly larger
diameter so that an essentially snug fit is created with lid 61
overlying dish 60.
A circular disc 63 of the plastic backed, cellulose acetate
material described above may be formed to conform to the inner
diameter of dish 60. A spot may be applied at one point on the edge
of the dish to orient the application of samples. Discs 63 may be
stored in individual dishes 60 ready for use.
Application of antiserum may be performed as described in the
aforementioned technique. The equilibration may be performed by
placing disc 63 into dish 60 and covering the dish with protective
lid. The precautions to prevent drying are described above.
Referring to FIG. 8, template lid 61 may be drilled with a series
of holes 64 in a spaced-apart relationship. This spacing is planned
so that when sample is applied through holes 64 onto disc 63, there
is sufficient clearance for the spreading of each precipitin ring
without contact with adjacent rings. Although the dishlike device
is described with reference to a circular walled dish, it is to be
understood that other shapes, such as rectangular, may be employed
to the same effect.
In the incubation stage, template lid 61 may be removed and mineral
oil directly spread over disc 63 in dish 60. The timing of
incubation is as described above. The remainder of the quantitation
procedure including washing, staining, and projecting the results
onto photographic paper, may also be performed as described
above.
Quantitating by means of holders 14 or 14a has certain advantages
over quantitation with dish 60. For example, specimen spacing, to
prevent overlapping of precipitin rings, is easier to plan on a
rectangular template and the corrolation of precipitin rings with
the proper specimens is also simpler with a rectangular shape.
Furthermore, the disc shaped sheet is somewhat more difficult to
handle during a procedure, e.g., during removal from dish 60.
It will be apparent from the foregoing that I have provided a
number of embodiments of a holding device with which accurate and
simple immunodiffusion quantitations may be performed on a flexible
plastic sheet covered with a cellulose acetate film. Quantitation
according to this invention is exceedingly accurate, easy and rapid
to perform, and economical of serum and antiserum. Although the
invention is described with respect to specifically constructed
holders, it is understood that equivalent holders are within the
present scope.
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