Radioimmunoassay apparatus

A coated bead, a gripper for rotatably engaging the bead, a carrier for carrying at least one gripper, test tray bearing at least one reaction chamber and an agitator for agitating the carrier in relation to the test tray are disclosed.

BACKGROUND OF THE INVENTION 
The present invention relates to a diagnostic apparatus having utility in 
the radioimmunoassay of antigens and their antibodies. More particularly, 
the invention relates to an apparatus for a two-site immunoradiometric 
assay for serum ferritin in human blood samples. 
Ferritin is a high molecular weight protein functioning primarily as a 
situs for iron storage. Ferritin can be found in almost every solid tissue 
of the human body, but it appears principally in the cytoplasm of hepatic 
and reticuloendothelial cells. Prior to about 1972, it had been assumed 
that ferritin only existed in extracellular fluid under abnormal 
conditions. It has since been discovered that the concentration of serum 
ferritin directly correlates with the content of iron in body stores in 
normal human beings. Thus, the measurement of serum ferritin levels 
provides an effective method by which the status of body iron stores may 
be evaluated and clinical diagnoses made therefrom. For example, an 
accurate measurement of the serum ferritin level will enable a clinician 
to differentiate between that anemia caused by iron depletion and other 
forms of anemia. 
Inasmuch as ferritin is an antigenically active macromolecule, the presence 
of ferritin can be detected by techniques which have been developed in the 
past for detecting various other antigenic materials. The radioimmunoassay 
procedure is an example of such a technique. 
Addison et al, Journal of Clinical Pathology, Volume 25, pages 326-329 
(1972) disclose a technique wherein human blood serum is treated with 
soluble purified radioactive ferritin antibodies. The radioactive complex 
produced remains in solution while unused radioactive antibodies, i.e., 
those which do not link up with the antigen, are removed by a second 
reaction with a solid phase antigen. 
Miles et al, Analytical Biochemistry, Volume 61, pages 209-224 (1974) 
suggests an improvement in this technique wherein the ferritin is first 
insolubilized and thereafter is made to react with soluble labeled 
antibody. The labeled complex is thus insoluble and any unreacted labeled 
antibody can be washed away. It will be appreciated that the amount of 
ferritin present will be directly proportional to the radioactivity in 
solid phase. 
Solid phase radioimmunoassay of antigens is believed to have been developed 
by Catt (see, e.g., U.S. Pat. No. 3,646,346 to Catt) and others (see for 
example, U.S. Pat. No. 3,790,663 to Garrison et al). Basically, the prior 
art teaches the solid phase antibodies are coated on a polystyrene 
substrate. The substrates are then placed in contact with serum containing 
ferritin and permitted to stand for a period of time to enable reaction to 
occur. After an incubation period, the substrates are washed, dried, and a 
radioactive count measurement is taken. The measurement is compared with 
standard values for different serum ferritin concentration levels. 
More recently, a two-site immunoradiometric assay for serum ferritin has 
been developed. This new test is basically a two stage reaction. In the 
first stage, human serum ferritin is bonded to a solid-phase anti-human 
ferritin. In the second stage, a purified, radiolabeled anti-human 
ferritin is bound to the first stage reaction product. Then the solid 
phase is washed and counted in a radiation counter. The concentration of 
serum ferritin may be calculated by comparing the unknown with a 
simultaneously run standard sample. 
Apparatus for performing a quantitative radioimmunoassay have been somewhat 
varied. The present invention is directed to an improvement over apparatus 
which includes the following components. Firstly there is provided a water 
insoluble polymeric substrate fashioned as a sphere or bead. This bead is 
coated with a previously formed human ferritin antibody. After the beads 
have been prepared, the beads are washed with deionized water. 
Later, each bead is washed in a container such as a beaker. After the 
washing is completed, the container is aspirated to remove the rinsing 
water. Immediately after aspiration of the water, a bead would have to be 
placed in a test tube having a diluted sample of patient serum and a test 
tube containing the test standard. The beads would remain in the test 
tubes at room temperature for six hours or for sixteen hours at 4.degree. 
C. Alternatively, the test tubes could be covered and placed on a 
horizontal rotating table and shaken for approximately two hours at a 
moderate speed at room temperature. 
In the prior art, after incubation, the reaction solution is aspirated from 
the test tube bearing the patient sample and the test tube bearing the 
standard. Then each bead is washed twice with a special washing solution. 
This washing solution is sprayed onto the bead and is almost immediately 
thereafter aspirated by an apparatus especially adapted for such use. Each 
bead is washed twice with approximately one minute between washings. The 
test tubes must be aspirated twice to remove any liquid that may have 
drained from the sides of the test tubes. 
After washing and aspirating, the radiolabeled antibody is introduced onto 
the beads in the test tubes. The test tubes are then refrigerated at 
4.degree. C. for sixteen hours. Alternatively, the test tubes can be 
covered and shaken on a horizontal shaking apparatus for 2 hours at a 
moderate speed at room temperature. 
After this second incubation, the radioactive solution is aspirated from 
each test tube utilizing a special apparatus. This relatively complex 
device is used to wash each bead three times with a washing solution. The 
washing solution, of course, must be aspirated after each wash. After the 
last wash, the test tubes are required to remain stationary for an 
additional minute to give liquid on the side of the test tube an 
opportunity to drain to the bottom. This additional fluid should be 
aspirated. 
Each of the beads is then removed from their respective test tubes and 
introduced into special counting tubes which were not used in the assay. 
The counting tubes are introduced into a gramma radiation counter set to 
detect the labeling isotope. The beads are counted for a time appropriate 
for adequate statistical significance, and the appropriate calculations 
are made in a now well-known manner to provide a quantitative 
determination of iron ferritin in the patient. 
While such arrangements have exhibited at least a degree of utility in 
radioimmunoassay, room for significant improvement remains. The beads 
utilized in at least some known iron ferritin analyses are 1/4" in 
diameter and are relatively difficult to handle. Handling by a pathologist 
or laboratory technician introduces the risk of contamination both to the 
test sample and to the personnel. Because the 1/4" diameter beads are 
difficult to handle, they may be dropped thus necessitating repeating the 
time-consuming first stage and second stage incubation periods. This time 
delay may have adverse consequences to the patient and results generally 
in an inefficient laboratory operation. 
Utilizing known apparatus, the beads would require handling after both the 
first and second phase incubations have occurred. If a test sample were 
spoiled at the second phase of the procedure, several days may be required 
to repeat the procedure. 
Of independent significance, is the relatively complex equipment required 
to wash and aspirate each test tube containing a test bead. In order to 
achieve satisfactory washing, the washing procedure must be repeated once 
and is preferably repeated another time. Aspiration is required between 
washings, and both a pressure pump and a suction pump are required. The 
pressure pump is required to introduce the washing solution into each test 
tube, and a suction pump is required to draw the washing solution out of 
the test tube through a specially adapted conduit. It will be appreciated 
that in the event of a failure of the relatively expensive washing or 
aspirating equipment, the assay might be terminated or spoiled. In 
addition, it is believed that with washing and aspirating, even three 
times and with a specially formulated washing solution, there may not be 
an adequate washing of each bead. 
The problems enumerated in the foregoing are not intended to be exhaustive 
but rather are among many which tend to impair the effectiveness of 
previously known radioimmunoassay apparatus. Other noteworthy problems may 
also exist; however, those presented above should be sufficient to 
demonstrate that radioimmunoassay apparatus appearing in the art have not 
been altogether satisfactory. 
OBJECT AND SUMMARY OF PREFERRED EMBODIMENT 
Recognizing the need for an improved radioimmunoassay apparatus, it is, 
therefore, a general object of the present invention to provide a novel 
radioimmunoassay apparatus which minimizes or reduces the problems of the 
type previously noted. 
It is a more particular object of the present invention to provide a novel 
ferritin assay diagnostic kit which eliminates or substantially minimizes 
direct contact with test beads which form the substrate for solid phase 
human ferritin antibodies. 
It is another object of the present invention to provide a novel ferritin 
assay diagnostic kit which is not dependent upon complex washing or 
aspirating apparatus. 
It is yet another object of the present invention to provide a novel 
ferritin assay diagnostic kit which improves control and facilitates 
handling of coated beads. 
It is still another object of the present invention to provide a novel 
ferritin assay diagnostic kit which improves efficiency of laboratory 
operations and minimizes test repeats. 
It is yet still another object of the present invention to provide a novel 
ferritin assay diagnostic kit which improves the affectiveness of a 
washing and wiping of coated beads. 
An apparatus for performing a quantitative radioimmunoassay according to a 
presently preferred embodiment of the invention intended to substantially 
accomplish the foregoing objects includes a bead for carrying a dry 
antibody, a gripper for gripping the bead, a carrier for carrying at least 
one gripper, and a reaction chamber or test tube. The gripper is comprised 
of an elongate handle portion and a clasp portion preferrably in the form 
of a trident. The particular arrangement of the trident facilitates a 
spinning of the bead while engaged by the gripper. The carrier includes a 
base and preferably a plurality of spaced apart collars carried thereon 
for receiving the elongate handle portion of the gripper. In preferred 
form, the reaction chambers are arranged in a 4.times.8 matrix and are 
spaced-apart to correspond to the spacing of the collars of the carrier. 
Examples of the more important features of this invention have thus been 
outlined rather broadly in order that the detailed description thereof 
that follows may be better understood and in order that the contribution 
to the art may be better appreciated. There are, of course, additional 
features of the invention that will be described hereinafter and which 
will also form the subject matter of the claims appended hereto. 
These features will become apparent with reference to the following 
detailed description of a preferred embodiment in connection with the 
accompanying drawings, wherein like referenced numerals have been applied 
to like elements.

DETAILED DESCRIPTION OF A PREFERRED EMBODIMENT 
With particular reference now to FIGS. 1 and 2 there may be seen a 
radioimmunoassay (RIA) apparatus in accordance with the present invention. 
The RIA apparatus 10 is comprised of a test tray 12 having a plurality of 
reaction chambers 14 arranged preferably in a 4.times.8 matrix. A 
plurality of gripper carriers 16 are arranged along the 8-unit rows of the 
test tray 12. Each carrier 16 is arranged to carry a plurality of bead 
grippers 18. 
Each bead gripper 18 is comprised of an elongate handle portion 20 and a 
clasp or trident 22 at one end 24 of the handle portion 20 (see FIGS. 3 
and 4). The trident 22 is comprised of three resilient or spring-like 
fingers 26 which are arranged to engage a coated bead 28. Each finger 26 
extends from the end 24 of the handle portion 20 to a point beyond a 
diameter of the bead transverse the longitudinal axis of the handle. That 
is, each of the fingers extends beyond halfway around the bead 28. Each 
finger 26 has an interior surface 30 which is contoured to correspond to 
the surface of the bead 28. The distal end 32 of each finger 26 has a 
rounded camming surface 34. A base 36 of the trident, i.e., the zone where 
the fingers 26 intersect, is arranged to lie out of contact with the 
surface of the bead 28. An outer surface 38 of each finger intersects the 
intercontoured surface at two edges 40, 42 which extend from about the 
camming surface 34 almost to the trident base 36. 
When it is desired to grip a bead 28 with the gripper 18, the gripper is 
held by the handle portion 20 and the trident 22 is positioned over the 
bead. The gripper is then pressed towards the bead. This pressing force 
causes each of the fingers 26 to be cammed outwardly by their respective 
camming surfaces 34. As the fingers travel over the surface of the bead, 
they are urged outwardly until the plane of the transverse diameter is 
intersected whereupon the fingers begin to converge. As the fingers begin 
to converge, they tend to "snap" the bead toward the base 36 of the 
trident. 
With the bead 28 thus gripped or engaged by the gripper 18, the bead may be 
manipulated with facility. The bead may be washed by positioning the bead 
(held with the gripper) beneath a stream of flowing water. The inside 
contoured surfaces 30 of the fingers 26 and the displacement of the 
trident base 36 out of contact with the bead facilitate a spinning of the 
bead when placed beneath a stream of flowing liquid. Moreover, the edges 
40, 42 of the fingers 26 tend to wipe the bead as the bead rotates. This 
wiping action along with the rotation of the bead provides an improved 
washing on all surfaces including those lying initially beneath each 
finger 26 and beneath the trident base 36. 
As may be seen in FIGS. 5 and 6, each carrier 16 comprises a base portion 
44, a handle 46 which extends along the entire length of the base portion 
44, and a lip 48 which also extends along the entire length of the base 
portion 44. At locations along the base portion 44 there are provide a 
plurality of gripper handle-engaging collars 50. Each collar 50 extends 
slightly above a top surface 52 of the base 44. Fashioned coaxially with 
respect to each collar 50 is a chamfer 54. The diameter of each collar 50 
is selected to provide an interference fit with the gripper handle 20. As 
may be seen in FIG. 2, a series of bead grippers 18 may be inserted into a 
plurality of collars 50 to facilitate handling several grippers at one 
time. 
As will be appreciated from FIGS. 1 and 2, the collars 50 are preferably 
arranged in the carrier 16 to correspond to the openings in a row of eight 
reaction chambers 14 in the test tray 12. The base 44 of the carrier 16 is 
of a length sufficient to accommodate an entire 8-unit row of reaction 
chambers 14. However, it will be appreciated that a single carrier could 
be provided which was of a length appropriate to position a collar above 
each reaction chamber in 8-unit rows of adjacent test trays. In addition, 
the carrier could be of a width appropriate to facilitate handling more 
than one row of reaction chambers in a test tray. That is, a single 
carrier might be provided to accommodate all of the reaction chambers 14 
in the test tray 12 of FIG. 1. 
In preferred form the carrier 16 will provide collars which correspond to a 
single row of reaction chambers 14 in a single test tray 12. It has been 
found that providing a signal or indicator of orientation for the carrier 
in addition to its structural components is desirable. In carrying out a 
procedure with the RIA apparatus of the present invention, proper labeling 
of each reaction chamber 14 and/or bead 28 is essential. Therefore, the 
letters "L" and "R", corresponding to "left" and "right" are preferably 
embossed or printed or otherwise affixed to each carrier 16 (see FIG. 5). 
As noted above, the test tray 12 comprises a matrix of reaction chambers 
14. Each reaction chamber 14 is defined by a generally cylindrical wall 54 
and a curved bottom 56 (see FIG. 7). The curved bottom portion 56 is 
provided with a foot 58. The generally cylindrical wall 54 of each 
reaction chamber 14 tapers slightly over the length of the reaction 
chamber. This taper is in the order of a half degree or so. The reaction 
chambers 14 are interconnected with a web 60. A peripheral channel 62 is 
provided below the level of the web 60 to facilitate control of spillage. 
A peripheral lip 64 which forms an outer wall of the peripheral channel 62 
may extend to or above the plane of the tops 66 of the reaction chambers 
14. An upper edge 68 of the peripheral lip 64 is operable to support each 
carrier 16 when it is desired to position the beads within the reaction 
chambers as shown in FIG. 2. 
Each bead 28 is preferably fashioned from a water insoluble polymeric 
material. This polymeric material forms the substrate for previously 
formed human ferritin antibodies. The substrate should be of a material 
which readily adsorbs the proteinaceous antibody material. It is known 
that most organic polymeric materials meet this criterion. 
Examples of organic polymers suitable for the manufacture of beads 28 are 
hydrocarbon polymers such as polystyrene, polyethylene, polypropylene, 
polybutylene, butyl rubber and other synthetic rubbers, as well as 
polyesters, polyamides, vinyl and acrylic polymers such as polyvinyl 
chloride, and polymethel methacrylate, cellulose and cellulose derivatives 
such as cellulose acetate. In general, any organic polymeric material 
which adsorbs protein in relatively large amounts will be acceptable. The 
preferred substrate is impact grade polystyrene. The coated beads are 
prepared by dipping them into the antigen or antibody solution desired and 
following a known procedure for suitable adsorption. 
In operation, the apparatus may be utilized to perform a two-site 
immunoradiometric assay as follows. An unknown sample of human serum to be 
tested for ferritin content is introduced into an appropriately labeled 
reaction chamber 14. A ferritin standard is similarly introduced into 
another appropriately labeled reaction chamber 14. 
Coated plastic beads 28 required for the assay preferably are placed in a 
petri dish (not shown) containing distilled water. A gripper 18 is then 
placed over the submerged or partially submerged bead 28 to engage the 
bead. After engagement of the bead, the pathologist or technician has 
absolute control over the bead, and the chances of spoiling the test are 
minimized. With the required number of beads engaged, the grippers may be 
inserted into the collars 50 on the carrier 16. As noted above, the 
grippers are interference fit into the collars. 
The carrier (with the grippers inserted into the collars) may be placed 
over the reaction chambers, and the beads are submerged in the serum 
samples. 
The reaction chambers may be permitted to stand at room temperature for six 
hours or at 4.degree. C. for sixteen hours. Alternatively, the test tray 
may be placed on a horizontal rotating table and shaken for two hours at a 
moderate speed at room temperature. 
It has been found desirable in some instances to position the test tray 12 
on a stationary surface and connect the carrier 16 to a vibrator or 
agitation apparatus schematically depicted at 70 (see FIG. 2) to 
facilitate incubation. Alternatively, the carrier 16 may be supported by a 
fixed object, and the test tray 12 may be vibrated. In any event, it will 
be appreciated that the apparatus of the present invention facilitates 
improved agitation of the beads with improved results in the quality of 
incubation. 
After a first stage incubation, the carrier 16 may be lifted away from the 
test tray 12, and the grippers 18 may be placed under a stream of, say, 
distilled water to wash the beads. As noted above, the beads 28 will 
rotate in the stream, and a wiping action will be produced by the edges 
40, 42 of the fingers 26. 
The carrier 16 is then positioned over the test tray to introduce the beads 
28 into a different row of reaction chambers 14 which contain a 
radio-labeled antibody or antisera. A second stage incubation is 
facilitated by refrigerating the test tray 12 with the beads in the 
reaction chambers at 4.degree. C. for sixteen hours. Alternatively, the 
incubation may be stimulated by agitating either the test tray 12 or the 
carrier 16 for two hours at a moderate speed at room temperature. 
Then each carrier 16 is removed from the test tray, and the beads are again 
rinsed under a stream of distilled water. Then each gripper 18 is removed 
from the carrier. The gripper may be used to move the bead to a counting 
tube (not shown) where the bead 28 will be forced out of the trident 
either manually or with an appropriate instrument. The beads are then 
counted in a gamma radiation counter set to detect the labeling isotope. 
All beads are counted for the amount of time required for adequate 
statistical results. The appropriate calculations are then made to 
evaluate the test results. 
SUMMARY AND SCOPE OF THE INVENTION 
According to the present invention there is provided an apparatus for 
performing a quantitative analysis for determining the concentration of 
serum ferritin in human blood. According to the invention, at least for a 
portion of the assay, a water insoluble organic polymeric bead such as a 
polystyrene bead, is held by a gripper which enables the bead to be 
rotated during washing. A group of such grippers are treated at one time 
by means of a special carrier. 
Agitation to enhance incubation may be facilitated by vibrating either the 
test tray in relation to the stationary grippers or vice versa. It will, 
of course, be appreciated that the test tray, carrier, gripper and bead 
are fashioned from materials having sufficient strength to withstand the 
forces of agitation imposed during incubation. 
In constructing a radioimmunoassay apparatus according to the present 
invention, certain significant advantages are provided. In particular, the 
test beads may be manipulated without direct contact with the beads and 
without a resulting likelihood of contamination. Moveover, the beads are 
securely gripped by the gripper of the present invention which greatly 
reduces the possibility of dropping or misplacing the bead. 
In addition, entire rows of beads may be handled at one time with the 
present apparatus. This results in increased laboratory efficiency and 
minimizes repeat testing. 
Further modifications and alternative embodiments of the apparatus of this 
invention will be apparent to those skilled in the art in view of this 
description. Accordingly, this description is to be construed as 
illustrative only and is for the purpose of teaching those skilled in the 
art the manner of carrying out the invention. It is to be understood that 
the forms of the invention herewith shown and described are to be taken as 
the presently preferred embodiment. Various changes may be made in the 
shape, size and arrangement of parts. For example, the trident portion of 
the gripper may be comprised of four, five, or more finger-like members, 
equivalent materials may be substituted for those described herein, some 
parts may be reversed, and certain features of the invention may be 
utilized independently of the use of the other features, all as would be 
apparent to one skilled in the art after having the benefit of this 
description of the invention.