Title:
Method and apparatus for radioimmunoassay with regeneration of immunoadsorbent
United States Patent 3896217
Abstract:
Method and apparatus for radioimmunoassay providing a continuously reusable short cycle analytical system of particular use in biochemical analysis such as the determination of steriods, polypeptides and the like. The method disclosed comprises admixing an unlabelled antigen sample with a known concentration of labelled antigen, bringing the mixture into contact with a mass of appropriate antibody that has been immobilized thereby to bind at least part of the mixed antigen to the antibody. The amount of bound and/or unbound labelled antigen is detected and counted and, by use of previously prepared standards, the unknown concentration of unlabelled antigen is found. The still immobilized antibody is regenerated for immediate reuse by rinsing it with a selected eluting solution that is capable of breaking the antigen-antibody bond without altering the characteristics of the antibody. Alternate ways of immobilizing the antibody are described. Several suitable eluting solutions are disclosed. Also disclosed is a novel apparatus by which the new method may be automatically carried out and the antibody reused many times. By proper substitution, the antibody and antigen may be reversed.
US Patent References:
/3555143.html
Axen et al. - January 1971 - 3555143
/3592888.html
Wolf - July 1971 - 3592888
/3720760.html
Bennich et al. - March 1973 - 3720760
/3555143.html
Axen et al. - January 1971 - 3555143
/3592888.html
Wolf - July 1971 - 3592888
/3720760.html
Bennich et al. - March 1973 - 3720760
Application Number:
05/342513
Publication Date:
07/22/1975
Filing Date:
03/19/1973
View Patent Images:
Export Citation:
Assignee:
Summa Corporation (Las Vegas, NV)
Primary Class:
Other Classes:
436/826, 436/529, 436/808, 436/532, 436/829, 436/804, 436/817
International Classes:
G01N33/543; G01N33/548; G01N33/544; A61K27/04; G01N23/00
Field of Search:
424/1,12 23/23B 250/303
Primary Examiner:
Padgett, Benjamin R.
Attorney, Agent or Firm:
Martella, Mario A.
Claims:
I claim
1. In an analytical method for biochemical analysis of an antigen wherein a mass of antibodies specific to a selected antigen is immobilized to form an immunoadsorbent and wherein a solution containing the selected antigen is flowed into and out of contact with the immunoadsorbent whereby at least part of said selected antigen is bound to said mass of antibodies immobilized by said immunoadsorbent and the remainder is not bound to said antibodies, the improvement which comprises the steps of:
2. In an analytical method as set forth in claim 1 wherein the immunoadsorbent is formed by co-valently bonding the said mass of antibodies to individual solids of a mass of particulate solids,
3. In an analytical method as set forth in claim 1 wherein said mass of antibodies is co-valently bonded to a cross-linked dextran polymer.
4. In an analytical method as set forth in claim 1 wherein the antibodies are specific to an antigen selected from the class consisting of steroids, polypeptides and thyroid hormones.
5. In an analytical method as set forth in claim 2 wherein the antibodies are specific to an antigen selected from the class consisting of steroids and thyroid hormones, and
6. In a radioimmunoassay analytical procedure in which a sample solution containing antigen to be assayed is mixed with a known concentration of labelled antigen, the mixed sample solution being brought into contact with an immunoadsorbent formed by immobilizing a mass of antibodies specific to said antigen thereby to effect binding of part of the labelled and unlabelled antigens in said mixed sample solution to the antibodies immobililzed by said immunoadsorbent, the remaining portion of the labelled and unlabelled antigens in said mixed sample solution passing through said immunoadsorbent and forming an unbound antigen fraction, the improvement which comprises:
7. In a radioimmunoassay procedure as set forth in claim 6 wherein said detecting step includes
8. In a radioimmunoassay procedure as set forth in claim 6 wherein said antibodies are specific to an antigen selected from the class consisting of steroids, polypeptides, thyroid hormones, and viruses, and
9. In a radioimmunoassay procedure as set forth in claim 6 in which said mixed sample solution is flowed into contact with and through said immunoadsorbent to effect binding a part of the labelled and unlabelled antigens,
10. In a radioimmunoassay procedure as set forth in claim 6 wherein said immunoadsorbent is formed by co-valently bonding said mass of antibodies to polymeric material, and wherein said procedure is continuous and involves repetitively for different samples the recited steps of
1. In an analytical method for biochemical analysis of an antigen wherein a mass of antibodies specific to a selected antigen is immobilized to form an immunoadsorbent and wherein a solution containing the selected antigen is flowed into and out of contact with the immunoadsorbent whereby at least part of said selected antigen is bound to said mass of antibodies immobilized by said immunoadsorbent and the remainder is not bound to said antibodies, the improvement which comprises the steps of:
2. In an analytical method as set forth in claim 1 wherein the immunoadsorbent is formed by co-valently bonding the said mass of antibodies to individual solids of a mass of particulate solids,
3. In an analytical method as set forth in claim 1 wherein said mass of antibodies is co-valently bonded to a cross-linked dextran polymer.
4. In an analytical method as set forth in claim 1 wherein the antibodies are specific to an antigen selected from the class consisting of steroids, polypeptides and thyroid hormones.
5. In an analytical method as set forth in claim 2 wherein the antibodies are specific to an antigen selected from the class consisting of steroids and thyroid hormones, and
6. In a radioimmunoassay analytical procedure in which a sample solution containing antigen to be assayed is mixed with a known concentration of labelled antigen, the mixed sample solution being brought into contact with an immunoadsorbent formed by immobilizing a mass of antibodies specific to said antigen thereby to effect binding of part of the labelled and unlabelled antigens in said mixed sample solution to the antibodies immobililzed by said immunoadsorbent, the remaining portion of the labelled and unlabelled antigens in said mixed sample solution passing through said immunoadsorbent and forming an unbound antigen fraction, the improvement which comprises:
7. In a radioimmunoassay procedure as set forth in claim 6 wherein said detecting step includes
8. In a radioimmunoassay procedure as set forth in claim 6 wherein said antibodies are specific to an antigen selected from the class consisting of steroids, polypeptides, thyroid hormones, and viruses, and
9. In a radioimmunoassay procedure as set forth in claim 6 in which said mixed sample solution is flowed into contact with and through said immunoadsorbent to effect binding a part of the labelled and unlabelled antigens,
10. In a radioimmunoassay procedure as set forth in claim 6 wherein said immunoadsorbent is formed by co-valently bonding said mass of antibodies to polymeric material, and wherein said procedure is continuous and involves repetitively for different samples the recited steps of
Description:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates generally to the analytical technique known as radioimmunoassay in which antigens are bound to specific antibodies and, through the use of tracers (labels) and predetermined behavior standards the concentration of antigen in a sample is determined. More particularly, the invention relates to improved methods and apparatus for radioimmunoassay in which a short-cycle time, hence more rapid analysis is achieved, the antibody mass (immunoadsorbent) is regenerated to be reused indefinitely rather than being wastes and the entire operation is automated.
2. State of the Art
Radioimmunoassay is an analytical technique which depends upon the competition (affinity) of antigen for antigen-binding sites on antibody molecules. In practice, standard curves are constructed from work on a plurality of samples each containing (a) the same known concentration of labelled antigen, and (b) various, but known, concentrations of unlabelled antigen. Antigens are labelled with a radioactive isotope tracer. The mixture is incubated in contact with an antibody, the free antigen is separated from the antibody and antigen bound thereto, and then, by use of a suitable detector, such as a gamma or beta radiation detector, the percent of either the bound or free labelled antigens is determined. This procedure is repeated for a number of samples containing various known concentrations of unlabelled antigens and the results plotted. The percent of bound tracer antigens is plotted as a function of the antigen concentration. Typically, as the total antigen concentration increases the relative amount of the tracer antigen bound to the antibody decreases. After the standard graph is prepared, it is thereafter used to determine the concentration of antigen in samples undergoing analysis.
In actual analysis, the sample in which the concentration of antigen is to be determined is mixed with a known amount of tracer antigen. Tracer antigen is the same antigen known to be in the sample but which has been labelled with a suitable radioactive isotope. The sample with tracer is then incubated in contact with the antibody. Thereafter, it may be counted in a suitable detector which counts the free antigen remaining in the sample. The antigen bound to the antibody or immunoadsorbent may also be similarly counted. Then, from the standard curve, the concentration of antigen in the original sample is determined. Afterwards, the antibody or immunoadsorbent mass is discarded.
In order to detect the percentage of antigen that is bound to the antibody (bound antigen) and/or the percentage that remains free or unbound it is necessary to first separate the sample into a fraction containing bound antigen and one containing only free antigen. One common method for doing this is to add a dextran coated charcoal to the mixture. The charcoal is allowed to adsorb the free antigen. The charcoal with adsorbed free antigen is then separated from the antibody (and bound antigen) by centrifugation. Another known procedure is to add to the mixture another antibody which selectively precipitates the first antibody (with the bound antigen) thus leaving in solution only free antigen. Classification into appropriate free and bound fractions is then effected by separating the precipitate from the supernatant by centrifugation or other suitable means. Some workers have resorted to the technique of binding the antibody to the inner walls of a plastic vessel, filling the vessel with the antigen bearing sample, allowing it to stand for an incubatioin period that typically ranges from 4 to 72 hours and then separating free antigen from bound antigen by draining and rinsing the vessel leaving therein only the antibody and bound antigen. A more recently developed technique is to prepare the immunoadsorbent by binding the antibodies onto an insoluble cross-linked dextran. The immunoadsorbent and antigen bearing sample are incubated then the dextran with bound antigen is separated from the solution by suitable means.
In all of the foregoing procedures, the percentage of labelled antigen in either or both the bound or free fractions is determined and the standard curve used to determine the antigen concentration. Thereafter, the immunoadsorbent is discarded.
Although the foregoing radioimmunoassay techniques have proven to be valuable tools and have gained widespread acceptance, they are still not all that are to be desired because the antibody (immunoadsorbent) is consumed with each analysis hence must be discarded. Moreover, prior practice is batch type and the several reagents are added to the antibody in test tubes in which the separate steps, such as incubation, rinsing and the like, are performed, thus resulting in a slow and costly operation.
SUMMARY OF THE INVENTION
The present invention provides improved method and apparatus for carrying out radioimmunoassay. In accordance with the invention the immunoadsorbent (antibody) is repeatedly and rapidly regenerated thus obviating the need and therefore the time and cost of constant replacement. According to the invention, steps are incorporated by which the method may be carried on continuously in constantly repeating cycles thereby eliminating the expensive time-consuming batch operation. A novel equipment arrangement for automating the method is also provided.
The invention is predicated on the discoveries that (1) by forming the immunoadsorbent mass as an immobilized mass of antibodies through which the antigen sample and other reagents may flow the procedure may be sequentially carried out without resort to the use of several manual steps and (2) the immobilized immunoadsorbent may be regenerated for repeated reuse by the rinsing with a solvent or eluting solution having particular characteristics. That is to say, bound antigens are released so that the antibody may be washed clean of bound antigen and thereby regenerated for reuse without affecting the essential characteristic of the antibody mass such as its ability to permit repeated flow of antigen solution therethrough and its antigen-binding efficiency during a large number of cycles.
As used herein, the term immobilized antibody mass refers to a mass of antibodies held in place in a liquid stream flow path in such a manner that the stream may flow over or through the mass while the latter remains in place.
A suitable antibody mass may be formed from solid surfaces such as glass or water insoluble polymers to which the antibodies are attached by co-valent bonds. A mass of such beads with attached antibodies is supported on a screen in a tube or other hollow column through which the antigen bearing sample and other reagents are flowed sequentially. Co-valent coupling of antibodies or antigens to said carrier is known. (It is disclosed for instance in U.S. Pat. No. 3,652,761).
In accordance with this invention, antibodies, specific to the antigen under analysis, are co-valently coupled to a solid support such as beads, a mass of the beads is immobilized, an antigen bearing solution is flowed therepast, the percentage of free labelled antigen remaining in the solution is detected, the bound antigen is released simultaneously with regeneration of the antibody mass and the percentage of released antigen is also detected. The percentages are determined with reference to the total antigen in the incoming sample. As noted, detection is of the tracer or labelled antigen.
Release of bound antigen and concommitant regeneration or reactivation of the antibody mass is effected by rinsing it with a solvent or eluting solution that breaks the bond between antigen and antibody, but does not break the co-valent bond between the antibody and its support. Moreover, the solvent must not alter either the flow characteristic or antigen affinity of the antibody mass.
Another type of immobilized antibody mass can be formed by encapsulating antibodies in a semi-permeable membrane. A quantity of such micro-capsules is then held in place in a column through which the antigen-bearing solution flows. The membranes are selected to block the outward passage of antibodies while permitting the free passage of antigens and their solvent in both directions.
In the case of membrane capsules, since there is no covalent bond, there is no need to be concerned with its destruction by the regenerating solvent. However, the limitation must still be observed that the solvent not adversely affect the flow characteristic or adsorption efficiency of the antibody. It is possible to increase efficiency of the membranes by coupling the antibodies to a soluble polymer thereby increasing the molecular weight of the combined antibody thus enabling the use of a relatively more permeable membrane.
In connection with flow characteristics in either system, the solvent should not cause swelling of the antibody support or the membrane wall to the point where flow of relatively small antigens is blocked or the loss of relatively larger antibodies through the wall occurs.
In summary then, the method of the invention comprises the steps of providing a mass of immobilized specific antibodies to selected antigen, holding such mass in a liquid flow path, preparing an antigen bearing sample by adding a known quantity of labelled antigen to a sample containing an unknown amount of the same antigen, flowing such sample along said liquid flow path over and in contact with said immobilized antibody, thereafter detecting the quantity of free labelled antigen remaining in the sample and/or that which is bound to the antibody.
The quantity of antigen bound to the antibody is determined by rinsing the antibody with a particular solvent to release bound antigens then detecting the antigens in the rinse solution.
The solvents found to meet the requirements of this invention have been hydrophobic in character. Typical solvents are methyl, ethyl and isopropyl alcohols as well as dimethylformamide.
In its most essential form, the apparatus invention comprises a contact chamber having an inlet and outlet and adapted to accommodate the flow of a liquid stream therethrough, a mass of antibodies, means for immobilizing said antibodies and holding them in position in said contact chamber in the path of antigen flow therethrough, a detection chamber having an inlet and outlet, a detector associated with said chamber for determining the quantity of a given tracer flowing through said chamber, conduit means for conductng liquid from the outlet of said contact chamber to the inlet of said detection chamber, a source of sample solution containing both labelled and unlabelled antigen, a source of regenerating solvent, means for supplying separately and sequentially to the inlet of said contact chamber for flow therethrough a quantity of said sample and of said solvent, and means for controlling the flow of said sample and solvent through said contact chamber and said detection chamber. Means are also provided for introducing a so-called scintillation cocktail into the system between the contact chamber and detector to flow through the latter and therein to convert radioactive decay impulses to light or photons for detection by a photo multiplier detector.
The required flow rates through the contact chamber and detector are determined emperically and are controlled by metering pumps. The flow sequence is controlled by valves. By proper timing the flow through the system may be continuous with the rates being selected to provide sufficient time at each station to achieve the results, such as binding, releasing or counting, sought at each station.
In order that the invention may be more readily understood and carried into effect, reference is made to the accompanying drawing and the description thereof which are offered by way of illustration and not in limitation of the invention, the scope of which is defined by the appended claims and equivalents thereof rather than by any illustrative description.
BRIEF DESCRIPTION OF THE DRAWINGS AND DESCRIPTION OF PREFERRED EMBODIMENT
The FIGURE is a schematic diagram of apparatus embodying the invention.
As illustrated, the system comprises a sample source 10, a source 11 of buffer rinse, a source 12 of regenerating solvent, a source 13 of scintillation cocktail, a rinse water reservoir 14, a contact chamber 16 filled with immobilized antibodies (immunoadsorbent) a mixing chamber 17, and a detector chamber or coil 18 with detector 19 adjacent thereto. The foregoing components are connected together by a series of conduits and flow directing valves which, together with metering pumps and a timing mechanism, regulate flow through the system.
Specifically, the sample source 10 connects via a conduit 21 to a two-position valve 22 which in one position connects to a sample loop 23. The sample loop terminates in a two-position valve 24 which in one position connects to a conduit 26 leading to another two-position valve 27 which in one position connects the conduit 26 and sample loop 23 to an aspirator pump 28 which discharges to waste. A rinse water inlet conduit 29 also connects to the pump 28 via the alternate position of valve 27 to keep the pump primed and rinsed when it is not drawing sample.
The displacement rinse flows from tank 11 to a two-position valve 31 which in one position connects through a pump 32 to a further two-position valve 33 whence it connects either to a conduit 34 leading to the valve 22 and sample loop 23 or to a conduit 36 leading to the contact chamber, mixer and detector. A conduit 37 connects the valve 24 to the main conduit 36. A branch conduit 38 connects to the main conduit 36 at a junction between the contact chamber and the detector at a location ahead of the mix chamber 17. A pump 39 is provided to move a reagent from the tank 13 through the conduit 38.
A central timer, generally designated 41, is employed to control sequencing of the valves and pumps. The timer may be custom assembled for the job in accordance with available technology hence, its structure need not be described in detail. It is sufficient to indicate, as is done in the drawing, that the valves, all of which are electrically actuatable solenoid valves, and the metering pumps, all of which are electric, are connected to the timer by suitable conductors 42.
OPERATION OF THE SYSTEM
At the start of a cycle rinse reagent from the tank 11 passes through valve 31, pump 32, valve 33 into and through conduit 36. This rinses the system. Sample from tank 10 is drawn by pump 28 through the valves into and through the sample loop 23 until the loop is rinsed and filled whereupon the valves 22, 24, 27 and 33 switch to the alternate positions whereupon there is a continuous flow from tank 11 through valve 31, pump 32, valve 33, valve 22, loop 23, valve 24 and conduit 37 to conduit 36. Liquid flowing from tank 11 thus displaces sample from the loop into and through the contact chamber 16. When the flow has continued long enough to displace all sample from the loop, the valves 22, 24 and 33 return to the initial positions shown in the drawing. This isolates the sample loop.
After the sample has passed through the contact chamber 16 and the detection coil, the valve 31 shifts to accept regenerating solvent from the tank 12 and directs it through pump 32 and valve 33 directly into conduit 36 to flow through the contact chamber where it releases the bound antigens and carries them to the mixer and detector. Simultaneously, the immunoadsorbent in the contact chamber is regenerated.
The scintillation cocktail in tank 13 typically comprises a primary scintillator, a secondary scintillator and a solubilizer all carried in toluene. Its function is to mix with the antigen bearing solution in the mix chamber 17 and then to convert the radioactive impulses of the tracer to light or photons which can be detected by a photo multiplier detector. Photons may be detected by either a gamma or beta scintillation spectrometer.
The tracer or label is selected from those conveniently available. A label of 3 H Tritium may be used with a beta scintillator detector with a coincidence circuitry counter whereas an Iodine label 125 I may be used with a gamma detector which does not require the coincidence circuitry. In the tests reported herein, a tritium label was used.
It is important that the sample loop be kept uncontaminated with the regenerating solvent since traces of that mixed with the sampler could reduce the bonding efficiency in the contact chamber.
Although a very elemental sample is illustrated, it will be appreciated that several sophisticataed samplers are readily available that may be adapted to use in this invention.
A series of basic tests were performed to confirm that immunoadsorbents when formed as immobilized antibodies could be consistently regenerated with suitable reagents.
Reagents used were as follows:
Buffer Rinse (Solution A)
0.02 m sodium phosphate, pH 7.5
0.05 m sodium chloride
0.01% merthiolate
0.02% sodium azide
Regenerating solvent (Solution B)
Solution A with the addition of
30% (v/v) of 95% ethyl alcohol.
Scintillating Solution (Solution C)
Five parts of a solution of
0.4% (w/v) 2.5 diphenyloxazole
0.008% (w/v) 1, 4-bis-2(4-methyl-5-phenyloxazolyl) benzene
toluene as solvent
one part of Scintisol-GP (manufactured by Isolab, Inc.)
EXAMPLE I
A contact chamber was prepared from a glass pipette plugged at one end with glass wool. A slurry of 0.04 ml of immunoadsorbent suspension was introduced. The suspension contained 20 mg/ml of solid support to which testosterone antibody had been covalently bonded. The chamber was washed with 5 ml of a buffer rinse (Solution A) then by 1 ml of regenerating solvent (Solution B) and finally by another 2 ml of buffer rinse (Solution A.) The cycle was repeated several times. Each fraction discharged from the contact chamber was separately collected in scintillation vials mixed with 15 ml of scintillation cocktail (Solution C) and counted for 5 minutes in a scintillation spectrometer. The results are shown in Table 1 in which for each of four cycles the percent of bound antigen and percent of free antigen are tabulated. In the case of the bound antigen, it was first released from the immobilized antibody by the regenerating solvent then it was detected and counted.
TABLE I ______________________________________ (Sample - 3 H-Testosterone) ______________________________________ Cycle Cycle Cycle Cycle 1 2 3 4 ______________________________________ Percent free* labelled Antigen 61.3 33.4 33.1 33.3 Percent Bound** Labelled Antigen 40.3 65.3 70.5 71.4 ______________________________________ The first two cycles were used to stabilize conditions. *Measured after passage through the immobilized antibody. **Measured after release from the immobilized antibody by the regeneratin solvent (Solution B).
EXAMPLE II
A separate series of tests were conducted on estriol antigen under conditions comparable to those reported in Example I. The data reported in Table II establishes that the antibody may be regenerated many times without loss of efficiency. In the tests, a standard sample always contained 0.28 ng/ml of labelled antigen (62.nC/ml of tritium) plus a known amount of unlabelled estriol. The concentration in the first column recites only the known unlabelled estriol concentration.
TABLE II ______________________________________ Conc. Unlabelled Number Average Estriol of Percent Standard ng/ml Cycles Bound** Deviation ______________________________________ 0 13 84.8 0.57 0* 22 84.6 1.02 .27 5 77.1 1.1 .54 14 70.6 2.2 1.08 10 57.3 1.1 2.7 13 36.1 1.9 5.4 8 24.3 .47 ______________________________________ *These two series were separated in time. **The bound fraction was measured after release by the regenerating solvent.
All runs were made on a single antibody mass. Twenty one actual clinical samples were run through the same mass, interspersed among the runs reported in Table II. Additionally, several hundred more standards and other samples were run through the same antibody mass so that it has actually been regenerated about 500 times without detrimental loss in antigen binding efficiency. The tests reported in connection with Table II were all run on the operating prototype hereinafter described in Example III.
A variety of regenerating solvents have been tested and found satisfactory for release of bound antigen and regeneration of antibody. Some solvents, particularly ethyl and isopropyl alcohols, have demonstrated almost indefinite regeneration capacity. In fact, as noted with Solution B, an antibody mass has been regenerated about 500 times. The required characteristics of the solvent are that it breaks the antibody-antigen bond without destroying the flow characteristics or antigen binding efficiency of the antibody. In practice it will be necessary empirically to determine the proper solvent for any given system.
EXAMPLE III
A prototype system was constructed which employed a turntable sampler holding a plurality of samples and having a lifter for moving the sample tube 21 into and out of samples as they are sequentially rotated into position by a timer. A cyclic timer 41 was employed. A commercially available rotary sample valve was used to perform the functions of valves 22, 24 and 33 in response to signals from the timer. The valves 27 and 31 were simple twoposition electrically operated valves. Suitable metering pumps 28, 29 and 32 were used. Flow lines were small bore Teflon plastic tubes typically of 0.0012 to 0.0062 inch I.D. The contact chamber consisted of a polypropylene tube 0.125 inch inside diameter by 0.188 inch long. As used, the colums contained 0.04 ml of a Sephadex G-25 suspension at a concentration of 20 mg Sephadex/ml of a solution of 0.02M sodium phosphate at pH 7.5, 0.05 M NaCl, 0.01% merthiolate and 0.02% sodium oxide. Antibody to the antigen was covalently bonded to the Sephadex. The sample loop 23 had a capacity of 0.2 ml. Flow rates were:
Through the sample loop --0.45 ml/minute for 2 minutes
From tank 11 or 12 via pump 32 --0.112 ml/minute
From tank 13 via pump 39 --1.0 ml/minute.
The coil forming the detector cell has a volume capacity of 2.2 ml. The mixer 17 has a capacity of approximately 1 ml. Detection and counting was accomplished by means of a beta scintillation detector adjacent the coil and coupled to a counter not shown but which is a conveniently available type.
The immunoadsorbent in the contact chamber was an antibody coupled to Sephadex beads which were in turn immobilized in the contact chamber by nylon screen (325-400 mesh). Sephadex is a cross-linked dextran made by Pharmacia A.B. of Uppsala, Sweden. Such beads or other particulate solids with the ability to hold antibodies by co-valent bonds make convenient supports on which to bind the antibodies for immobilization.
As previously mentioned the tests reported in connection with Table II were all run in apparatus of the foregoing type.
In operation, a typical total cycle time for some tests was 28 minutes. This total included a three minute rinse cycle during which rinse from tank 11 flushed the system free from regeneration solvent. The sample loop, which does not require preliminary rinse, is at the same time flushed and filled with sample. The first three minute period is followed by a ten minute period during which reagent from tank 11 flows through the sample loop displacing sample therefrom into and through the contact chamber, the mixer and the detector. All of this is followed by a 15 minute regeneration period during which solvent from tank 12, by-passing the sample loop as previously described, flows through the contact chamber to release bound antigens thereby regenerating the immunoadsorbent. The released antigens are subjected to detection as they move through the flow cell to eventual discard.
In other tests, the cycle time has been reduced to less than fifteen minutes. In general, the cycle time can be reduced by increasing the concentration of radioactive tracer.
As noted, a suitable reagent for regeneration of the immunoadsorbent is one which will break the bond between the antigen and antibody but does not adversely affect the antibody. That is, it does not loosen it from its support nor reduce either its permeability or affinity for antigen. So far identification of suitable reagents has been empirical on the basis of behavior. However, once a suitable reagent for a given antibody-antigen system has been identified, it becomes a permanent reagent for that system.
Thus, the unexpected discovery that regeneration is possible and the identification of suitable reagents makes possible rapid analysis at greatly reduced costs. Moreover, the system is capable of automation thereby reducing human error with a concomitant increase in accuracy.
Throughout the specification reference has been made to bound antigen. This refers to the antigen bound to the antibody mass. The bound antigen is measured only after it has been released from the antibody by the regenerating solvent. If the percent recovery of bound (released) antigen is added to the percent of free antigen measured in any cycle, the total is consistently at 100% for practical purposes. This is significant because it confirms that substantially all bound antigen is released from the antibody and that the antibody is completely regenerated.
Although the invention has been described with particular reference to steriods, it is not intended to be so limited as it has been used with systems for the determination of polypeptides, thyroid hormones and some viruses. It is clear then, that the invention has broad application to radioimmunoassay procedures.
1. Field of the Invention
This invention relates generally to the analytical technique known as radioimmunoassay in which antigens are bound to specific antibodies and, through the use of tracers (labels) and predetermined behavior standards the concentration of antigen in a sample is determined. More particularly, the invention relates to improved methods and apparatus for radioimmunoassay in which a short-cycle time, hence more rapid analysis is achieved, the antibody mass (immunoadsorbent) is regenerated to be reused indefinitely rather than being wastes and the entire operation is automated.
2. State of the Art
Radioimmunoassay is an analytical technique which depends upon the competition (affinity) of antigen for antigen-binding sites on antibody molecules. In practice, standard curves are constructed from work on a plurality of samples each containing (a) the same known concentration of labelled antigen, and (b) various, but known, concentrations of unlabelled antigen. Antigens are labelled with a radioactive isotope tracer. The mixture is incubated in contact with an antibody, the free antigen is separated from the antibody and antigen bound thereto, and then, by use of a suitable detector, such as a gamma or beta radiation detector, the percent of either the bound or free labelled antigens is determined. This procedure is repeated for a number of samples containing various known concentrations of unlabelled antigens and the results plotted. The percent of bound tracer antigens is plotted as a function of the antigen concentration. Typically, as the total antigen concentration increases the relative amount of the tracer antigen bound to the antibody decreases. After the standard graph is prepared, it is thereafter used to determine the concentration of antigen in samples undergoing analysis.
In actual analysis, the sample in which the concentration of antigen is to be determined is mixed with a known amount of tracer antigen. Tracer antigen is the same antigen known to be in the sample but which has been labelled with a suitable radioactive isotope. The sample with tracer is then incubated in contact with the antibody. Thereafter, it may be counted in a suitable detector which counts the free antigen remaining in the sample. The antigen bound to the antibody or immunoadsorbent may also be similarly counted. Then, from the standard curve, the concentration of antigen in the original sample is determined. Afterwards, the antibody or immunoadsorbent mass is discarded.
In order to detect the percentage of antigen that is bound to the antibody (bound antigen) and/or the percentage that remains free or unbound it is necessary to first separate the sample into a fraction containing bound antigen and one containing only free antigen. One common method for doing this is to add a dextran coated charcoal to the mixture. The charcoal is allowed to adsorb the free antigen. The charcoal with adsorbed free antigen is then separated from the antibody (and bound antigen) by centrifugation. Another known procedure is to add to the mixture another antibody which selectively precipitates the first antibody (with the bound antigen) thus leaving in solution only free antigen. Classification into appropriate free and bound fractions is then effected by separating the precipitate from the supernatant by centrifugation or other suitable means. Some workers have resorted to the technique of binding the antibody to the inner walls of a plastic vessel, filling the vessel with the antigen bearing sample, allowing it to stand for an incubatioin period that typically ranges from 4 to 72 hours and then separating free antigen from bound antigen by draining and rinsing the vessel leaving therein only the antibody and bound antigen. A more recently developed technique is to prepare the immunoadsorbent by binding the antibodies onto an insoluble cross-linked dextran. The immunoadsorbent and antigen bearing sample are incubated then the dextran with bound antigen is separated from the solution by suitable means.
In all of the foregoing procedures, the percentage of labelled antigen in either or both the bound or free fractions is determined and the standard curve used to determine the antigen concentration. Thereafter, the immunoadsorbent is discarded.
Although the foregoing radioimmunoassay techniques have proven to be valuable tools and have gained widespread acceptance, they are still not all that are to be desired because the antibody (immunoadsorbent) is consumed with each analysis hence must be discarded. Moreover, prior practice is batch type and the several reagents are added to the antibody in test tubes in which the separate steps, such as incubation, rinsing and the like, are performed, thus resulting in a slow and costly operation.
SUMMARY OF THE INVENTION
The present invention provides improved method and apparatus for carrying out radioimmunoassay. In accordance with the invention the immunoadsorbent (antibody) is repeatedly and rapidly regenerated thus obviating the need and therefore the time and cost of constant replacement. According to the invention, steps are incorporated by which the method may be carried on continuously in constantly repeating cycles thereby eliminating the expensive time-consuming batch operation. A novel equipment arrangement for automating the method is also provided.
The invention is predicated on the discoveries that (1) by forming the immunoadsorbent mass as an immobilized mass of antibodies through which the antigen sample and other reagents may flow the procedure may be sequentially carried out without resort to the use of several manual steps and (2) the immobilized immunoadsorbent may be regenerated for repeated reuse by the rinsing with a solvent or eluting solution having particular characteristics. That is to say, bound antigens are released so that the antibody may be washed clean of bound antigen and thereby regenerated for reuse without affecting the essential characteristic of the antibody mass such as its ability to permit repeated flow of antigen solution therethrough and its antigen-binding efficiency during a large number of cycles.
As used herein, the term immobilized antibody mass refers to a mass of antibodies held in place in a liquid stream flow path in such a manner that the stream may flow over or through the mass while the latter remains in place.
A suitable antibody mass may be formed from solid surfaces such as glass or water insoluble polymers to which the antibodies are attached by co-valent bonds. A mass of such beads with attached antibodies is supported on a screen in a tube or other hollow column through which the antigen bearing sample and other reagents are flowed sequentially. Co-valent coupling of antibodies or antigens to said carrier is known. (It is disclosed for instance in U.S. Pat. No. 3,652,761).
In accordance with this invention, antibodies, specific to the antigen under analysis, are co-valently coupled to a solid support such as beads, a mass of the beads is immobilized, an antigen bearing solution is flowed therepast, the percentage of free labelled antigen remaining in the solution is detected, the bound antigen is released simultaneously with regeneration of the antibody mass and the percentage of released antigen is also detected. The percentages are determined with reference to the total antigen in the incoming sample. As noted, detection is of the tracer or labelled antigen.
Release of bound antigen and concommitant regeneration or reactivation of the antibody mass is effected by rinsing it with a solvent or eluting solution that breaks the bond between antigen and antibody, but does not break the co-valent bond between the antibody and its support. Moreover, the solvent must not alter either the flow characteristic or antigen affinity of the antibody mass.
Another type of immobilized antibody mass can be formed by encapsulating antibodies in a semi-permeable membrane. A quantity of such micro-capsules is then held in place in a column through which the antigen-bearing solution flows. The membranes are selected to block the outward passage of antibodies while permitting the free passage of antigens and their solvent in both directions.
In the case of membrane capsules, since there is no covalent bond, there is no need to be concerned with its destruction by the regenerating solvent. However, the limitation must still be observed that the solvent not adversely affect the flow characteristic or adsorption efficiency of the antibody. It is possible to increase efficiency of the membranes by coupling the antibodies to a soluble polymer thereby increasing the molecular weight of the combined antibody thus enabling the use of a relatively more permeable membrane.
In connection with flow characteristics in either system, the solvent should not cause swelling of the antibody support or the membrane wall to the point where flow of relatively small antigens is blocked or the loss of relatively larger antibodies through the wall occurs.
In summary then, the method of the invention comprises the steps of providing a mass of immobilized specific antibodies to selected antigen, holding such mass in a liquid flow path, preparing an antigen bearing sample by adding a known quantity of labelled antigen to a sample containing an unknown amount of the same antigen, flowing such sample along said liquid flow path over and in contact with said immobilized antibody, thereafter detecting the quantity of free labelled antigen remaining in the sample and/or that which is bound to the antibody.
The quantity of antigen bound to the antibody is determined by rinsing the antibody with a particular solvent to release bound antigens then detecting the antigens in the rinse solution.
The solvents found to meet the requirements of this invention have been hydrophobic in character. Typical solvents are methyl, ethyl and isopropyl alcohols as well as dimethylformamide.
In its most essential form, the apparatus invention comprises a contact chamber having an inlet and outlet and adapted to accommodate the flow of a liquid stream therethrough, a mass of antibodies, means for immobilizing said antibodies and holding them in position in said contact chamber in the path of antigen flow therethrough, a detection chamber having an inlet and outlet, a detector associated with said chamber for determining the quantity of a given tracer flowing through said chamber, conduit means for conductng liquid from the outlet of said contact chamber to the inlet of said detection chamber, a source of sample solution containing both labelled and unlabelled antigen, a source of regenerating solvent, means for supplying separately and sequentially to the inlet of said contact chamber for flow therethrough a quantity of said sample and of said solvent, and means for controlling the flow of said sample and solvent through said contact chamber and said detection chamber. Means are also provided for introducing a so-called scintillation cocktail into the system between the contact chamber and detector to flow through the latter and therein to convert radioactive decay impulses to light or photons for detection by a photo multiplier detector.
The required flow rates through the contact chamber and detector are determined emperically and are controlled by metering pumps. The flow sequence is controlled by valves. By proper timing the flow through the system may be continuous with the rates being selected to provide sufficient time at each station to achieve the results, such as binding, releasing or counting, sought at each station.
In order that the invention may be more readily understood and carried into effect, reference is made to the accompanying drawing and the description thereof which are offered by way of illustration and not in limitation of the invention, the scope of which is defined by the appended claims and equivalents thereof rather than by any illustrative description.
BRIEF DESCRIPTION OF THE DRAWINGS AND DESCRIPTION OF PREFERRED EMBODIMENT
The FIGURE is a schematic diagram of apparatus embodying the invention.
As illustrated, the system comprises a sample source 10, a source 11 of buffer rinse, a source 12 of regenerating solvent, a source 13 of scintillation cocktail, a rinse water reservoir 14, a contact chamber 16 filled with immobilized antibodies (immunoadsorbent) a mixing chamber 17, and a detector chamber or coil 18 with detector 19 adjacent thereto. The foregoing components are connected together by a series of conduits and flow directing valves which, together with metering pumps and a timing mechanism, regulate flow through the system.
Specifically, the sample source 10 connects via a conduit 21 to a two-position valve 22 which in one position connects to a sample loop 23. The sample loop terminates in a two-position valve 24 which in one position connects to a conduit 26 leading to another two-position valve 27 which in one position connects the conduit 26 and sample loop 23 to an aspirator pump 28 which discharges to waste. A rinse water inlet conduit 29 also connects to the pump 28 via the alternate position of valve 27 to keep the pump primed and rinsed when it is not drawing sample.
The displacement rinse flows from tank 11 to a two-position valve 31 which in one position connects through a pump 32 to a further two-position valve 33 whence it connects either to a conduit 34 leading to the valve 22 and sample loop 23 or to a conduit 36 leading to the contact chamber, mixer and detector. A conduit 37 connects the valve 24 to the main conduit 36. A branch conduit 38 connects to the main conduit 36 at a junction between the contact chamber and the detector at a location ahead of the mix chamber 17. A pump 39 is provided to move a reagent from the tank 13 through the conduit 38.
A central timer, generally designated 41, is employed to control sequencing of the valves and pumps. The timer may be custom assembled for the job in accordance with available technology hence, its structure need not be described in detail. It is sufficient to indicate, as is done in the drawing, that the valves, all of which are electrically actuatable solenoid valves, and the metering pumps, all of which are electric, are connected to the timer by suitable conductors 42.
OPERATION OF THE SYSTEM
At the start of a cycle rinse reagent from the tank 11 passes through valve 31, pump 32, valve 33 into and through conduit 36. This rinses the system. Sample from tank 10 is drawn by pump 28 through the valves into and through the sample loop 23 until the loop is rinsed and filled whereupon the valves 22, 24, 27 and 33 switch to the alternate positions whereupon there is a continuous flow from tank 11 through valve 31, pump 32, valve 33, valve 22, loop 23, valve 24 and conduit 37 to conduit 36. Liquid flowing from tank 11 thus displaces sample from the loop into and through the contact chamber 16. When the flow has continued long enough to displace all sample from the loop, the valves 22, 24 and 33 return to the initial positions shown in the drawing. This isolates the sample loop.
After the sample has passed through the contact chamber 16 and the detection coil, the valve 31 shifts to accept regenerating solvent from the tank 12 and directs it through pump 32 and valve 33 directly into conduit 36 to flow through the contact chamber where it releases the bound antigens and carries them to the mixer and detector. Simultaneously, the immunoadsorbent in the contact chamber is regenerated.
The scintillation cocktail in tank 13 typically comprises a primary scintillator, a secondary scintillator and a solubilizer all carried in toluene. Its function is to mix with the antigen bearing solution in the mix chamber 17 and then to convert the radioactive impulses of the tracer to light or photons which can be detected by a photo multiplier detector. Photons may be detected by either a gamma or beta scintillation spectrometer.
The tracer or label is selected from those conveniently available. A label of 3 H Tritium may be used with a beta scintillator detector with a coincidence circuitry counter whereas an Iodine label 125 I may be used with a gamma detector which does not require the coincidence circuitry. In the tests reported herein, a tritium label was used.
It is important that the sample loop be kept uncontaminated with the regenerating solvent since traces of that mixed with the sampler could reduce the bonding efficiency in the contact chamber.
Although a very elemental sample is illustrated, it will be appreciated that several sophisticataed samplers are readily available that may be adapted to use in this invention.
A series of basic tests were performed to confirm that immunoadsorbents when formed as immobilized antibodies could be consistently regenerated with suitable reagents.
Reagents used were as follows:
Buffer Rinse (Solution A)
0.02 m sodium phosphate, pH 7.5
0.05 m sodium chloride
0.01% merthiolate
0.02% sodium azide
Regenerating solvent (Solution B)
Solution A with the addition of
30% (v/v) of 95% ethyl alcohol.
Scintillating Solution (Solution C)
Five parts of a solution of
0.4% (w/v) 2.5 diphenyloxazole
0.008% (w/v) 1, 4-bis-2(4-methyl-5-phenyloxazolyl) benzene
toluene as solvent
one part of Scintisol-GP (manufactured by Isolab, Inc.)
EXAMPLE I
A contact chamber was prepared from a glass pipette plugged at one end with glass wool. A slurry of 0.04 ml of immunoadsorbent suspension was introduced. The suspension contained 20 mg/ml of solid support to which testosterone antibody had been covalently bonded. The chamber was washed with 5 ml of a buffer rinse (Solution A) then by 1 ml of regenerating solvent (Solution B) and finally by another 2 ml of buffer rinse (Solution A.) The cycle was repeated several times. Each fraction discharged from the contact chamber was separately collected in scintillation vials mixed with 15 ml of scintillation cocktail (Solution C) and counted for 5 minutes in a scintillation spectrometer. The results are shown in Table 1 in which for each of four cycles the percent of bound antigen and percent of free antigen are tabulated. In the case of the bound antigen, it was first released from the immobilized antibody by the regenerating solvent then it was detected and counted.
TABLE I ______________________________________ (Sample - 3 H-Testosterone) ______________________________________ Cycle Cycle Cycle Cycle 1 2 3 4 ______________________________________ Percent free* labelled Antigen 61.3 33.4 33.1 33.3 Percent Bound** Labelled Antigen 40.3 65.3 70.5 71.4 ______________________________________ The first two cycles were used to stabilize conditions. *Measured after passage through the immobilized antibody. **Measured after release from the immobilized antibody by the regeneratin solvent (Solution B).
EXAMPLE II
A separate series of tests were conducted on estriol antigen under conditions comparable to those reported in Example I. The data reported in Table II establishes that the antibody may be regenerated many times without loss of efficiency. In the tests, a standard sample always contained 0.28 ng/ml of labelled antigen (62.nC/ml of tritium) plus a known amount of unlabelled estriol. The concentration in the first column recites only the known unlabelled estriol concentration.
TABLE II ______________________________________ Conc. Unlabelled Number Average Estriol of Percent Standard ng/ml Cycles Bound** Deviation ______________________________________ 0 13 84.8 0.57 0* 22 84.6 1.02 .27 5 77.1 1.1 .54 14 70.6 2.2 1.08 10 57.3 1.1 2.7 13 36.1 1.9 5.4 8 24.3 .47 ______________________________________ *These two series were separated in time. **The bound fraction was measured after release by the regenerating solvent.
All runs were made on a single antibody mass. Twenty one actual clinical samples were run through the same mass, interspersed among the runs reported in Table II. Additionally, several hundred more standards and other samples were run through the same antibody mass so that it has actually been regenerated about 500 times without detrimental loss in antigen binding efficiency. The tests reported in connection with Table II were all run on the operating prototype hereinafter described in Example III.
A variety of regenerating solvents have been tested and found satisfactory for release of bound antigen and regeneration of antibody. Some solvents, particularly ethyl and isopropyl alcohols, have demonstrated almost indefinite regeneration capacity. In fact, as noted with Solution B, an antibody mass has been regenerated about 500 times. The required characteristics of the solvent are that it breaks the antibody-antigen bond without destroying the flow characteristics or antigen binding efficiency of the antibody. In practice it will be necessary empirically to determine the proper solvent for any given system.
EXAMPLE III
A prototype system was constructed which employed a turntable sampler holding a plurality of samples and having a lifter for moving the sample tube 21 into and out of samples as they are sequentially rotated into position by a timer. A cyclic timer 41 was employed. A commercially available rotary sample valve was used to perform the functions of valves 22, 24 and 33 in response to signals from the timer. The valves 27 and 31 were simple twoposition electrically operated valves. Suitable metering pumps 28, 29 and 32 were used. Flow lines were small bore Teflon plastic tubes typically of 0.0012 to 0.0062 inch I.D. The contact chamber consisted of a polypropylene tube 0.125 inch inside diameter by 0.188 inch long. As used, the colums contained 0.04 ml of a Sephadex G-25 suspension at a concentration of 20 mg Sephadex/ml of a solution of 0.02M sodium phosphate at pH 7.5, 0.05 M NaCl, 0.01% merthiolate and 0.02% sodium oxide. Antibody to the antigen was covalently bonded to the Sephadex. The sample loop 23 had a capacity of 0.2 ml. Flow rates were:
Through the sample loop --0.45 ml/minute for 2 minutes
From tank 11 or 12 via pump 32 --0.112 ml/minute
From tank 13 via pump 39 --1.0 ml/minute.
The coil forming the detector cell has a volume capacity of 2.2 ml. The mixer 17 has a capacity of approximately 1 ml. Detection and counting was accomplished by means of a beta scintillation detector adjacent the coil and coupled to a counter not shown but which is a conveniently available type.
The immunoadsorbent in the contact chamber was an antibody coupled to Sephadex beads which were in turn immobilized in the contact chamber by nylon screen (325-400 mesh). Sephadex is a cross-linked dextran made by Pharmacia A.B. of Uppsala, Sweden. Such beads or other particulate solids with the ability to hold antibodies by co-valent bonds make convenient supports on which to bind the antibodies for immobilization.
As previously mentioned the tests reported in connection with Table II were all run in apparatus of the foregoing type.
In operation, a typical total cycle time for some tests was 28 minutes. This total included a three minute rinse cycle during which rinse from tank 11 flushed the system free from regeneration solvent. The sample loop, which does not require preliminary rinse, is at the same time flushed and filled with sample. The first three minute period is followed by a ten minute period during which reagent from tank 11 flows through the sample loop displacing sample therefrom into and through the contact chamber, the mixer and the detector. All of this is followed by a 15 minute regeneration period during which solvent from tank 12, by-passing the sample loop as previously described, flows through the contact chamber to release bound antigens thereby regenerating the immunoadsorbent. The released antigens are subjected to detection as they move through the flow cell to eventual discard.
In other tests, the cycle time has been reduced to less than fifteen minutes. In general, the cycle time can be reduced by increasing the concentration of radioactive tracer.
As noted, a suitable reagent for regeneration of the immunoadsorbent is one which will break the bond between the antigen and antibody but does not adversely affect the antibody. That is, it does not loosen it from its support nor reduce either its permeability or affinity for antigen. So far identification of suitable reagents has been empirical on the basis of behavior. However, once a suitable reagent for a given antibody-antigen system has been identified, it becomes a permanent reagent for that system.
Thus, the unexpected discovery that regeneration is possible and the identification of suitable reagents makes possible rapid analysis at greatly reduced costs. Moreover, the system is capable of automation thereby reducing human error with a concomitant increase in accuracy.
Throughout the specification reference has been made to bound antigen. This refers to the antigen bound to the antibody mass. The bound antigen is measured only after it has been released from the antibody by the regenerating solvent. If the percent recovery of bound (released) antigen is added to the percent of free antigen measured in any cycle, the total is consistently at 100% for practical purposes. This is significant because it confirms that substantially all bound antigen is released from the antibody and that the antibody is completely regenerated.
Although the invention has been described with particular reference to steriods, it is not intended to be so limited as it has been used with systems for the determination of polypeptides, thyroid hormones and some viruses. It is clear then, that the invention has broad application to radioimmunoassay procedures.