Immunoassay Procedures

My invention relates to improvements in immunoassay and, more particularly, to the separation of a radioactive or otherwise tagged bound material from an unbound material.

Radioimmunoassay (RIA) and similar procedures are a relatively recent development whereby difficult to measure constituents are analyzed through the use of radioisotopes, etc. The general procedure usually requires a specific antibody, a radiolabeled or otherwise tagged antigen, a pure sample of the antigen to serve as a reference standard and a means of separating the free antigen from the antibody bound antigen. Competitive binding occurs between the labeled ("tagged") and the unlabeled antigen for a fixed number of antibody binding sites. When equilibrium is reached between the antigen antibody reaction, the free and bound components of the mixture are separated and the relative amounts of each are determined by measuring the radioactivity or other tagging (e.g., fluorescence) of the separated components. This can be done by comparing the results of the unlabeled antigen in the sample to a standard curve prepared from known amounts of the unlabeled antigen.

This separation step is the key to an accurate and timely assay.

Presently, the majority of these separations are performed manually and, of course, these manual procedures are time consuming and prone to error because of the meticulous measurements that are required. Automation of these techniques has taken place but the separation step has continued to lead to pitfalls in automated laboratory RIA equipment.

Charcoal has generally been the sorbent used to make the separation. The various forms of charcoal presently in use tend to plug the automatic equipment, thereby rendering this equipment ineffectual for continuous assay analysis. Therefore, analysts continue to rely on manual procedures rather than risk the continuous flow procedures. The following patents are exemplary of RIA procedures using charcoal and other sorbents in their presently utilized forms. Mead U.S. Pat. No. 3,615,222; Mead et al. U.S. Pat. Nos. 3,721,528 and 3,768,979; Eisentraut U.S. Pat. No. 3,775,615; Eisentraut U.S. Pat. No. 3,666,854; Wolf U.S. Pat. No. 3,843,775; Johnson U.S. Pat. No. 3,896,217.

I have now found that the inadequacies of automated procedures can be overcome without sacrificing any accuracy in result or delay in analysis. The form of the sorbent which I use permits continuous filtering so that the filtrate can be counted to give an immediate readout of the results. The automation of the technique reduces the turnaround time of these tests and minimizes errors inherent in the procedure.

My improvement provides for the separation of a radioactive or other tag material and untagged bound material from that which has not been bound by utilizing a clathrate sorbent. The clathrate sorbent presently preferred is charcoal. The preferred sorbent is composed of a mixture of hydrate zinc sulfate, sodium hydroxide and charcoal powder. This mixture is present in relative amounts of 3 grams of ZnSO.sub.4.sup.. 7H.sub.2 O, 15 ml. of 1 N NaOH, 365 ml of H.sub.2 O and 2.48 grams of charcoal powder. The charcoal powder can also be combined to a clathrate sorbent with other salts of metals which produce a gelatinous substance with alkaline hydroxides. Other sorbents such as magnesium trisilicate can also be used.

A clathrate compound is generally considered as an inclusion complex in which molecules of one substance are completely caged within the other. Specifically, the molecules of one component are held within the crystal lattice framework of the other component. The larger molecule remains dominant and the inclusion molecule is positioned in cavities of the larger molecule defined by the larger molecule's crystal lattice. This spotty attachment allows for exposure of substantial surface area of the dominant molecules.

I have found that by using charcoal and other sorbents to form a clathrate compound, I have arrived at a substance which retains the absorptive qualities of the sorbent while, at the same time, making it free flowing so that it can be used in continuous flow separations. I have successfully prepared a clathrate charcoal composition by mixing the following chemicals and substances:

Preparation of clathrate compound formed of charcoal with zinc hydroxide.

3 grams ZnSO.sub.4.sup.. 7H.sub.2 O

15 ml 1 N NaOH

365 ml H.sub.2 O

2.48 grams charcoal powder of the type presently used in RIA procedures.

The mixture is made as follows: The hydrate zinc sulfate is dissolved in the water. Then the sodium hydroxide is added and the preparation is thoroughly mixed and the charcoal powder is added. The pH of the preparation is then measured with a glass electrode. If it is below 6.8, more sodium hydroxide is added dropwise until pH 6.8 is attained. The charcoal powder is thereafter added. The entire procedure is carried out at room temperature. The preparation is stored in the refrigerator.

I have also successfully formed other clathrate sorbents.

Clathrate magnesium trisilicate.

The same weight (2.48 grams) of magnesium trisilicate (U.S.P. grade) is added to the chemicals of Example I instead of the RIA grade charcoal.

Preparation of a clathrate compound formed of charcoal with aluminum hydroxide. Ammonium alum, N. F., having twice the number of moles than the hydrate zinc sulfate of Example I was dissolved and heated with 40/15 times the amount of sodium hydroxide to drive off the ammonia. The mixture was cooled, diluted to the usual volume, and the charcoal was added in the normal manner. The pH was adjusted, if necessary, to 6.8.

I have successfully employed the clathrate charcoal in an automated RIA assay equipment. This has permitted the use of continuous flow filtering which speeds up the assay time, see Table 1. As a secondary benefit, I have noted that the clathrate charcoal has substantially improved precipitation properties and thus can speed up centrifugation where such procedures are necessary for assay.

Table 1 illustrates the amount of radioactivity bound to a fixed amount of antibody. Duplicate results show reproducibility. The decrease in radioactivity is correlated with increase in the amount of untagged antigen.

Table 2 demonstrates the results obtained during a five minute centrifugation with clathrate charcoal in a titer demonstration.

I have thus been able to automate completely the assay of serum digoxin through a continuous flow procedure. Specifically, the samples plus the radioactive reagent are picked up and mixed with an antiserum and incubated in the delay coil for one half hour. The clathrate charcoal made in accordance with my invention is then added, mixed and filtered continuously. The clear filtrate is counted by a two inch crystal, pierced horizontally in a rate meter with associated recorder, power supply amplifier and spectrometer. All of this latter equipment is known and presently used and does not form a part of my invention.

In the digoxin test, I obtained confidence limits of .+-.3.9%. I have also had conducted thyroxine assays using my method and have obtained confidence limits of .+-.4.9. These results compare favorably with the standard manual techniques presently in use.

It is presently believed that the hydrate zinc sulfate, as well as other metal salts, and the sodium hydroxide or other alkaline hydroxide, react to form a zinc or other hydroxide which is held in lattice sites of the material. The gelatinous hydroxide acts somewhat like a lubricant, thereby preventing the charcoal from clogging the equipment necessary for the automatic RIA or other immunoassay procedures. At the same time, effectiveness of the charcoal or other sorbent used in separating the bound from the free material is not affected because of the large surface area of the charcoal or other sorbent which remains in contact with the mixture to be separated.