Means for accelerating precipitation in immunoassay and immunoassay method

A serum factor extracted through dialysis of an animal serum or anti-serum has been found to accelerate precipitation in radioimmunoassays.

This invention relates to the radioimmunological field. More particularly, 
this invention concerns immunological analysis and, still more 
particularly, a means for accelerating precipitation reactions in 
immunoassay and the relevant immunoassay method. 
Radioimmunoassay (RIA) is an analytical technique permitting infinitesimal 
amounts of hormones or other substances which are present in biological 
fluids to be assayed. 
Even though this technique is very precise and sensitive, it suffers from 
several disadvantages, for instance in that it requires rather lengthy 
operational times. For example, in one of the basic operations in RIA, 
such as, the separation of the immunocomplex (antigen-I-Antibody) from the 
excess free antigen, a precipitation reaction of the immunocomplex is 
commonly carried out which requires periods of about 24 hours. This kind 
of reaction occurs by addition of a second antibody (anti-gammaglobulin 
serum) to the incubation medium where the immunocomplex is found, and 
renders it precipitable as it forms a new antigen-I antibody-II antibody 
insoluble complex. 
One main purpose of this invention is that of shortening the operational 
times for immunoassay by reducing the precipitation time of the 
immunocomplex. 
Another object of this invention is a means of accelerating the 
insolubilization reaction and, therefore, the precipitation of the 
immunocomplex in radioimmunoassay. 
Still another purpose of this invention is the relevant immunoassay method 
in which the new means for accelerating the precipitation is used. One 
further object of this invention is a process for preparing (that is, 
extracting) the above quoted accelerating means. Other objects and 
purposes of this invention will be understood from the following 
specification and examples. 
As is known, the precipitation reaction of the immunocomplex is influenced 
by several elements such as: 
(a) the quality of the anti-gammaglobulin serum; 
(b) the temperature of reaction 
(c) the volume of reaction 
(d) pH and protein concentration of the incubation medium 
(e) the presence of certain amount of salts in the reaction medium 
(f) the gamma-globulin concentration of the animal species in which the I 
antibody was raised. 
In the experiments which lead to this invention, the following operational 
conditions were used, which are listed below according to the same listing 
of the several factors mentioned above. 
(a) a specific antiserum for gamma-globulin of the animal species in which 
the first antibody was raised is employed having a preestablished antibody 
titer and pre-established affinity for gammaglobulin. 
(b) all reactions are performed at 20.degree.-22.degree. C. 
(c) all reactions are performed in a final reaction volume of 0.4 
milliliters. 
(d) all reactions are performed at pH 7.5 and at a protein concentration of 
3.3 mg/ml of bovine serum albumin. 
(e) the following salts are present in the incubation medium: 
0.01 molar phosphates 
the sodium salt of ethylene diaminotetracetic acid (12.5 mg/ml) 
(f) the gamma-globulin concentration is 16 ug per ml of the first 
anti-serum. 
Among all these factors whose standardization is necessary in order that 
the precipitation reaction is carried out, there is none permitting the 
reaction to be accelerated. 
This invention is based on the discovery that a "serum factor" exists 
which, when added to the incubation medium, has the property of 
accelerating the precipitation reaction. This serum factor (which has 
enzymatic activity) was found to be present in sera and anti-sera of 
numerous animal species such as man, rabbit, sheep, guinea-pig, and horse. 
The extraction of the above mentioned serum factor from serum is 
accomplished by means of a dialysis procedure. The dialysis procedure is 
carried out as follows: 
20 milliliters of human serum are placed in a dialyzing tube (Visking 
Tubing 24/32). The tube is accurately sealed and placed in a two liter 
beaker full of distilled water and left for 20 hours to dialyze, the 
distilled water being maintained constantly stirred. After 20 hours, the 
contents are centrifuged at approximately 2000 g. The supernatant is 
discarded and the solid matter (serum factor) is washed with distilled 
water, redissolved in 0.05 phosphate buffer and then lyophilized. 
The so obtained serum factor is a protein having the aspect of a white 
powder and having the following chemical-physical characteristics: 
(1) has a molecular weight higher than 50000 (Sephadex G-100) 
(2) is the Euglobulin type, completely insoluble in distilled water 
(3) has an electrophoretic mobility which is exactly the same as that of 
serum alpha or beta globulins. 
(4) cannot be assimilated to any component of the serum complement 
(5) is homogeneous on Sephadex G-100 
(6) has enzymatic activity 
(7) is thermolabile, that is, loses 90% of its activity when heated to 
60.degree. C. for 30 minutes. 
The accelerating effect determined by the serum factor on the precipitation 
reaction is a phenomenon depending upon the concentration of the serum 
factor in the reaction medium. In fact, the amount of serum factor 
expressed in mg or in equivalent ml of serum to be added to the incubate, 
depends on how much the precipitation reaction is to be accelerated. 
Sometimes, an accelerating effect can be obtained even using untreated 
serum. In this case, however, other side reactions may intervene in the 
assay making the use of untreated serum undesirable.

EXAMPLES 
The following examples were carried out using RIA for determining human 
follicle-stimulating hormone (hFSH). Alternatively, other RIA models could 
be used to give similar results. 
What follows is the general outline of examples 1(a), 1(b), 1(c) and 1(d). 
To get formation of the immunocomplex (first reaction), the operational 
scheme followed was that explained below: 0.2 ml of anti-hFSH raised in 
rabbit and diluted 1:200,000 were reacted with 0.1 ml, equivalent to 0.08 
nanograms, of I-125 labeled human FSH (specific radioactivity 150 
microcurie per microgram). Once the equilibrium was reached (after 24 
hours), the immunocomplex anti-hFSH-I-125-hFSH was made insoluble by 
adding to the reaction medium 0.1 ml of rabbit anti-gamma-globulin serum 
raised in sheep and diluted 1:300 (precipitating anti-serum). This 
operation was carried out in the following four different ways for the 
purpose of giving clear examples of this invention. 
Example 1(a): Conventional way. At the end of the first reaction, only 0.1 
ml of the precipitating anti-serum were added. Maximum precipitation was 
obtained after 24 hours reaction. 
Example 1(b): According to this invention. At the end of the first 
reaction, serum factor in an amount equivalent to 0.01 ml human serum was 
added in addition to the precipitating anti-serum. Maximum precipitation 
was reached after 15 hours reaction. 
Example 1(c): According to this invention. At the end of the first 
reaction, serum factor in an amount equivalent to 0.05 ml human serum was 
added in addition to the precipitating anti-serum. Maximum precipitation 
was obtained after 4 hours reaction. 
Example 1(d): According to this invention. At the end of the first 
reaction, serum factor in an amount equivalent to 0.10 ml human serum was 
added in addition to the precipitating anti-serum. 
Maximum precipitation was obtained after one hour reaction. 
Analogous results were obtained using a serum factor raised in other animal 
species, such as those listed above. 
In addition, it has been experimentally noticed that the precipitation 
reaction of the immunocomplex is accelerated even when the serum factor is 
added to the reaction medium containing the immunocomplex, before the said 
first reaction has reached equilibrium. It is well understood that the 
above described examples should not be considered limitative, but 
illustrative, of the invention, possible modifications being comprised in 
the wide ambit thereof.