Assay for hepatitis A antigen

Liquid containing hepatitis A antibody is adsorbed on a surface. The surface is then coated with a proteinaceous material and incubated in the presence of the sample to form a complex of hepatitis A antigen and hepatitis A antibody. The complex is incubated again in the presence of excess radioactively labelled hepatitis A antibody, and the resulting radionuclide is measured.

BACKGROUND OF THE INVENTION 
Presently known assays for detection of hepatitis A antigen in biological 
liquids consist of the immune adherence hemagglutination assay described 
by Miller et al., Proc. Soc. Exp. Biol. Med., 149, 254-261 (1975) and the 
immune electron microscopic assay described by Feinstone et al., Science, 
182, 1026-1028 (1973). Both of these methods are insufficiently sensitive 
to detect hepatitis A antigen in serum or plasma, and require a subjective 
evaluation to determine the end point. 
OBJECTS OF THE INVENTION 
It is, accordingly, an object of the present invention to provide an assay 
capable of detecting hepatitis A antigen in serum or plasma as well as 
other biological liquids. Another object is to provide an assay in which 
the end point is determined objectively. These and other objects of the 
present invention will be apparent from the following description. 
SUMMARY OF THE INVENTION 
Liquid containing hepatitis A antibody is adsorbed on a surface. The 
surface is then coated with a proteinaceous material and incubated in the 
presence of the sample to form a complex of hepatitis A antigen and 
hepatitis A antibody. The complex is incubated again in the presence of 
excess radioactively labelled hepatitis A antibody, and the resulting 
radionuclide is measured. 
DETAILED DESCRIPTION 
The present invention relates to an assay for hepatitis A antigen and, more 
particularly, to a highly sensitive radioimmune assay for hepatitis A 
antigen. 
According to the present invention, a solution of hepatitis A antibody of 
known titer is adsorbed on a surface capable of adsorbing the antibody. 
The surface may be, for example, finely divided glass beads, or a plastic 
such as polyethylene, polypropylene, polystyrene or polyvinylchloride. 
The adsorption conditions may be varied as to time, temperature, and 
concentration of the antibody. Typical adsorption conditions employ 
contacting the surface for at least about 2 hours, preferably for from 
about 8 to about 36 hours, at from above about 0.degree. to about 
60.degree. with a solution of hepatitis A antibody of known titer. After 
the adsorption of the antibody the surface is coated with a material 
effective to be adsorbed by sites on the surface which are unoccupied by 
the antibody. Such a material is preferably a proteinaceous material, such 
as serum albumin, preferably bovine serum ablumin or human serum albumin. 
The coating may also be any non-proteinaceous chemical which is able to 
associate with the bead surface. This coating may be carried out under the 
same range of conditions employed to adsorb the antibody. 
The coated surface is then incubated in the presence of the sample to be 
tested for hepatitis A antigen under conditions effective to form a 
complex of hepatitis A antigen and hepatitis A antibody. Typically this 
incubation takes from about 4 hours to about 48 hours or more at 
temperatures of from above about 0.degree. to about 45.degree.. 
After the complex is formed it is incubated with radioactively labelled 
purified hepatitis A antibody under conditions effective to attach the 
radioactively labelled purified hepatitis A antibody to the hepatitis A 
antigen portion of the hepatitis A antigen-hepatitis A antibody complex. 
This incubation typically requires from about 30 minutes to about 8 hours 
at a temperature of from about 25.degree. to about 60.degree.. The 
radionuclide residual is then measured in an appropriate counting device.

The following example illustrates the present invention without, however, 
limiting the same thereto. All temperatures are expressed in degrees 
Celsius. 
EXAMPLE 1 
A quantity (about 100) of polystyrene beads having a diameter of about 0.25 
inch (0.64 cm) are placed in a 50 ml beaker. Hepatitis A antibody positive 
serum diluted 1:100 in physiologic saline containing 0.1% sodium azide 
(bacteriacide) is added in a quantity sufficient to cover the beads. A 
sheet of plastic is stretched over the top of the beaker which is then 
incubated at 5.degree. for 24 hours. The supernatant liquid is decanted 
and the beads are placed in a 250 ml beaker to which 100 ml of saline 
azide (0.9% NaCl and 0.1% sodium azide in sterile distilled water) 
solution is added. The mixture is washed by slurrying for about 1 minute 
and the liquid then decanted. This slurry wash is repeated three more 
times. After the final wash the beads are transferred to a 50 ml beaker 
and 1% bovine serum albumin dissolved in saline azide solution is added in 
sufficient quantity to cover the beads. The beaker is allowed to stand at 
5.degree. for 24 hours. The liquid is decanted and the beads placed in a 
250 ml beaker and slurry washed with saline azide solution four times as 
previously described. These washings are followed by two washes in 
distilled water. The beads are then placed on a sheet of filter paper and 
air dried for four hours at room temperature. The beads are then placed in 
a stoppered flask and stored at 20.degree. until used. 
Using 1.0 ml pipet, 1.0 ml of sample to be tested for hepatitis A antigen 
is placed in a 5 cm tube having an inside diameter of 0.8 cm. A coated 
bead is added and the tube is tapped to submerge the bead in the sample. 
The tube is sealed and incubated at room temperature overnight with 
continuous inversion of the tube. After incubation, the bead is washed 
with 2-5 ml amounts of distilled water. To the washed, damp beads 1.0 ml 
of radioactively labelled (.sup.125 I) hepatitis A antibody is pipetted 
into a 5 cm.times.0.8 cm tube. The tube is tapped to submerge the bead. 
The tube is covered and incubated 4 hours at 37.degree. C. After 
incubation the bead is washed several times with 2-5 ml amounts of 
distilled water. The bead is then transferred into a counting tube and the 
radioactivity is counted in a Gamma counter.