Freeze-dried composition containing enzyme-labeled anti-human interferon-.beta. antibody and enzyme immunoassay kit containing the composition

The present invention relates to the stabilization of enzyme-labeled anti-human interferon-.beta. antibody for use in an enzyme immunoassay of human interferon-.beta. and provides a freeze-dried composition containing an anti-human interferon-.beta. antibody which has been freeze-dried in the presence of trehalose as a nonreducing disaccharide. The freeze-dried composition according to the present invention whose reduction in enzymatic activity remains minimized even when stored for a long period of time is conveniently incorporated into an EIA kit. No conventional non-volatile buffer solution such as a phosphate buffer solution is added to the original freeze-dried liquid of the present invention.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
The present invention relates to the stabilization of an enzyme-labeled 
anti-human interferon-.beta. antibody which may be used for an 
immunological microassay of human interferon-.beta., and more particularly 
to a freeze-dried composition which is capable of stabilizing an 
enzyme-labeled anti-human interferon-.beta. antibody over a long period of 
time. The present invention further relates to an enzyme immunoassay kit 
comprising such a freeze-dried composition. 
2. Prior Art 
When a trace amount of substance such as a protein, a peptide hormone etc. 
contained in a biological material such as serum, urine or the like will 
be measured, the method for measuring it requires a high sensitivity and 
high specificity. As a method for measuring used for this purpose, an 
immunoassay which utilizes an antigen-antibody reaction is well known. 
The immunoassay may be classified into a competitive method and a 
non-competitive method. In the former, a sample liquid to be measured 
containing the antigen or antibody, which is a substance to be measured, 
is mixed with a substance to be measured which was pre-labeled and had a 
known concentration, and then the antibody or antigen is added to the 
mixture to form an antigen-antibody complex. By measuring a ratio of the 
labeled substance to be measured and the substance to be measured, each of 
which was involved in the complex formation, a content of the substance to 
be measured can be calculated. In the latter method known as a sandwich 
method, on the other hand, a first antibody is first bound to a solid 
phase, and then a sample liquid to be measured containing an antigen, 
which is a substance to be measured, is brought into contact therewith to 
bind the antigen to the first antibody on the solid phase, the solid phase 
being thereafter separated from the liquid phase. By the antigen-antibody 
reaction, a labeled second antibody is then bound to the antigen which is 
a substance to be measured and has been bound to the first antibody on the 
solid phase, and thereby an amount of the antigen to be measured can be 
determined after measuring an amount of the bound labeled antibody. 
In accordance with the labeled substance used, the immunoassay may be 
divided into, for example, radio immunoassay utilizing a labeling agent of 
radioactive substance and enzyme immunoassay utilizing a labeling agent of 
enzyme. The enzyme immunoassay has recently come into wide use for such as 
ordinary clinical examinations because the assay has a sufficiently 
measurable sensitivity and a simple operability and is exempt from 
troublesome disposal after use. 
However, an enzyme-labeled antibody or antigen has a problem of the 
long-term stability in a freeze-dried state for practical use. Therefore, 
the development of an enzyme-labeled antibody or enzyme-labeled antigen in 
which the enzyme activity is not lowered during the long storage has been 
demanded. Generally, an activity which is determined immediately after the 
preparation of the enzyme-labeled anti-human interferon-.beta. antibody as 
the enzyme-labeled antibody is lost by 50% at room temperature about 2 
days after the preparation thereof, and substantially all the activity is 
lost 7 days thereafter. 
SUMMARY OF THE INVENTION 
It is an object of the present invention to provide a method for 
stabilizing an enzyme-labeled antibody for use in an enzyme immunoassay, 
hereinafter referred to as "EIA", in particular an enzyme-labeled 
anti-human interferon-.beta. antibody which may be used for a microassay 
of the human interferon-.beta. in a living body fluid by EIA. 
It is another object of the present invention to provide a freeze-dried 
composition of an enzyme-labeled anti-human interferon-.beta. antibody 
whose reduction in enzymatic activity remains minimized even after the 
storage for a long period of time. 
It is still another object of the invention to provide an enzyme 
immunoassay kit, in particular, a non-competitive EIA kit, which comprises 
the above-described freeze-dried composition having a preferred stability 
over a long period of time. 
It has been found that the above objects and other objects may be achieved 
by freeze-drying an enzyme-labeled anti-human interferon-.beta. antibody 
in the presence of trehalose which is a nonreducing disaccharide. 
The present invention, therefore, provides a freeze-dried composition 
substantially consisting of trehalose and an enzyme-labeled anti-human 
interferon-.beta. antibody. 
The freeze-dried composition according to the present invention exerts a 
good effect on the storage stability of an enzyme-labeled anti-human 
interferon-.beta. antibody over a long period of time and is capable of 
maintaining the activity by not less than 70% at room temperature even 
after 30 days and by 100% at 4.degree. C. even after 500 days. 
The present invention also provides an enzyme immunoassay kit comprising 
such a freeze-dried composition because the composition having excellent 
long-term storage stability is advantageously utilized for the 
immunological microassay of the human interferon-.beta..

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT 
The human interferon-.beta., which is a subject of measurement in the 
present invention, includes a natural human interferon-.beta. produced by 
human diploid cells and a recombinant human interferon-.beta. produced by 
a microorganism such as Escherichia coli and yeast or by animal cells such 
as hamster and monkey, in which the structural gene of the human 
interferon-.beta. has previously been recombined using a recombinant DNA 
technology. 
For the immunoassay measuring the trace amount of the human 
interferon-.beta. with high sensitivity and high accuracy, a monoclonal 
antibody which specifically recognizes only the specific antigenic 
determinant of an antigen molecule is preferably used as an antibody, 
i.e., an anti-human interferon-.beta. antibody to be labeled by the 
enzyme. The monoclonal antibody may be produced, for example, by 
monoclonal antibody-forming cells which are obtained in accordance with a 
known cell fusion method (e.g., "Monoclonal Antibodies-Hybridomas: A New 
Dimension in Biological Analysis", edited by Roger H. Kennet, Thomas J. 
Mckearn, Kathleen B. Bechtol et al., Plenum Press, New York and London, 
1980). That is, splenic cells from a mouse, rat etc. which have been 
immunized by the human interferon-.beta. are fused with myeloma cells 
having an infinite proliferation potential to obtain hybridomas of the 
mouse, rat etc. which have both a potential of antibody formation and a 
proliferation potential, and the target monoclonal antibody-forming cells 
are then obtained by cloning. The human interferon-.beta. used for 
immunization may be either of the natural type or of the recombinant type. 
If a monoclonal antibody is from an ascites fluid of the mouse or the like 
which is obtained by proliferating the hybridoma as an ascites type, the 
monoclonal antibody suitably diluted in protein concentration of 
approximately 5-500 .mu.g/ml is sufficiently usable. It is also naturally 
possible to use an immunoglobulin fraction purified from the ascites 
fluid. When the hybridoma is cell-cultured in vitro to collect the 
monoclonal antibody, the purity of the monoclonal antibody in protein 
ingredients is too low as compared with that of the ascites fluid. In such 
a case, it is necessary to purify and concentrate the immunoglobulin 
fraction from a supernatant of the in vitro culture by using an ammonium 
sulfate precipitation, a protein-A column or an affinity column (i.e., 
antigen column) which uses the human interferon-.beta. as a ligand, and 
the like. Although in this case bovine immunoglobulins from the bovine 
serum ingredients which are used for the culture of the hybridoma may 
sometimes contaminate the above-described immunoglobulin fraction, there 
is no problem in practical use. Various serum-free culture media have 
recently been developed (for example, T. H. Chang et al., J. Immunol. 
Methods, 39 (1980), 369-375), and the use of the hybridoma cultured on 
such a culture medium is convenient because it may produce the monoclonal 
antibody in a high purity. The anti-human interferon-.beta. monoclonal 
antibody of the present invention is preferably obtained from the 
hybridoma 1H12 strain described in Japanese Patent Application Laid-Open 
(KOKAI) No.59-144796. The disclosures of Japanese Patent Application 
Laid-Open (KOKAI) No.59-144796 are hereby included as reference. 
As the monoclonal antibody of the present invention, a Fab'fragment is 
preferably used which may be obtained by cleaving the Fc portion by a 
known pepsin-treatment (Y. Hamaguchi et al. (1979), J. Biochem. 85, 
1289-1300) after purified to an immunoglobulin fraction, and then by 
cleaving reductively with mercaptoethylamine (S. Yoshitake et al. (1979), 
Scandy J. Immunol. 10, 81-86). 
Then, the resulting antibody is labeled by an enzyme. For the preparation 
of the enzyme-labeled antibody, a known reagent can be used. The reagent 
having two functional groups, for instance N-(.epsilon.-maleimide 
caproyloxy)succineimide, bismaleimide, glutaraldehyde, carbodiimide and 
the like, or an aldehyde group obtained by oxidizing the saccharide of 
peroxidase with periodic acid may be effectively used. By using such a 
reagent, the enzyme is labeled by a conventional method while maintaining 
the reactivity of the antibody. A preferred enzyme-labeling method used in 
the present invention is to bind the antibody Fab' having a free Cys 
residue to a maleimide-bound peroxidase. 
The freeze-dried composition according to the present invention is 
generally prepared by mixing 0.01-1 .mu.g of the enzyme-labeled antibody 
with 0.5-10% aqueous trehalose solution to obtain an original liquid to be 
freeze-dried, and then by freeze-drying the original liquid by a 
conventional method. The trehalose is 
.alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside, which is a 
disaccharide considered to naturally occur in a living body in relation to 
the protection of cells. 
The freeze-dried composition according to the present invention 
substantially contains only the trehalose and the enzyme-labeled antibody, 
but not a substance which disturbs the storage stability. In the case of 
adding a conventional buffer solution such as a phosphate buffer solution 
into the original liquid to be freeze-dried, the salts and the like from 
the buffer solution remain in the composition after freeze-drying and 
disadvantageously disturb the storage stability. However, a volatile 
buffer such as ammonium bicarbonate buffer can be added into the original 
liquid to be freeze-dried because such a buffer, even if it is added into 
the original liquid, does not disturb the storage stability after 
freeze-drying. 
The thus-prepared freeze-dried composition comprising the enzyme-labeled 
anti-human interferon-.beta. antibody according to the present invention 
retains the enzyme activity thereof stable over a long period of time and 
can be advantageously utilized for an enzyme immunoassay kit to 
quantitatively determine a trace amount of human interferon-.beta.. 
An EIA kit according to the present invention comprises: (a) a solid phase 
reagent prepared by binding an anti-human interferon-.beta. antibody, 
which is a first antibody, to a solid phase carrier; and (b) a 
freeze-dried composition substantially comprising an anti-human 
interferon-.beta. antibody, which is an enzyme-labeled second antibody, 
and trehalose. 
As the first antibody, a polyclonal antibody may be used which is obtained 
from antiserum of a human interferon-.beta.-immunized animal. More 
specifically, the conventional antiserum which is obtained by immunizing 
an animal such as mouse, guinea pig, rat, rabbit, goat, sheep, horse etc. 
with a preparation (not necessarily a pure preparation) containing the 
human interferon-.beta. in accordance with the usual method is preferably 
purified and concentrated as an immunoglobulin fraction by the usual 
method before use. 
The purified conventional antibody is bound to, for example, a 96-wells 
microplate, then blocked with bovine serum albumin, casein, gelatin or a 
commercially available blocking agent, treated with a solution containing 
polyvinyl pyrrolidone and sucrose, and dried to obtain a solid phase 
reagent of the microplate. 
The second antibody, which is an enzyme-labeled anti-human 
interferon-.beta. antibody described above in detail, is freeze-dried in 
the presence of trehalose as described hereinbefore. 
The EIA kit according to the present invention is conveniently used for a 
non-competitive sandwich method. 
The procedure for the sandwich method, in which the freeze-dried 
composition of the enzyme-labeled antihuman interferon-.beta. antibody 
according to the present invention is used, is described hereinafter. 
(A) The first antibody directed against the interferon-.beta., which is a 
substance to be measured, forms the solid phase thereof on a carrier. As 
the carrier, any carrier may be used which is generally used in the 
sandwich system of immunoassay. Some examples of the carrier include a 
microplate which is commercially available as an immunoassay plate, 
plastic beads or iron beads coated with a plastic, glass beads, a plastic 
tube, a paper disk, cross-linked dextran particles, cross-linked agarose 
particles, and the like. As a method for forming the solid phase of the 
first antibody on such a carrier, such a method as physical adsorption and 
chemical linkage may appropriately be selected in accordance with the 
carrier to be used. 
(B) The sample to be measured is brought into contact with the first 
antibody on the solid phase, and then the human interferon-.beta. to be 
measured is bound to the first antibody by an antigen-antibody reaction. 
The conditions (temperature, time etc.) for contact may appropriately be 
adjusted. 
(C) Then, the freeze-dried enzyme-labeled second antibody dissolved in the 
buffer is brought into contact therewith and is subsequently bound to the 
human interferon-.beta. which has been bound to the first antibody by the 
antigen-antibody reaction. In this way, the human interferon-.beta. is 
sandwiched between the first and the second antibodies. The conditions 
(temperature, time etc.) for contact may also appropriately be adjusted. 
It is also possible to effect a one-step process in which the steps (B) and 
(C) are carried out simultaneously. That is, the coexistence of a sample 
to be measured and the second antibody is capable of sandwiching the human 
interferon-.beta. between the first and the second antibodies. In 
addition, the measurement with higher sensitivity is expected from the 
one-step process. 
Labelling is carried out with an enzyme by a conventional method. The 
enzyme generally used includes alkaline phosphatase, .beta.-galactosidase, 
a peroxidase such as horseradish peroxidase and the like. The experimental 
procedures are described in detail in some literature (for example, 
"Enzyme Immunoassay" edited by Ishikawa, Kawai, Miyai et al., Igakushoin, 
1978, Tokyo; "Selected Methods in Cellular Immunology" edited by B. B. 
Mishell, S. M. Shiigi et al., Freeman and Comp.: 1980, San Francisco; 
"Fluorescent Antibody Techniques and their Applications" vol. 2 edited by 
A. Kawamura, Univ. Tokyo Press, 1977, Tokyo), and so it is easy to know 
the method for preparing the labeled antibody in accordance with the 
object. 
(D) The human interferon-.beta. sandwiched between the first and the second 
antibodies is quantified by measuring the amount of the second antibody. 
For example, in using an enzyme-labeled second antibody, the decomposition 
of an added enzyme substrate by the enzyme reaction is measured by 
colorimetry. If a calibration curve is formed by using the human 
interferon-.beta. having known concentrations, it is possible to know a 
target concentration of the sample. 
Examples of the sample include a culture liquid, a biological material such 
as a body fluid and urine, and the like. Human serum or plasma may also be 
used, and IFN-.beta. present in human blood is also measurable. The level 
of the endogenous IFN-.beta. has been reported to vary in the blood of 
patients infected with various viruses such as HIV, HB, HTLV-1 and HTLV-2, 
and accordingly the assay kit according to the present invention is 
expected as a diagnostic reagent for AIDS, hepatitis, ATL or the like. 
The present invention is further illustrated with the aid of the following 
non-limited Example. 
EXAMPLE 
A freeze-dried composition of an enzyme-labeled antibody for use in an 
enzyme immunoassay of human interferon-.beta., which is excellent in 
storage stability: 
An enzyme-labeled antibody was prepared and human interferon-.beta. was 
measured in accordance with the method described in Japanese Patent 
Application Laid-Open (KOKAI) No.62-206447. 
a) Preparation of an enzyme-labeled antibody: 
To a mouse anti-human interferon-.beta. monoclonal antibody disclosed in 
Japanese Patent Application Laid-Open (KOKAI) No. 59-144796, 4% pepsin 
based on the weight of the antibody was added to digest the antibody at 
37.degree. C. for 20 hours, and then a F(ab').sub.2 fragment thereof was 
obtained by a gel filtration through a Sephacryl S-200 column. The 
fragment thus obtained was reduced by mercaptoethylamine, and then a Fab' 
fragment was obtained by a gel filtration through a Sephacryl S-200 
column. On the other hand, N-(.epsilon.-maleimidecaproyloxy)succineimide 
was added to horseradish peroxidase, hereinafter referred to as "HRP", 
reacted at 30.degree. C. for 60 minutes, and then fractionated by passing 
through a Sephadex G-25 column to obtain maleimide-bound HRP. The antibody 
Fab' prepared in the above, in an amount of 1.5 mg, was added to 1.2 mg of 
the maleimide-bound HRP and then reacted at 4.degree. C. for 20 hours. An 
enzyme-labeled antibody, HRP-antibody Fab', was then obtained by a gel 
filtration through a Ultrogel AcA-44 column. 
b) Measurement of human interferon-.beta.: 
After a polyclonal antibody, which had been affinity-purified from a rabbit 
anti-human interferon-.beta. antiserum, was bound to a microplate, the 
plate was treated with a PBS solution containing 4% polyvinyl pyrrolidone 
and 10% sucrose and then dried. Thereafter, the plate was washed once with 
the buffer containing a detergent, and 100 .mu.l of a sample containing 
human interferon-.beta. and 50 .mu.l/well of the enzyme-labeled antibody 
diluted appropriately, which had been prepared in the above, were added 
thereto and reacted at 2.degree. to 10.degree. C. overnight. The plate was 
washed on the next day, and then 100 .mu.l/well of an enzyme substrate 
liquid consisting of a phosphate-citrate buffer solution, (pH 5.0), which 
contains 40 mg of o-phenylenediamine and 20 .mu.l of aqueous hydrogen 
peroxide, was added thereto and reacted for 60 minutes at room temperature 
at a dark place. The reaction was then stopped by adding 100 .mu.l/well of 
4.5N sulfuric acid as a reaction terminator, and an absorbance of the 
reaction product was measured at a wavelength of 490 nm using 405 nm as 
reference. 
c) Stability of the freeze-dried composition according to the present 
invention: 
The stability of the freeze-dried composition containing the enzyme-labeled 
antibody prepared in the below way was measured by following the 
measurement described in the foregoing. 
6 .mu.l (about 0.2 .mu.g) of the enzyme-labeled antibody prepared in a) was 
charged into a 10-ml brown vial, and then 1 ml of 1% aqueous solution of 
trehalose or a buffer solution (0.1M phosphate buffer, pH 7.0) containing 
1% trehalose was added thereto. Two different mixtures were separately 
freeze-dried to obtain two kinds of freeze-dried composition. When an 
enzyme immunoassay of human interferon-.beta. was carried out, the 
freeze-dried composition in a vial was dissolved in 6 ml of buffer 
solution (0.1M phosphate buffer, 0.1% bovine serum albumin (BSA), 0.05% 
Tween-20, pH 7.0), and then 50-.mu.l portions of the solution were added 
to each well for the measurement b). These two kinds of the freeze-dried 
compositions containing the enzyme-labeled antibody were stored at 
37.degree. C. for 30 days in order to compare the storage stabilities 
between the two. There was no difference between the residual activities 
of two different freeze-dried compositions, when measured immediately 
after the freeze-drying. A change in the storage stability was indicated 
by the residual activity on an assumption that the activity determined 
immediately after the freeze-drying was 100%. The results are shown in 
FIG. 1. 
As shown in FIG. 1, the residual activity of the freeze-dried composition 
containing the buffer solution was very low on the 16th and the 30th days, 
while the freeze-dried composition substantially comprising trehalose and 
the enzyme-labeled antibody exhibited a prominent effectiveness on the 
storage stability. 
d) Quantitative determination of the human interferon-.beta.: 
Use of the freeze-dried composition of the enzyme-labeled antibody 
according to the invention makes possible a highly sensitive measurement 
of human interferon-.beta.. The result of measurement which was obtained 
in accordance with the measurement b) using the serial dilutions (200 
.mu.l each) of human interferon-.beta. is shown in FIG. 2. 
In the calibration curve shown in FIG. 2, the line is linear between 0.25 
IU/ml and 50 IU/ml, and the concentration of measurable limit was 0.25 
IU/ml. The sensitivity of the measurement is not less than 20 times as 
high as that of a bioassay. Thus, the method of the present invention is 
effective for the measurement of the endogenous IFN-.beta. in human blood 
and may provide important information on a relationship between an 
etiology and an IFN-.beta. level in the human blood.