DE2853859A1 - REAGENTS FOR DETECTION OF HEPATITIS B E ANTIGENTS AND ANTIBODIES - Google Patents

Description

DR.-ING. WALTER ABITZ

DR. DIETER F. MORF ³

DIPL.-PHYS. M. GRITSCHNEDER

Patent attorneys

Munich,

December 13, 1978

Postal address Postfach 860109, 8000 Munich

PienzenauerstraQs Telephone 983222 Telegrams: Chemlndus Munich Telex: (O) 523992

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ABBOTT IABOEATOEIES North. Chicago, Illinois 60064

Eeagentien for the detection of hepatitis B e antigen and antibodies

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DR.-ING. WALTER ABITZ DR. DIETER F. MORF DIPL.-PHYS. M. GRITSCHNEDER

Patent attorneys

Munich,

Postal address Postfach 860109, 80OO Munich 8β

PienzenauerstraOe 28

Telephone 98 32 23

Telegrams: Chemlndus Munich

Telex: CO) S23993

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1972 Magnius and Espiaark described in J. Immunol., Vol. 109, Pp. 1017-1021 describe a new precipitating antigen in sera from patients positive for hepatitis B surface antigen. This antigen was called the e antigen. It was also found that the antibody against e-antigen in other hepatitis B surface antigen (HBeAg) sera present, is. Subsequent investigations suggested that the presence of hepatitis B e antigen (HBeAg) included Signs of chronic liver disease and viral infectivity. Correspondingly, the detection of e-antigen is a valuable prognostic indicator of the chronic character considered of hepatitis B virus infections. One of those Evidence would provide valuable information regarding the infectivity of HBsAg positive individuals. Logically would be the presence of e antigen in such sera is an indication

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for a poor prognosis and for progressive liver damage. In contrast, the presence of e-antibodies would be an indication of a good prognosis.

The methods currently used to detect e-antigen and e-antibodies make use of agar gel immunodifusion and antagonist electrophoresis. These procedures are due relative to their low sensitivity and reproducibility unreliable. It can also be assumed that this process is due to indefinite and multiple precipitation patterns lacks the necessary objectivity.

According to the invention, reagents were made available which they prove to be valuable in various immunoassays for the detection of HbeAg and anti-HBe ala. In particular, the reagents for detecting HBeAg have solid supports coated with high titer human or animal anti-HBe immunoglobulin. Reagents for detecting anti-HBe include solid supports coated with aati-HBeAg and HBeAg alone ode # pre-coated on a fixed "with anti-HBe carriers adsorbed" JPüe άχ © immunoassays here "described are aucli marfcie3 tes _t meii £ isfeli © li © s odes titrisclies aosti-HBe immunoglobulin Odey HBeAg -. uses organic label can can be carried out according to any known marking process, for example with enamines, fluorescent or radioactive markers.

1 _a manufacturer; ^ en aati-HBeAfi; -imiauno ^ lobulin

Meneöhliches plasma containing anti-HBe or durek Animal antiserum induced by HBeAg immunization al® output fragrances are used. The plasma is recalculated

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and saturated with 50 percent (LiH ^^ SO ^. The Immunoglobulinniederschlag is collected and dissolved in a minimum volume of 0.01 M potassium phosphate buffer, pH 8.0. The solution overnight at 4 ⁰ C against the The same buffer is dialyzed, changing the buffer twice, then chromatographed on a column packed with DEA-E cellulose (DE-52, Whatman) equilibrated with the same buffer, and the eluate fractions containing IgG are collected No IgM or albumin can be detected in them by immunodiffusion methods.

2. Preparation of anti-HBe reagents

Polystyrene beads of 6 mm. Diameter are coated with anti-HBe by being biert 24 hours at temp tree he a door in a diluted solution of anti-HBe IgG from pH 9 ₅ O incubators.

These anti-HBe IgG preparations are made with iodine-125 using the ghloramin-T method with a specific badioactivity of

125 about 20 μ Gi protein per pig radiolabeled. The ^ J-anti-HBe solution is diluted to about 3 p · Gi per ml with a diluent consisting of 10 percent normal human serum and 50 percent calf serum in 0.01 M Tris-HGl buffer, pH 7.5 , diluted.

3. Test for HBeAp:

To detect HBeAg, 0.2 ml of the sample is incubated with the antibody-coated bead overnight at room temperature. After washing with Wasstr, the ball is further incubated in 0.2 ml of ¹²⁵ I-anti-HBe solution for 4 hours at 45 ° C. The bead is then washed and determined

12 * 5 of his ^ J-anti-HBe recordings. Si · count is in

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Relation to the amount of HBeAg in the sample.

4-. Test for anti-HBe

Anti-KBe is determined according to the following neutralization procedure. 0.1 ml of sample is mixed with 0.1 ml of diluted HBeAg positive serum containing a predetermined amount of HBeAg. An antibody coated bead becomes the mixture given and incubated for 20 hours. After washing with water or the pearl is kept in an aqueous buffer for 4 hours Incubated 45 ° C in 0.2 ml of anti-HBe solution. The amount of HBeAg in the first incubation is chosen so that in a serum that is negative for anti-HBe, 5000 to 10,000 cpm (counts per minute) due to ^ J anti-HBe uptake result in «Significant (50 percent) reductions

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the ^ J-anti-HBe uptake indicate the presence of anti-HBe in the rehearsal. If a sample contains HBeAg, a Increase in count observed. This method is therefore suitable for the detection of HBeAg and anti-HBe, depending on whether the Count. Increases or decreases.

5 «Purification of HBeAg and anti-HBe

A raw HBeAg preparation is made from HBeAg serum. precipitation obtained with ammonium sulfate and then on an anti-HBe affinity column, those by coupling purified anti-HBe immunoglobulin has been obtained on activated Sepharose B, put on. After applying the sample and at least once The column is washed with recirculation. The adsorbed HBeAg is then mixed with an aqueous, dissociating solution eluted to release the HBeAg. The fractions with HBeAg activity are determined and pooled.

Purified anti-HBe antibodies are _s explained above obtained by affinity chromatography. In this case

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are the affinity columns made of HBeAg, which is activated. Sepharose B, is coupled. The eluted fractions containing anti-HBe are collected and used as reagents.

6. Preparation of HBeAg reagents

The HBeAg purified by affinity chromatography is determined by the method described above for labeling anti-

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HBeAg with ^ J. marked. Both ligands can also be labeled with enzymes such as horseradish peroxidase if enzyme immunoassay is preferred. When using fluorescence methods, fluorescent compounds are used.

A solution of HBeAg in 0.01 M Tris-HCl with a pH of 9.0 is adsorbed on the surface of a styrene bead. The adsorption can also be used on solid surfaces of objects such as tubes or holes (wells) made of plastic or glass be performed. Treatment of the solid surface with the ligand can be made 1/2 to 24 hours. Afterward becomes the solid phase with a neutral buffer washed and dried at room temperature. The use of HBeAg reagents for the direct determination of anti-HBe is possible explained in example 2.

7. Production of HBeAg antiserum in animals

Purified HBeAg is used as an immunogenic product in animals. Guinea pigs are immunized by injection with HBeAg solution mixed with an equal volume of Freund's adjuvant. The sera of these immunized animals react with purified HBeAg and with HBeAg-positive sera. After absorption with normal human serum and HBsAg, the hyperimmune serum acts as an anti-HBe source for the determination or purification of antibodies.

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example Λ

Carrying out a hadioimmuno test for the detection of anti-HBe. Competitive gest-phase radioimmuno test. An aliquot of labeled immunoglobulin with a content of anti-HBe and the solution to be determined are incubated for a while with a bead coated with HBeAg. With each thorough washing, the bead is counted to determine the amount of marker adhered. As a negative control sample, a sample negative for anti-HBe is determined in the same way. A sample is positive for anti-HBe if the count or other measurement number of the sample is significantly below the corresponding value of the negative control sample.

The above procedure can be varied as follows:

1) Beads coated with HBeAg are first treated with the Incubate sample before adding labeled anti-HBe.

2) Proceed according to (1), with the modification that the beads washed before adding labeled anti-HBe.

Example 2

Solid phase direct determination: Homologous sandwich type for the detection of anti-HBe

Beads coated with HBeAg can be used in connection with labeled HBeAg can be used in the direct radioimmunoassay of the sandwich type. An aliquot of a sample is made incubated for a period of time with an HBeAg coated bead, tube, or other object. Then the coated article washed and further incubated with a solution of labeled HBeAg. If anti-HBe in the sample is present, a sandwich structure of HBeAg-anti-HBe-labeled arises HBeAg. After washing shows the bead

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a significantly higher count "or a different measurand as control beads containing an anti-HBe negative sample can be determined.

Example 3 Solid phase direct determination

Beads coated with HBeAg are first placed in a Incubated aliquots of appropriately diluted human serum. If the sample contains anti-HBe, the anti-HBe specifically bound to the HBeAg on the beads. After washing the beads with water or a buffer solution a labeled anti-human immunoglobulin solution was added. One Increase in the count bzxtf. another measured quantity of the globules compared to a negative control sample indicates the presence of anti-HBe in the sample. With this one Method, anti-HBe can be distinguished from IgG or IgM if labeled specific anti-human IgG or IgM is used will.

Example 4-

Solid phase direct determination: Homologous sandwich type for the detection of HBeAg

Beads coated with anti-HBe that are conjugated with labeled anti-HBe can be tested in a direct radioimmunoassay from Sandwich type can be used. An aliquot of a sample is made with beads, tubes, or objects containing anti-HBe are coated, incubated. The items are then washed and further treated with a solution of labeled anti-HBe incubated. If HBeAg is present in the sample, a sandwich structure of anti-HBe-HBeAg-labeled anti-HBe formed. After washing, the beads show a significantly higher count or a different measured variable than con— troll beads tested for anti HBe negative samples will.

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A collection of sera from different populations is made with respect to HBeAg and anti-HBe for comparison purposes the immunodiffusion test and the radioimmunoassays according to the invention examined.

The results of such a study on HBsAg positive female carriers are shown in Table I. According to the immunodiffusion test, 6.9 percent of HBsAg carriers are positive for HBeAg. Further 13 »8 percent are positive for anti-HBe. The remainder (79 »3 percent) turn out to be negative for HBeAg and anti-HBe. On the other hand, when the same sera are analyzed after the radioimmuno test, the following results are obtained: 48.4 percent HBeAg positive and 50.2 percent anti-HBe positive. Only 1.4 percent turn out to be negative in terms of both HBeAg as well as anti-HBe. Thus, the detectability of HBeAg is about a factor of 7 uxicL the detectability of anti-HBe about a factor of 4 compared to the immunodiffusion test elevated,-

Table I.

Comparison of a rheophoresis test and a radioimmuno test for the Detection of HBeAg and anti-HBe in female HBsAg carriers

Rheophoresis radioimmuno test

Sample- _{0 /} sample-. _{0 /} number ^{/ 0} number ^{/ o}

HBeAg + 15th 6th , 9 105 48 Λ anti-HBe + 30th 13th ,8th 109 50 , 2 HBeAg / anti-HBe negative 172 79 , 3 3 1

Total 217 100.0 217 100.0

End of description

Claims (1)

Patent to s, ρ r ü c h 1.] Reagent for determining an immunological component from the group HBeAg and anti-HBeAg, characterized by a "'content of a ligand from the group HBeAg. Aiti-HBeAg and anti-HBeAg-HBeAg complex in insoluble ^T .orm. 2. Reagent according to claim 1, characterized in that the insoluble form consists of a solid coated with HBeAg Material pre-coated with anti-HBe is made. 3. Reagent for the detection of anti-HBeAg, characterized by a content of a ligand from the group HBeAg, anti-HBeAg and anti-HBeAg-HBeAg complex in insoluble form. 4. ' Reagent for the determination of HBeAg, characterized in that that it contains HBeAg in insoluble form. 5. Reagent according to claim 4, characterized in that it is in the form of polystyrene beads coated with anti-HBeAg is present. 909826] / D73O INSPECTED 6. Reagent for determining an immunological
characterized by a content of a ligand
from the group HBeAg and anti-HBeAg, the ligand being marked to enable detection. 7. Reagent according to claim 6, characterized in that the ligand is radioactively labeled. 8. reagent according to claim 7, characterized in that as 125
radioactive label ^ J is present. 9- reagent according to claim 6, characterized in that the Labeling with an enzyme has been made. .0. Reagent according to claim 9, characterized in that as Enzyme horseradish peroxidase has been used. 11. Reagent according to claim 6, characterized in that it is anti-HBe of animal origin, which by immunization
has been induced with HBeAg contains. 12. reagent according to claim 6, characterized in that the Ligand is a component of an isolated serum fraction. 0-98 21/07 30