DE3021891C2 - Method for the simultaneous detection of hepatitis B surface antigen and antibodies against hepatitis B core antigen - Google Patents

Description

The invention relates to a method according to the preamble of claim 1. ⁴ p

There are at least 2 different types of viral hepatitis. It is believed that hepatitis A is caused by the hepatitis Α antigen (HAVAg). It is generally characterized by an incubation period of 2 to 6 weeks, slight prodromas, and a relatively mild clinical course. This disease is commonly transmitted through contaminated food or liquids. However, transmission by systemic inoculation has also been demonstrated. Hepatitis A is ⁵ S often referred to as "infectious hepatitis". In the United States, the number of people sick each year is more than 50,000.

The most likely etiological agent for "serum hepatitis" is believed to be the hepatitis B virus. A & o Hepatitis B infection is generally transmitted through blood products or contaminated instruments, for example Needles, but transmission in other ways is also possible.

An incubation period of 6 weeks to 6 months was previously assumed for a hepatitis B infection. It however, there are also reports of incubation times as short as 2 weeks. The disease can be mild or are asymptomatic. If the course is symptomatic however, the clinical picture is particularly severe. as Prodromata can cause joint pain, arthritis, rashes, Having fever, anorexia, fatigue and pruritus or occur without jaundice.

At least 3 antigen-antibody systems can be associated with hepatitis virus B: surface (HB ₅ Ag: anti-HB ₅ ), core (HBcAg: anti-HB _c ) and e-antigen (HB _£ Ag: anti -HB _e ) - The hepatitis B surface antigen (HB ₅ Ag) is found in the blood in the form of 22 nxn beads and elongated tubes of 22 nro diameter and variable length. It is believed to be the protein envelope from the hepatitis B virus

A 42 nm particle containing DNA and DNA polymerase is considered to be the infectious virus (Dane particle). The surface of the Dane particles is similar or identical to HB ₅ Ag. Under the influence of detergents, the Dane particle is _{broken down into a 27 nm electron-dense nucleus, HB c} Ag. This nucleus occurs in the cell nuclei of hepatocytes from patients with serum hepatitis during the acute stage of infection.

It can thus be expected that patients with viral hepatitis of type B will develop antibodies against the surface antigen (anti-HB ₅ ) and also against the protein core (anti-HB _c ). HB ₅ Ag is a steady indicator of the presence of the hepatitis B virus. Anti-HB _C generally occurs during early viraemia often together with antigenemia (HB ₂ Ag) at the peak of the liver dysfunction and long before the onset of anti-HBj. Anti-HB _C is generally associated with prolonged circulation of HB2Ag, suggesting that anti-HB _c is produced in response to active virus replication. In 1972 the hepatitis e antigen (HB _e Ag) was detected and characterized. The e indicator was found in HB ₅ Ag positive serum. It is believed that it occurs more frequently in the serum of chronic HBjAg carriers with active liver disease than in healthy carriers in patients at the stage of hepatitis B-incubation period, it was found that the e antigen immediately after the occurrence of HB ₅ Ag and before clinical manifestation of liver damage occurs Logically, the presence of e-antigen in such sera can be taken as an indication of a poor prognosis and progressive liver damage. Conversely, the presence of e-antigen (anti-HB _e ) can be taken as an indication of a ^ i / th prognosis. While these relationships are not absolute, they can provide valuable clinical guidelines.

As the serological indicators (markers) of hepatitis in a logical order over the course of Infection, acute illness and recovery period would occur an analysis of 2 or more Such indicators are valuable in determining the course of the disease over time represent.

A determination of more than one indicator could also be an important double check for HB ₅ Ag in blood donors or patients in an effort to reduce the incidence of B-type hepatitis. Such a need was addressed by Goldfield et al. Am. J. Med. Sci., Vol. 270 (1975), pp. 335 to 342., after careful examination of recipients of _{blood negative for HB 1} Ag in 7 of 465 patients, exposure to hepatitis antigen

proven. A method of detecting more than an indicator of the .hepatitis B virus could Reduce or prevent false negative results from occurring. Similarly would in many If the reaction to two tests is positive, there is a possibility of false positive results may occur during a test.

In the older application DE-OS 29 43 648 a method for the simultaneous detection of at least described two different indicators of exposure to hepatitis B virus. Included however, the labeled reagent will have the same labels for the different immunoreactants on.

From DE-OS 28 03154 is the simultaneous Detection of folate and vitamin B ^ using known from folate and vitamin Bi2 tracers, whereby the both tracers are marked by different radioactive isotopes.

DE-AS 22 62 413 relates to a radioimmunological process for the detection of individuals Ligands indicative of hepatitis. An indication of the simultaneous detection of two different ones Immunoreactants in a sample are not found in this publication

The registration is based on the task of creating a new procedure that provides simultaneous evidence of hepatitis B surface antigen and antibodies against hepatitis B core antigen

The solution to this problem is characterized in claim 1

An advantageous embodiment of the method and a reagent for carrying out the process result from claims 2 and 3.

The following examples illustrate the production process and the use of special reagents according to the invention. The first example illustrates a procedure for the production of a solid support that can be used with 2 different non-complementary immunoreactants, which are complementary to the indicators indicative of exposure to hepatitis, coated is.

In particular, a solid support (bead) is used 2 different, non-complementary immunoreactants are coated in such a way that both their Retain reactivity and are able to deal with complementary indicators in an unknown Connect sample.

In the first example, polystyrene beads are coated with an antibody against HBjAg and with core antigen, HB _c Ag. The antibody retains its reactivity and reacts with HB ₂ Ag present in the unknown sample. The fixed core antigen retains its antigenic effect and reacts with antibodies against the core (anti-HB _c ) in the unknown sample. The special feature of the invention is that both immune reactions occur simultaneously on a single solid phase reagent.

example 1
Production of a coated carrier

An HB _c Ag preparation is made by adding Dane particles to a solution of 2 percent 2-mercaptoethanol (2-ME) and 5 percent Tween 80 (sorbimacrogoleate) in Tris-EDTA saline buffer (TSE) for 2 hours Treated at 37 ° C. This solution is diluted tenfold in 5 percent 2 ME-TSE and allowed to stand overnight before diluting to the final concentration of 1: 8000 in TSE. Separately: Polystyrene beads (634 mm; 1/4 in.) Are coated with a 1: 2000 dilution of anti-HB2 serum (guinea pigs) in phosphate buffered saline (RBS) by soaking for 2 hours. The beads are then washed away and coated dried. The core preparation is then poured over the coated beads. By £ day

Allowing the core antigen to adhere to the beads is allowed to stand at room temperature. Then the beads are removed from the buffer solution and washed to stabilize the adhering components coated with 10 percent sucrose solution and air-dried

Polystyrene spheres are preferred because they are easy to coat and handle. It can but also any other carrier with macro or micro dimensions made of different plastics, Metals or glass coated and in the invention Process are used.

It is possible to coat the beads first with the core antigen and then with anti-HB _s. It was found, however, that prior coating of the beads with guinea pig _{anti-HB s} serum markedly improves the adhesion of the core antigen.

It is also important to point out that other combinations of non-complementary Immunoreactants related to hepatitis virus can be used. the Of course, immunoreactants do not have to be complementary, as they have a complementary relationship would reduce the antigenic activity and antibody reactivity of the reagents.

The following example illustrates the preparation of a liquid phase reagent containing a radioactively ( ¹²³ J) labeled immunoreactant, anti-HB ₅ and an enzyme (horseradish peroxaase) labeled indicator, anti-HB _c . Although the terms "indicator" and "immunoreactant" can be used interchangeably, the term "indicator" (marker) is preferably used to designate the antigen to be detected in the unknown sample or

Antibodies or their equivalents are used. The term "immunoreactant" is used to define antigens or antibodies that are complementary to the indicators to be detected
Accordingly, in the following examples, the antigens and antibodies used on the solid support are always complementary to one of the unknown indicators and are therefore referred to as immunoreactants.
In the liquid phase reagent there is; The surface antibodies marked here are complementary to the unknown indicator (HB ₅ Ag). It is therefore called a labeled immunoreactant. The labeled core antibody is identical to the indicator to be detected and is therefore referred to as a labeled indicator.

Example 2
Labeled antibody reagent

For iodination ( ¹²⁵ J) of the antibody against HB ₅ Ag

fi5 (anti-HBj) about 0.1 ml of 0.5 M phosphate buffer with a pH of 7.5, a small volume of 5 to 6 mCi Na ¹²⁵ I and 100 mg of purified anti-HB ₅ in a vial of 3, Given 7 cm ³ (1 dram). The pH will be

set to 7.5 to 8.0. All of the ingredients are mixed together. The mixture is with 50 μ! a freshly prepared solution of 35 mg of chloramine T in 10 ml of 0.05 M phosphate buffer with a pH of 7.5. After further mixing, the reaction is allowed to proceed for 60 seconds at room temperature. Then, _t the reaction mixture with 50 μΐ a freshly prepared solution of 35 mg sodium metasulfite in 10 ml of 0.05 M phosphate buffer, pH 7.5 was added to reduce the chloramine T and thereby stop the reaction.

The reaction is monitored by placing 5 μl of the reaction mixture on a strip of Whatman No. 1 paper and chromatographing the mixture in 70 percent methanol. The iodinated protein remains at the contact point, while free ¹²⁵ J migrates with the solvent. The percentage of ^t25 J in the protein is regarded as the percentage of conversion.

The crude iodinated antibody is obtained using a three-dimensionally crosslinked D-aetran (Sephadex G-50 gel) packed column using 0.1 m Tris-Puifer containing 03 percent Purified sodium chloride from pH 7.8. The column is made with a small volume of 30 percent aqueous solution of bovine serum albumin and then with a corresponding volume of 0.1 m Tris buffer with a content of 0.9 percent sodium chloride of pH 7.8 pretreated The iodized Antibody is placed on the top of the column and eluted using the saline Tris buffer. The labeled antibody is obtained as the first eluate.

Peroxidase is used to bind antiquity against hepatitis B core to horseradish peroxidase (HRP) activated using sodium metaperiodate. The excess periodate and by-products are activated by the active peroxidase through gel filtration to a? with three-dimensionally cross-linked dextran (Sephadex G-25) packed column. The activated peroxidase is then combined with the antibody (anti-HBc) implemented. The reaction is spontaneous. Then sodium borohydride is added to the Stabilize the bond between the peroxidase and the antibody. By adding acetone this becomes residual sodium borohydride decomposes

When used according to the invention, the two labeled antibodies are in a diluent diluted with the following ingredients:

50% fetal calf serum
ii 15% normal human serum
;: 0.00SmEDTA

0.1% sorbimacrogollaurate (Tween-20)
0.01% thimerosal in PBS

'i B e i s ρ i e 1 3

; Simultaneous proof of

HBsAg and anti-HBc

Serum samples containing unknown hepatitis indicators are added to the individual wells of a reaction plate. A polystyrene bead coated according to Example 1 is then added to each sample. After incubation at 45 ° C. for 2 hours, the beads are removed from the well, washed with water to remove excess reagent and diluted 0.2 ml; of the liquid phase reagent prepared according to Example 2 was added. The solid and liquid phases are incubated for 1 hour at 45 ° C. The solid phase is then separated from the antibody reagent and washed 4 times with water to remove excess reagent. The washed beads are placed in test tubes containing o-phenylenediamine (30 mg in 10 ml of 0.1 M citrate buffer, pH 5.5) and incubated for 30 minutes. The enzymatic reaction is then stopped by adding 1 ml of 1 M HCl are visually inspected for the presence or absence of coloration due to the reaction of the enzyme label and the substrate. The lack of coloration or a slight coloration shows that anti-HB _{c is present} in the unknown sample and has competed with the labeled indicator for the reactive sites on the coated carrier. The radioactivity on the beads is then measured in a y-counter counted. The counts per minute (cpm) are determined. Radioactivity found on the beads shows that HB ₅ Ag is present in the unknown sample, which adheres to the fixed antibody and provides a binding site for the radioactively labeled antibody.

Claims (3)

Patent claims: 1. Method for the simultaneous detection of hepatitis B surface antigen and antibodies against hepatitis B core antigen in a sample, characterized in that one a) the sample with a solid phase containing both hepatitis B core antigen and antibody is coated against hepatitis B surface antigen, brings into contact, b) the sample is incubated with the coated solid phase for 1 to 24 hours, c) the coated solid phase from the sample separates, d) washes the coated solid phase to remove unbound sample, e) bringing the washed solid support into contact with a liquid reagent which radiolabelled antibodies against hepatitis B surface antigen and with a Contains enzyme-labeled antibodies against hepatitis B core antigen f) separating the solid phase from the labeled antibody reagent and g) the presence of labeled antibodies on the solid support by detecting the different markings determined 30th 2. The method according to claim 1, characterized in that the radioactive label ^'25 J and the enzymatic label horseradish peroxidase are used. 3. reagent for performing the method according to claim 1 or 2, characterized in that it contains a solid support containing hepatitis B core antigen and antibodies against hepatitis B surface antigen is coated. 40