NL8001661A - METHOD AND REAGENTS FOR SIMULTANEOUSLY DISPLAYING DIFFERENT MARKS ABOUT HEPATITIS. - Google Patents

Description

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Title: Method and reagents for the simultaneous detection of various markings concerning hepatitis.

The invention relates to an improvement of the solid phase immunoassay methods for the detection and determination of antigens and antibodies (labels) concerning hepatitis.

At least two different types of virus hepatitis exist.

Hepatitis A is believed to be caused by the hepatitis A antigen (HAVAg) and is usually characterized by a 2-6 week incubation period, mild prodromata and a relatively mild clinical disease. This disease is usually passed on through contaminated food or drinks, but can also be passed on through systemic contamination. Hepatitis A is commonly referred to as infectious hepatitis and is quite common.

The hepatitis B virus is believed to be the most likely causative agent of serum hepatitis. A hepatitis B infection is usually passed on through blood products or contaminated instruments, such as needles, but can also be passed on by other means. It was previously believed that the incubation period for hepatitis B was 6 weeks to 6 months. However, incubation periods of only 2 weeks have also recently been described. The disease can be mild or asymptomatic, but also symptomatic, even with severe symptoms. Prodromata include arthralgia, arthritis, rash, fever, anorexia, fatigue and pruritis with or without jaundice.

At least three different antigen-antibody systems can be associated with hepatitis virus: the surface (HB_Ag: anti-HB), the core (HB Hg: anti-HB), and the e-antigen (HB Ag: anti-HB). The hepa-

Cv 6 6, 25 titis B surface antigen (HBgAg) is found in blood in the form of 22 nm spheres and as elongated ovals, with a diameter of 22 nm and variable lengths, the protein coating of the hepatitis B virus is believed to be .

An h2 nm particle with DM and a DM polymerase is considered the infectious virus (Dane particle). The surface of one

Dane particle is almost identical or even identical to HB Ag. In surfactants, the Dane particle is degraded to an electron-dense 27 nm core, HBcAg. The latter is observed in hepatocyte nuclei in patients with serum hepatitis during the acute infection period.

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For example, patients with virus hepatitis type B can be expected to produce antibodies against surface antigen (anti-HBs) and also against the protein core (anti-EB). Serum HB_Ag is a cs consistent marker for the presence of hepatitis B virus and anti-EB usually appears during early viraemia, often accompanied by antigenemia (HBsAg), at the peak of liver dysfunction and long before anti-HB appears . Anti-HB is usually trafficked with a prolonged circulation of HB_Ag suggesting that anti-HBc is always produced in response to an active proliferation of the virus.

In 19T2, the hepatitis e antigen (HBgAg) was discovered and described. This e-labeling was found in HB Ag positive serum and is more common in serum or chronic HBsAg carriers with active liver disease than in healthy carriers. In patients during the incubation period of hepatitis B, this e-antigen appeared to occur just after the appearance of HBsAg and for a clinically detectable liver damage. His presence in. this kind of sera would therefore logically indicate a poor prognosis and progressive liver damage. Conversely, the presence of e-antibody (anti-HB) € 20 could indicate a good prognosis. These correlations are not absolute, but still useful as a clinical guide.

Since the serological markers for hepatitis appear in a consistent order throughout the course of the infection, the acute illness and recovery, an analysis of two or more of these markers may be a valuable tool to monitor the course of the disease.

A test for more than one marker may also be an important control of HBsAg in blood donors or patients in attempts to reduce the number of hepatitis B cases. Such a need has been mentioned by Goldfield in Am. J. Med. Sci., 270, -35- ^ 2 (1975), who nevertheless received evidence of exposure to exposure to recipients of blood responding negatively to HB Ag.

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hepatitis antigen in 7 of b65 patients. It follows that a method which makes it possible to detect more than one mark concerning hepatitis 35 B reduces the occurrence of false negatives or even prevents 80 0 1 661 t -3-. Likewise, in many cases, a positive response to two trials reduces the possibility of false positives.

Although the reagents and methods described in more detail below correspond to "known commercial products and" known procedures for detecting the various markers of virus hepatitis, it has never previously been "described or suggested that these markers can be detected simultaneously. invention "now relates to reagents and a method for simultaneously detecting in a sample at least two different markers proving exposure to hepatitis virus.

This method involves contacting a sample with a solid support coated with at least two different non-complementary immuno-reagents that are complementary to the unknown labels; incubating this sample with the coated support for up to 2¼ hours; separating the coated solid support from the sample; washing the coated solid support; contacting the washed solid support with a liquid reagent containing at least two different hepatitis markers or immuno-reactants, each selected so as to either react or compete with one of the unknown hepatitis markers and each is labeled with readily detectable labels; separating the solid phase from the liquid reagent and determining the presence of labeled labels on the solid support by detecting each specific label.

The following examples illustrate the preparation and use of characteristic reagents within the invention. The first example describes the method of preparing a solid support coated with two different non-complementary immuno-reagents that are complementary to markers proving exposure to hepatitis. More specifically, a solid support (grain) is coated with two different non-complementary immuno-reactants such that both retain their reactivity and can combine with complementary labels in an unknown sample.

In the first example, a polystyrene bead is coated with an antibody to HB Ag and a core antigen, HB Ag. The anti-sc 800 t 6 61 -u body hereby retains its appeal and can react well with any HB_Ag in the unknown male and the attached core antigen also retains its antigenic activity and can react well with any antibody to the nucleus (anti-HBc ) in the unknown monster. The crux of the invention is that both immunoreactions occur simultaneously with a single solid phase reagent.

Example I

Preparation of a coated support.

A preparation with HB Ag was obtained by exporting Dane particles to a liquid of 2 # 's 2-mercaptoithanol (2-ME) and 5%

Tween 80 in tris-EDTA salt (TSE) buffers for 2 hours at 37 ° C. This solution was diluted 10-fold with 5% 2ME-TSE and left overnight for dilution to the final concentration of 1: 8000 in TSE.

Separately, 6 mm polystyrene beads were coated with a 1: 2000 dilution of anti-HB serum (guinea pig) in phosphate buffered saline (PBS) by impregnation for 2 hours. The granules were removed, washed and dried. The core preparation was then poured over the coated granules and the core antigen was allowed to adhere to the granule at room temperature for 2 days. After this, the grains were removed from the. 20 buffer solution removed, washed, coated with a 10% sucrose solution to stabilize the adhered material and air dried.

Although polystyrene granules are preferred for their ease of coatability and handling, any solid support of macro or micro dimensions made from a range of plastics, metal or glass can be used equally well.

Although it is also possible to coat the granules first with core antigen and then with anti-HB_, it has been observed that first coating with guinea pig anti-HBs serum clearly enhances the adhesion of core antigen.

It is also important that other combinations of non-complementary hepatitis virus immunoreagents may also be used. The immunoreactants should, of course, not be complementary and that complementary attraction would reduce the antigenic properties and the attractiveness of the reagent.

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The following example illustrates the preparation of an oily phase reagent containing an immunoreactant, anti-HBs, bearing a radioactive label (125 J) and an antigen-HBc, bearing a reactive enzyme label (horseradish peroxidase).

Although the terms "tag" and immunoreactant "are interchangeable, preferably the term" tag "is used for the antigen or antibody and their equivalents in the unknown sample. In contrast, the term "immunoreactant" is most preferably used for the antigen or antibody on the support.

Thus, in the following examples, the antigens and antibodies on the solid supports are always complementary to any of the unknown markers and are thus referred to as immunoreactants.

In the liquid phase reagent, the labeled surface antibody is complementary to the unknown marker HBsAg and is therefore also referred to as labeled immunoreactant. The labeled core antibody, on the other hand, is identical to the mark that one wishes to demonstrate and is therefore referred to as the labeled mark.

Example II

Labeled Antibody Reagent 20 The iodination (125 J) of antibodies to HBsAg (anti-HBs) includes the addition of about 0.1 cm -1 of a 0.5 M phosphate buffer with 7.5 µl, a small volume of 5-6 May After 125 Js and 100 mg of purified anti-HB_ in a vial, adjusting the pH to 7.5-8.0 and mixing

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all components. An additional 50 microliters of fresh was added to this mixture

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Prepared solution of chloramine T (35 mg in 10 cm 0.05 M phosphate buffer with pH 7s5) was added. After mixing again, the reaction was allowed to proceed at room temperature for 60 seconds. 50 microliters of a freshly prepared

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sodium metasulfite solution (35 mg in 10 cm 0.05 M phosphate buffer pH 7.5) was added to this mixture to reduce the chloramine T 30 and thereby stop the reaction.

The effectiveness of the reaction was verified by applying 5 microliters of the mixture to a strip of Whatman No. Chromatograph 1 paper and the mixture in 70% methanol. The iodinated protein remained at the origin while the free 125 J migrated with the solvent. The 125 J percentage in the protein is counted as the 800-1661V -6 percent reaction efficiency.

The crude iodinated antibody was purified with Sephadex G-50 gel columns using 0.1 mol of tris buffer containing 0.9% sodium chloride and 7.8 µl. This colan was pretreated with a small volume of a 30% aqueous solution of bovine serum albumin followed by an equal volume of 0.1 mole tris buffer solution with 0.9% sodium chloride and pH 758. The iodinated antibody was added to the top of the kolan added and washed with a buffered tris-zcut solution. The labeled antibody was first collected from the 10 colan.

The conjugation of antibody to hepatitis B kera to horseradish peroxidase (HRP) involved activation of the peroxidase with sodium metaperiodate. The excess periodate and by-products were separated from the active peroxidase by gel filtration (Sepha-15 dex C-25) kolan. The activated peroxidase was then reacted with the anti-EB antibody. This reaction was spontaneous. Sodium borohydride was then added αα to stabilize the bond between peroxidase and antibody, then acetone was added to decompose the residual sodium borohydride.

When used according to the invention, the two labeled antibodies were diluted in a diluent containing 50% fetal calf serum, 15% normal human serum, -0.005M EDTA, 0.1% Tween-20 and 0.01% thiomersal in PBS.

It will be understood that a range of detectable labels can be used. The only requirement, of course, is that they must be clearly demonstrable in different ways. Each of a series of isotopes can be used as well as 125 J. It is not necessary for one label or immunoreactant to be radiolabelled and the other enzymatic or fluorescent and that different isotopes themselves be differently detectable. Likewise, all labeled reagent components can equally easily employ different enzymes that require different substrates in order to be detected.

Importantly, any of the antibodies and antigens of hepatitis A and B can be labeled for use in the invention. The only immunochemical requirement is that the labeled 800 1 6 61 -7 components are not complementary to each other or the markings to be demonstrated.

Example III

Simultaneous demonstration of HB Ag and anti-HB

s c 5 Serum samples containing the unknown hepatitis labels were added to individual wells in a reaction dish and a polystyrene bead coated according to Example I was added to each sample and incubated for 2 hours at U5 ° C. The granules were then removed from the wells, washed with water to remove excess reagent and added to 0.2 cm of a liquid phase reagent prepared according to Example II. The solid and liquid phase were incubated at k5 ° C for 1 hour. The solid phase was then removed from the antibody reagent and washed four times with water to remove excess reagent. The 15 washed granules were then placed in test tubes with o-phenylene

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diamine (30 mg in 10 cm 0.1 M citrate buffer with pH 5> 5) and incubated with it for 30 minutes. After this, the enzyme reaction was quenched by the addition of 1 cm 1 M HCl and the tubes visually examined for the presence or absence of color resulting from a reaction of the enzyme label with the substrate. An absence or a slight color indicated the presence of anti-HB in V »the unknown sample which had anti-HBc competed with the labeled mark for the reactive sites on the coated support. Then the radioactivity on the grain was counted in a gamma counter and the CEM numbers recorded. Grain radioactivity indicated presence of HB_Ag in the unknown - _______ S - sample that had adhered to the attached antibody and thus a binding gap for the radiolabeled antibody.

The invention can be practiced in any manner prescribed.

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Claims (12)

A method for simultaneously detecting in a sample of at least two different markers proving exposure to hepatitis virus, the method comprising: a) contacting a sample with a solid phase coated with at least two different, non-complementary immunoreactants that are complementary to the unknown labels; b) incubating this sample with the coated solid phase for 1-2¼ hours; c) separating the coated solid phase from the sample; D) washing the coated solid phase to remove unbound sample; e) contacting the washed solid support with a liquid reagent containing at least two different hepatitis markers or immunoreactants, each selected to either reacts or competes with any of the unknown hepatitis markings, each bearing a demonstrably clear label; f) separating the solid phase from the labeled reagent; g) and determining the presence of labeled tags or immunoreactants on the solid support by demonstrating the appropriate labels. 2. Method for the simultaneous detection in a sample of hepatitis B surface antigen and antibody against hepatitis B core antigen, characterized in that a) the sample is contacted with a solid phase3 coated with both hepatitis B core antigen and antibody to hepatitis B surface amigen, * b) the sample net incubates the coated solid phase for 1 to 2 hours; c) separating the coated solid phase from the sample; D) washing the coated solid phase to remove unbound sample; e) contacting the washed solid support with a liquid reagent containing antibodies to hepatitis B surface antigen as well as hepatitis B core antigen, each labeled with demonstrably different labels, 35 f) separating the solid phase from the labeled antibody reagent and 800 1 6 61 * G -9- g) the presence of labeled antibodies on the solid support "determined by detection of the individual labels. Method according to claim 2, characterized in that the antibody is radiolabeled against surface antigen and the antibody is labeled with enzyme antigen. 4. A method according to claim 3, characterized in that the radioactive label is 125 J and the enzyme horseradish peroxaidaseis. 5. Reagent for the simultaneous detection in a sample of at least 2 different markers indicating exposure to hepatitis virus, characterized in that a solid support is coated with at least two different, non-complementary immunoreactants which are complementary to the unknown marks. 6. Reagent according to claim 5, characterized in that the solid support is coated with hepatitis B core antigen and with an antibody against hepatitis B surface antigen. 7. Reagent for the simultaneous detection in a sample of at least two different markers proving exposure to hepatitis virus, characterized in that this reagent contains at least two different hepatitis markers or immunoreactants, each selected so that they either respond or compete with an unknown hepatitis mark and each is marked with demonstrably different labels. Reagent according to claim 1, characterized in that the reagent contains antibodies against hepatitis B surface antigen and hepatitis B core antigen. Reagent according to claim 8, characterized in that the anti-hepatitis B surface antigen antibody is radiolabelled. 10. Reagent according to claim 9, characterized in that the radioactive mark is 125 J. Reagent according to claim 8, characterized in that the antibody to hepatitis B core antigen is labeled with an enzyme. Reagent according to claim 11, characterized in that this enzyme is horseradish peroxidase. 800 1 6 61