JPH0220945B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to an improved solid phase immunoassay for the detection and measurement of hepatitis antigens and antibodies (nukers).

There are at least two distinct types of viral hepatitis. Hepatitis A is said to be caused by hepatitis A antigen (HAVAg) and is generally characterized by a 2- to 6-week incubation period, mild premonitory, and relatively mild clinical disease. The disease is generally transmitted by contaminated food or liquids, but it is also said to be transmitted by systemic inoculation. Hepatitis A is often referred to as “infectious hepatitis” and is reported to occur annually in the United States.
There are over 50,000 reported cases.

Hepatitis B viruses are believed to be the likely causative agent of "serum hepatitis." Hepatitis B infections are commonly transmitted by blood products or contaminated equipment such as needles, but may also be transmitted by other methods. Traditionally, hepatitis B infectious diseases require incubation periods ranging from 6 weeks to 6 months. However, short-term culture such as 2 weeks has been demonstrated. The disease may be mild or without symptoms, but if symptoms are present, the condition may be particularly severe.
Pre-symptoms include joint pain, arthritis, rash, fever, anorexia, fatigue, and pruritus, which may or may not be accompanied by jaundice.

At least three different antigen-antibody systems _: surface _{( HBsAg} : anti- _HBs ), "core" ( _HBcAg : anti-
_HBc ), and e-antigen _{( HBeAg} : anti- _HBe ) can associate with hepatitis virus B. Hepatitis B surface antigen (HB _s A _g ) is found in the blood as 22 nm spheres and tubules 22 nm in diameter and varying in length and is believed to represent the protein membrane of the hepatitis B virus.

The 42 nm particles with DNA and DNA polymerase appear to represent a contagious virus (Dain particles). The den particle surface is similar or the same as HB _s A _g . Dein particles in surfactants
A core with an electron density of 27 nm, dissociated into HB _c A _g .
The latter is found in the hepatocyte nuclei of patients with serum hepatitis during the true infection stage.

Patients with viral hepatitis B are therefore expected to produce antibodies against surface antigens (anti- _HBs ) and antibodies against the protein core (anti- _HBc ).
_{Serum HBsAg} is consistent with the presence of hepatitis B viruses, and anti- _HBc is also commonly found in severe liver failure during early viralemia.
It often appears with antigenemia (HB _s A _g ) before the appearance of HB _s . Anti-HB _c is generally accompanied by prolonged circulation of HB _s A _g , suggesting that anti-HB _c is produced in response to viral activation.

In 1972 hepatitis e antigen (HB _e A _g ) was detected and characterized. The e marker has been found in _{HBsAg -} positive serum and occurs more commonly in the serum of chronic _HBsAg patients with active _liver disease than in normal healthy individuals. During the culture of hepatitis B, the e-antigen in patients was found to appear immediately after the appearance of _HBsAg or before the _appearance of clinically obvious liver damage. Its presence in the serum would logically indicate poor prognosis and progression of liver damage. Conversely, the presence of e antibodies (anti-HB _e ) may be an indicator of good prognosis. Although these relationships are not absolute, they may be useful for clinical guidance.

2 because serological markers of hepatitis appear in a consistent and sequential order during the course of infection, illness, and recovery.
Alternatively, analysis of three or more markers may be valuable in investigating the time course of the disease.

Testing for more than one marker can also be used to _{evaluate HBs} in blood donors or patients in efforts to reduce the impact of hepatitis B.
Enables an important double check of A _g . This required Goldfield et al. Am.J.Med.Sci.
270:335–342 (1975), the authors carefully investigated recipients of blood negative _{for HBsAg} and found evidence of exposure to hepatitis antigens in 7 of 465 patients. did. Clearly, testing for more than one marker for the hepatitis B virus should reduce or prevent the occurrence of false negatives. Similarly, in many cases 2
A positive response to a test minimizes the chance of a false positive found in one test.

Although the reagents and methods of the present invention are similar to known commercially available products and methods for testing various markers for viral hepatitis, no method has previously been published or suggested for simultaneously testing the aforementioned markers. It is.

Accordingly, this specification details reagents and methods for simultaneously detecting at least two different markers that establish exposure to hepatitis viruses in a sample.

The method involves contacting a sample with a solid support coated with at least two different non-complementary immunoreactants that are complementary to the unknown marker, and allowing the sample to remain with the solid support for up to 24 hours. separate the coated solid support from the sample, wash said support, and incubate the coated solid support for a time of contacting the washed support with a liquid reagent consisting of a hepatitis marker or an immunoreactant, separating the solid phase from the reagent liquid;
and determining the presence of labeled markers on the solid support, each with a different label detection.

In the above, complementary refers to antigens and antibodies that react in the presence of each other, and hepatitis B
The core antigen is complementary to the hepatitis B core antibody. Non-complementary is the opposite of the above,
Hepatitis B surface antigen is non-complementary to hepatitis B core antibody.

The following examples demonstrate the preparation and use of representative reagents within the scope of this invention. The example describes the preparation of a solid support coated with two different non-complementary immunoreactants complementary to a marker for establishing exposure to hepatitis.

Specifically, a solid support (granule) is coated with two different non-complementary immunoreactants to retain their reactivity and to bind complementary markers in an unknown sample.

In the example, polystyrene pellets are HB _s A _g
coated with antibodies against and core antigen, _{HBsAg .} The antibody retains its reactivity and reacts with any HB _s A _g in the unknown sample, and the attached core antigen retains its anti-reactivity and reacts with any antibody against the core (anti-HB _c ) in the unknown sample. . The key to the invention is that both immune reactions occur simultaneously with a single solid phase reagent.

EXAMPLE Preparation of Coated Support Dein particles were exposed to a solution of 2% 2-mercaptoethanol (2-ME) and 5% Tween 80 in Tris-EDTA-salt solution (TSE) buffer for 2 hours at 37°C to produce HB. _s A _g generation was performed. This solution was diluted 10 times with 5% 2ME-TSE and left overnight before being diluted in TSE to a final concentration of 1:8000.
Separately, polystyrene pellets (1/4″) were coated by soaking them in a 1:2000 dilution of anti- _HBs serum (guinea pig) in phosphate-buffered salt solution (PBS) for 2 hours. The core preparation was then injected onto the coated particles and left at room temperature for 2 days to allow the core antigen to adhere to the globules.The granules were removed from the buffer and washed with water to stabilize the deposits. It was coated with 10% sucrose solution and air dried.

Although polystyrene granules are preferred for their ease of coating and handling, any solid support, both large and small, of a variety of plastics, metals, and glasses can be readily coated and used in the demonstration of this invention.

Although globules can be coated first with core antigen and then coated with anti- _HBs , it has been observed that coating the globules first with Guinea pig anti- _HBs serum significantly increases the adhesion of core antigen.

Importantly, other combinations of non-complementary immune reactants for hepatitis viruses can also be used. Complementary affinity is the antigenicity of the reagent.
The immunoreactants must of course be non-complementary as this will reduce reactivity.

The following example describes immunoreactants, radioactive labels (
¹²⁵ I) Anti- _HBs and markers, reactive yeast labeled (wasabi peroxidase) anti-
This shows a method for producing a liquid phase reagent containing _HBc .

Although the terms "marker" and "immunoreactant" can be used interchangeably, "marker" is preferably used to refer to the antigen or antibody to be tested in an unknown sample and its equivalent. The term "immunoreactant" is used to describe either an antigen or an antibody that is complementary to the marker being tested.

Therefore, in the following examples, the antigen and antibody used on the solid support are always relative to one of the unknown markers and are therefore referred to as immunoreactants.

The labeled surface antigen antibody in the liquid phase reagent is complementary to the unknown marker ( _{HBsAg )} and is therefore referred to as a labeled immunoreactant. However, the labeled core antigen is identical to the marker being tested and is therefore referred to as a labeled marker.

Example Labeled Antibody Reagent 0.5M phosphate buffer (PH7.5) in 1/8 oz bottle
Add about 0.1 ml of 5-6 mci of Na ¹²⁵ I and 100 mg of purified anti- _HBs , mix all ingredients and adjust to pH 7.5.
8.0, an antibody _{against HBsAg} (anti- _HBs ) was iodinated ( ^125I ). To this mixture was added 50 μ of a solution of freshly formed chloramine T (35 mg in 10 ml of 0.05 M phosphate buffer, pH 7.5). After addition and mixing, the mixture was reacted at room temperature for 60 seconds. 50μ of freshly generated sodium metasulfite solution (35mg in 10ml of 0.05M phosphate buffer, pH 7.5) was added to the reaction mixture to reduce the chloramine T and stop the reaction.

The reaction efficiency was tested by placing 5μ of the reaction mixture on a piece of Whatman No. 1 paper and chromatographing the mixture in 70% methanol. The iodinated protein remained intact, but the free ¹²⁵ I was transferred to the solvent. The percent of ¹²⁵ I in the protein is determined as the percent reaction efficiency.

Using a Sephadex G-50 gel column, prepare crude iodinated antibodies at pH 7.8 containing 0.9% sodium chloride.
Purified with 0.1M Tris buffer. After pre-treating the column with a small amount of 30% aqueous solution of bovine serum albumin
Treated with an additional equal volume of 0.1M Tris buffer, pH 7.0 containing 0.9% sodium chloride. The iodinated antibody was added to the top of the column and washed out using a Tris solution buffered with a salt solution. The labeled antibody was first obtained from the column as an eluent.

The antibody against hepatitis B core was conjugated with wasabi peroxidase (HRP) by activating peroxidase using sodium metaperiodide. Excess periodide and by-products were separated from active peroxidase by gel filtration (Sephadex G-25) column. The active peroxidase was then reacted with an antibody (anti-HB _c ) and reacted naturally. Sodium borohydride was added to stabilize the binding between peroxidase and antibody, and then acetone was added to decompose the residual sodium borohydride.

When using two antibodies labeled according to the method of the invention, they are mixed with the following composition: 50% fetal bovine serum, 15% normal human serum, 0.005M EDTA, Tween-
20 in a diluent containing 0.1% thiomersal and 0.01% thiomersal in PBS.

It is clear that a variety of detectable labels can be used. The only requirement, of course, is that they can be detected separately.

As ¹²⁵ I is readily available, a variety of isotopes can be used. There is no need for one marker or immunoreactant to be radiolabeled and the other to be labeled with an enzyme or protein since the heterologous isotopes can themselves be detected separately. Similarly, the labeled components of the reagents can all be easily used with different enzymes that require different substrates to yield reaction products that can be detected separately.

Importantly, both hepatitis A and B antibodies and antigens can be labeled by the present invention. The only immunochemical requirement is that the labeled components are not complementary to each other or to the marker being measured.

EXAMPLE Simultaneous detection of HB _s A _g and anti-HB _c Serum samples containing “unknown” hepatitis markers were added to individual wells of a reaction toilet and polystyrene globules coated according to the example were added to each sample at 45°C. It was cultured for 2 hours. The pellets were removed from the reaction wells, washed with water to remove all excess reagent, and added to 0.2 ml of the liquid phase reagent prepared in the example. solid phase and liquid phase
The cells were cultured at 45°C for 1 hour. The solid phase was then separated from the antibody reagent and washed four times with water to remove excess reagent. O
- Washed globules were placed in a test tube containing phenylenediamine (30 mg in 10 ml of 0.1 M citrate buffer, pH 5.5) and incubated for 30 minutes. The enzymatic reaction was then stopped by adding 1 ml of 1M HCl, and the tubes were examined for the presence of color resulting from the reaction of the labeled enzyme with the substrate.
No or faint color indicated that anti-HB _c was present in the unknown sample and competed with the labeled markings of the reaction sites on the coated support. Next, the radioactivity on the small particles was examined using a gamma counter and CMP was recorded. The radioactivity on the pellets indicated that there was _HBsAg in the unknown sample that was attached to the attached antibody and provided the binding site for the radiolabeled _antibody .

The invention may now be carried out in a variety of ways as detailed above, all of which are pointed out in the claims.

Claims (1)

[Claims] 1. (a) A sample is brought into contact with a solid phase coated with both hepatitis B core antigen and an antibody against hepatitis B surface antigen on the same surface; (c) separating the solid phase from the sample; (d) washing the solid phase to remove unbound sample; and (e) individually labeling each hepatitis B sample with a detectable label.
contacting the washed solid support with a liquid reagent containing an antibody to the surface antigen and an antibody to the hepatitis B core antigen; (f) separating the solid phase from the labeled antibody reagent; and (g) attaching a separate label. Hepatitis B surface antigen, characterized in that the presence of labeled antibodies on the solid support is determined by detecting, and Hepatitis B
A method for simultaneously detecting antibodies against core antigens in a sample. 2. The method of claim 1, wherein the antibody to the surface antigen is radioactively labeled and the antibody to the core antigen is enzyme labeled. 3. The method according to claim 1, wherein the radioactive label is ¹²⁵ I and the enzyme is wasabi peroxidase. 4. A solid-phase immunoreagent consisting of a solid support whose same surface is coated with an antibody against hepatitis B core antigen and hepatitis B surface antigen.