DE3202559A1 - Method for the detection of IgM antibodies against hepatitis A virus and reagent for carrying out immunoassays - Google Patents

Description

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The invention relates to the field of immunoassays for detection of IgM against hepatitis Α virus. According to the invention, a method for improving the specificity of the detection is provided provided by IgM against hepatitis Α virus in human serum or plasma.

During the acute phase of a hepatitis Α infection, antibodies of the IgM type against hepatitis appear in the patient's serum Α virus (anti-HAV IgM). These antibodies persist even during the early recovery phase at. They cannot be detected in the serum of normal patients. This enables detection of anti-HAV IgM a diagnosis of acute hepatitis A infection.

There are different ways of detecting class-specific Known antibodies; See, for example, U.S. Patents 4,020,151, 4,048,298, and 3,904,367, Bradley et al., Journal of Clinical Microbiology, May 1977, pp. 521-530, Lancet, 23- September 1978, p. 684, Hepatitis Scientific Memorandum, November 1978, p. 3 and Hepatitis Scientific Memorandum, February 1979, p. 2. All of the aforementioned references indicate possibilities for the detection of class-specific antibodies. In DE-OS 29 30 562 is a 3-step solid phase immunoassay for the detection of IgM is described. In the latter determination, an anti-human IgM (specific for the u chain) coated Plastic beads one after the other with the serum sample, hepatitis A virus antigen and labeled antibody against hepatitis A virus antigen incubated. According to the invention, this method is improved by using the for the, u chain specific anti-human IgM coated solid support or the test sample with a dilute aqueous solution of human hepatitis negative serum, plasma or an IgG containing fraction thereof treated.

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The invention thus relates to an improved method for Detection of the acute phase of a hepatitis Α infection by measuring the IgM antibodies against hepatitis A virus antigen. As mentioned earlier, the improvement is that you can test the test sample with an aqueous solution that is hepatitis negative Contains serum, plasma or an IgG containing fraction thereof, diluted. It was found according to the invention that a 100- to 200-fold dilution of the test sample with a diluent that is about 0.1 percent Hepatitis negative human serum or plasma contains the specificity of a solid phase determination method in which a solid support coated with anti-human IgM is used as a reagent, is significantly improved. It will Reagent in the form of the solid carrier reacted with the diluted test sample, washed, reacted with hepatitis Α antigen, washed, reacted with labeled hepatitis Α antibodies, washed and then the amount of marking on the solid support is measured. Another option is to use the fixed Carrier with a dilute aqueous solution of hepatitis negative human serum, plasma or an IgM containing Fraction from pre-treating. In the latter embodiment, the treatment is with an IgG containing Group preferred.

Be for the preferred embodiment of the invention manufactured the following reagents:

With anti-; u coated beads:

Goat antiserum against human IgM, specific for the u chain, was obtained from Litton Bionetics. Polystyrene beads (6 mm) were treated with the antiserum for 2 hours, which was in 0.01 m Tris buffer was diluted from pH 9.0 to 10 "" * * coated, then washed and air dried. The beads were stored at 4 ° C until used.

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Hepatitis Α virus (HAV) antigen solution:

HAV was derived from the liver of acute stage tamarin marmosets obtained from hepatitis A '. Liver extracts were made. The viral infectivity was inactivated with formalin. The extracts were mixed with 0.01 M phosphate buffer (pH value 7.0) diluted in normal saline (PBS) containing 5 millimolar EDTA and 0.1 percent sodium azide, so that suitable counting results were obtained during the determination.

¹²⁵ J-anti-HAV:

IgG was determined from sera by chromatography on DEAE cellulose obtained from patients in the recovery stage who had high titers of anti-HAV. It was carried out according to the procedure of

125
Greenwood J radiolabelled. The iodinated antibodies were diluted to approximately 5 x 10 counts per minute (cpm) in beef serum containing PBS, normal human serum, EDTA and sodium azide.

First dilution solution;

The samples to be examined were initially each in normal saline solution (0.9 percent sodium chloride) or with Phosphate buffered saline solution (PBS, 0.01 M sodium phosphate, pH 7.2 in 0.9 percent saline solution) to the Diluted 200 times.

Second dilution solution (sample dilution):

A second solution was used to further dilute the samples. An aliquot of the first solution of the sample in Saline or PBS was added to the reaction well and 21 times with a Diluted solution of the following composition:

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0.1 percent normal human serum or plasma, 10 percent fetal calf serum, 0.05 m EDTA (ethylene diamine tetraacetate), 0.2 percent Tween-20 and 0.1 percent sodium azide in 0.01 PBS, pH 7.4.

Assay controls:

Positive controls were obtained from sera from patients with acute or early recovery hepatitis A. Negative control samples were obtained in the same manner from sera negative for anti-HAV.

Test procedure:

The test procedure can be summarized as follows:

1. Bead * anti μ + IgM -> Bead * anti ^ -IgM

2. beads * anti; u-IgM + HAV -> Beads * anti; u-IgM-HAV

3. Beads * anti, jj-IgM-HAV + ¹²⁵ J anti-HAV -> beads * anti, U-IgM-HAV- ¹²⁵ J-anti-HAV.

The tests including positive and negative controls were carried out according to the following procedure:

1. Add 10 ^ 1 sample to 2 ml of normal saline solution.

2. Each 10 ^ jI control or diluted sample in a well pipette into a reaction plate.

3- Add 20O _x Ul sample dilution to the wells and mix by gently poking the plate.

4. In each well one coated with antibody to human IgM Give beads.

5. Cover the plate and mix by tapping. Incubate for 2 hours at room temperature.

6. Aspirate the liquid and then wash each bead 2 times with 5 ml of water.

7. Add 200 ^ 1 HAV solution to each well. Cover up and Incubate for 18 to 22 hours at room temperature.

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8. Repeat step 6.

9. Add 200 μΐ anti-HAV J to each well. Cover and incubate for 4 hours at 45 ° C.

10. Repeat step 6.

11. Put the beads in counting glasses and put them in a suitable one Count the counter for 1 minute.

12. Average cpm of positive and negative Determine control. Calculate the positive-negative Gutoff by taking 1 / 1.0 of the mean of the positive Control added to negative control, e.g.

χ PO + x NC = cutoff. (PC = positive control, NC = 10 negative control).

A sample is judged positive if the cpm values correspond to or exceed the cutoff value. 13 · To check the test, the positive / negative ratio Determine (P: N) using the net cpm values for each control. On a valid attempt the P: N value must be> 5.0.

The influence of sample dilution and the presence of normal human plasma (NHP) on hepatitis A IgM specificity is shown in Table I.

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Table I. Sample dilution

high content of IgM anti-HAV

low content of IgM anti-HAV

Patient I IgG anti-HAV

Patient II IgG anti-HAV

no NHP in the diluent

1:50

6.32

5.17

1: 200

0.1% NHP in the diluent

1:50

5.1

6.61 +

1: 200

* S: CO + / o S: CO + / o S: CO + / o S: CO + / o

2.69 + 4.80 + 2.33 +

1.10 + 1.03 + 0.85 ο 0.54 ο

1.10 + 0.84 ο 0.96 ο 0.63 ο

* S: C0 = sample / cutoff ratio

In patients I and II it was found that high or increased IgG anti-HAV concentrations lead to false positive Results can give false positive results but by diluting with a diluent, containing 0.1 percent normal human plasma can be eliminated. Thus, by diluting 50 to 500 times, one becomes Sample with normal saline solution diluted 10 to 25 fold with a solution containing 0.1 percent Hepatitis-negative human serum or plasma reduces the sensitivity of the IgM / IgG ratio of that coated with human anti-IgM solid support increased · Those skilled in the immunoassay art will understand that those with anti-human IgM coated solid supports with the aqueous solution of hepatitis-negative serum, plasma or an IgG fraction of which can be implemented before the start of the test procedure or, as described above, during the test procedure.

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In general, only very small amounts of hepatitis negative serum or plasma are required to achieve the IgM / IgG sensitivity ratio to increase the reagent.

End of description

Claims (3)

Method for the detection of IgM antibodies against hepatitis Α virus and reagent for implementation of immunoassays Claims / 1.y Method for the detection of IgM antibodies against hepatitis A virus, in which an anti-human IgM coated solid carrier reacted with the test sample, washed, reacted with hepatitis Α antigen, washed, reacted with labeled antibodies against hepatitis Α antigen, washed and the amount of to the solid support Bound labeled antibody is measured, characterized in that the test sample with a diluted aqueous solution of hepatitis-negative human serum, plasma or an IgG-containing fraction thereof, - 1 - I * V «■». _w "*,". ",. """» - ² - 2. Method of improving the IgM / IgG sensitivity ratio a reagent for the detection of IgM against hepatitis Α virus in a test sample, wherein as a reagent a solid support coated with anti-human IgM is used is, characterized in that the anti-human IgM coated solid support with an aqueous Bringing solution of hepatitis-negative serum, plasma or an IgG-containing fraction thereof into contact. 3. Immunoassay reagent containing one with anti-human IgM coated solid support, characterized in that it is additionally treated with a dilute aqueous solution of a Hepatitis negative human serum, plasma or an IgG containing fraction thereof has been treated.