A Novel Ultra Sensitive HIV-1 p24 Antigen Assay
Franz Steindl, Thomas Schlederer#, Martin Purtscher, Ali Assadian, Andrea Buchacher,
Christine Schmatz,Willibald Steinfellner, Christa Tauer, Hermann W.D. Katinger
Institut für Angewandte Mikrobiologie
Universität für Bodenkultur
Nußdorfer Lände 11 A- 1190 Wien
Fax: +43 1 3692924-400 E.mail: iam@mail.boku.ac.at
# MEDIATORS Diagnostika Ges mbH, Dragonerweg 21 1220 Wien
INTRODUCTION
Determination of HIV-1 p24 antigen is a way to evaluate the amount of HIV-1 virus in blood at the time of sampling. Recently published data of two independend groups show that HIV-1 virions are produced countinuously in the blood of HIV-infected persons. Nevertheless the amount of circulating viral antigen is kept low most time and for a long period by the immunodefense mechanism compared to other infectious diseases. An further problem is, that the humoral response to the HIV-1 core proteins is individually and time dependend different. Therefore the availability of p24-protein in an assay (in collected plasma or serum samples, etc.) is also dependend on the amount of formed immuno-complexes and the characteristics of the antibodies involved. To date there are no satisfying methodes published to dissolve such immuno-complexes quantitative in all cases. We developed a new highly specific and sensitive monoclonal HIV-1 p24 antigen assay and a new method which enables a quantitative dissociation of immuno-complexes suitable for different sample matrixes.
PRINCIPLE OF THE TEST
The IAM HIV-1 p24 Antigen Assay is based on the IAM HIV-1 Antigen Assay. The detection of p24 Antigen is performed as double antibody sandwich ELISA. The assay involves two different monoclonal antibodies, one (IAM-24 M01) coated on the ELISA solid phase and the other (IAM-24 37G12) conjugated with biotin. In the next step streptavidin- ß-galactosidase is bound to the biotinylated antibody. ß-galactosidase sets a fluorophor free that gives red colour and shows the presence of HIV-1 antigen.
Fig.1: Schematically representation of the test design and test procedure
MATERIALS AND METHODS
Developement of the IAM-HIV1 p24 antigen Assay
Out of a number of monoclonal IAM anti HIV-1 p24 antibodies (hybridoma cell lines were established by Andrea Buchacher and Renate Predl (3)) the most affine combination of antibodies recognizing different epitopes on p24 antigen was selected. The standard p24-protein is derived from cell culture (H9 cells infected with MN-isolate) and inactivated according to the FDA and NIH guidelines. The second antibody IAM-24 37G12 is biotinylated using NHS-Biotin (Fa. Amersham). As detection system StrepAvidin*ß-Galactosidase (Fa. Boehringer Mannheim) and as substrate Resorufin-ß-D- Galactopyranoside (Fa. Boehringer Mannheim) is used. The design of the test and the procedure are schematically shown in Fig.1.
Developement of a new immuno-complex (IC) dissociation procedure (F.Steindl):
A lot of different substances was tested for their potency to free HIV-1 p24 antigen from antibody- antigen in a non denaturated form. This intensive screening resulted in a aqueous solution of two components named Miraculum (patent pending). To dissolve IC`s quantitatively samples are prediluted 1:3 with Miraculum and incubated 4 min in an boiling water bath. Ajusted to the protein content of samples the Miraculum is used e.g.: undiluted for human plasma 1: 2 prediluted with distilled water for human sera or 1:8 prediluted for cell culture supernatants containig 10% fetal calf serum.
Acid dissociation (AD) (4):
The samples were diluted 1:2 (100µl sample+100µl buffer) with 1,5 M Glycine/HCl buffer pH = 2,0; mixed thoroughly and incubated for 1h at 37oC. Then the samples were neutralized by adding 100µl 1,5M Tris/HCl buffer pH = 8,5.
Heat treatment (HT) (5):
Samples were prediluted 1:3 with a 0,5% Triton X-100 solution and 5 min incubated in a boiling water bath.
P24-spiking:
HIV-1 negative plasma, sera and cell culture medium containing 10% FCS were spiked with 600 pg cell culture derived p24 protein/ml.
IC-formation:
To aliquotes of the p24-spiked plasma, sera and cell culture medium different p24-specific monoclonal antibodies were added (to a final concentration of 1µg/ml). These samples were mixed thoroughly and incubated for 2h at 37oC to form IC`s.
Results and Discussion
The IAM HIV-1 p24 antigen assay is an ultra sensitive and specific ELISA. The chromogenic ELISA-Version gives a sensitivity of at least 2pg p24 protein/ml, the Luminescence Version 0,2 pg/ml. The use of monoclonal antibodies guarantees a persistent consistency of this Test Kit. The good linearity within the standard range (200 -1,5 pg p24 /ml) (Fig.2, Fig.3 and Fig.4) proves the high quality of the antibodies and an optimal test design. The new invented method for a nearly quantitativ freeing of p24 protein bound in IC`s is general effective and enables to detect total p24 antigen present in patient sera or plasma within the sensitivity range of this test. To illustrate the individually differences of free and in IC`s bound p24 present in HIV-1 antibody positive sera results of number of sera tested untreated and Miraculum treated is listed in table 1. To demonstrate the potency of the Miraculum treatment we compared this method with other methods AD and HT (AD = Acid dissociation and HT = simple boiling of samples 1:3 diluted in destilled water or 0,5% Triton X-100 solution) used to date. For HIV- positive sera or plasma are undefined samples, human HIV-1 negative plasma, sera and cell culture medium containing 10% FCS were spiked with cell culture derived p24 protein. The amount of p24 recovered after treatment with the 3 different methodes is shown in Fig. 5. Fig.6 and Fig. 7. To demonstrate the sucess of the 3 method in freeing antigen bound in IC`s we added to aliquotes of the p24-spiked plasma, sera and cell culture medium different p24-specific monoclonal antibodies (1µg/ml). These samples were mixed thoroughly and incubated for 2h at 37oC to form IC`s. The recovery of p24 in human serum using the different methods is shown in Fig.8 - Fig.11. Results obtained with human plasma and cell culture medium as sample matrix showed that the HT-method is less suitable for plasma and the AD-method works not good in cell culture samples. The Miraculum treatment avoids the dissadvantages of AD method (acid protein precipitation, uncomplete dissociation, possible rebinding after neutralization, etc.) and of the simple HT method (protein coagulation).
CONCLUSION
The IAM HIV-1 p24 antigen assay is an easy to perform and sensitive test. The new Miraculum treatment enables to recover at least 90% of p24-antigen bound in IC`s in human plasma, sera or cell culture supernants independent of the characteristics of the antibodies involved. We think that our developements presented here may increase the relevancy of p24-antigen as parameter or surrogate marker in HIV-1 infection and progression of the disease.
References
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Rapid turnover of plasma virons and CD4 lymphocytes in HIV-1 infection. Nature 373, 123-126 (1995).
3.Buchacher, A., Predl. R., Strutzenberger, K., Steinfellner, W., Trkola, A., Purtscher, M., Gruber, G., Tauer, C., Steindl, F., Jungbauer, A., Katinger. H.W.D.;
Generation of Human Monoclonal Antibodies against HIV-1 Proteins; Electrofusion and Epstein-Barr Virus Transformation for Peripheral Blood Lymphocyte
Immortalization. AIDS Research and Human Retroviruses 10, 359-369 (1994).
4. Ascher, D., Roberts, C. and Fowler, A.;
Acidification-modified p24 antigen capture assay in HIV seropositives. Journal of. Aquired Immune Deficiency Syndromes 5, 1080-1083 (1992).
5.Schüpach, J. and Böni, J.;
Quantitative and sensitive detection of immune- complexed and free HIV antigen after boiling of serum. Journal of Virological Methods, 43, 247-256 (1992).
Distributor
The IAM HIV-p24 antigen assay is distributed by:
MEDIATORS Diagnostika Ges mbH,
1220 Wien Dragonerweg 21
Tel.: +43 1 280 4950 or + 43 1 369 292 4447 Fax.: +43 1 280 4950 or + 43 1 369 292 4457
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03/17/95 by IAM
