EIA test using nondenatured HIV antigen for early detection of HIV infection

Abstract

Recombinant human immunodeficiency virus antigens capable of immunologically identifying the presence of early anti-HIV antibodies are stably expressed in a number of cell lines. These antigens have several clinically important applications as non-hazardous tools in the detection of human immunodeficiency virus exposure/infection, and in screening methods for HIV infection in idiopathic chronic lymphopenia (ICL). These techniques are improved over existing immunologically based and PCR based detection methods, as they provide for the detection of infection/exposure in samples determined to be negative by conventional forms of these types of assays that do not detect anti-HIV gp16O antibodies that react to conformational epitopes of HIV. The invention finds particular application in the detection of human immunodeficiency virus exposure/infection in infants. The earlier detection of the described methods is provided through the preserved immunoreactivity of the described recombinant conformationally intact human immunodeficiency virus that is capable of detecting a class of “early” anti-human immunodeficiency virus antibody not previously detectable by standard Western Blot or ELISA methods. The human immunodeficiency virus gp16O envelope antigen comprises one of the specific recombinant antigens examined with clinical human samples in these improved screening methods.

What is claimed is:  
     
         1 . A recombinant protein comprising a recombinant human immunodeficiency virus envelope protein capable of immunologically binding an early anti-HIV antibody, said protein further defined as having a molecular weight of about 160,000 Daltons as determined by SDS/PAGE.  
     
     
         2 . The recombinant protein of  claim 1  further defined as prepared by a process comprising: 
 preparing a restriction fragment comprising human immunodeficiency virus nucleic acid encoding an envelope protein gp160;  
 inserting said restriction fragment into a vector capable of expressing in a eukaryotic cell;  
 transfecting a eukaryotic cell with said recombinant vector; and  
 collecting a recombinant protein capable of immunologically binding an early anti-HIV antibody.  
 
     
     
         3 . The recombinant protein of  claim 2  wherein the eukaryotic cells in a mammalian cell line, such as CEM or Mu-1-Lu.  
     
     
         4 . The recombinant protein of  claim 3  wherein the eukaryotic mammalian cell is CEM or Mu-1-Lu.  
     
     
         5 . The recombinant protein of  claim 2  wherein the recombinant vector is pLNSX-env.  
     
     
         6 . A recombinant vector comprising a nucleic acid molecule having a sequence encoding a human immunodeficiency virus gp160 protein capable of binding an early anti-human immunodeficiency virus antibody.  
     
     
         7 . A mammalian cell comprising the recombinant vector of  claim 6 , said recombinant vector including a sequence encoding a gp160 HIV protein.  
     
     
         8 . The mammalian cell of  claim 7 , wherein the sequence encoding a gp160 HIV protein is a SalI-XhoI restriction fragment from HIV 213 , HIV AC-1 , HIV C , or a combination thereof.  
     
     
         9 . The mammalian cell of  claim 7  further defined as a CEM or Mu-1-Lu cell.  
     
     
         10 . The mammalian cell of claim  87 , wherein the sequence encoding a gp160 HIV protein is an SalI-XhoI restriction fragment from HIV 213  (ATCC VR2247).  
     
     
         11 . A screening method for detecting early infant infection to human immunodeficiency virus comprising: 
 exposing a biological sample obtained from an infant to a nondenatured human immunodeficiency virus antigen to form an incubation mixture;    monitoring the incubation mixture for the presence of immunocomplex formation with the human immunodeficiency virus; and    identifying the presence of IgA or IgM anti-HIV antibody immunobinding to the recombinant antibody,    wherein the presence of nondenatured human immunodeficiency virus antigen-IgM or IgA antibody immunocomplex formation indicates infection by human immunodeficiency virus in the infant.    
     
     
         12 . The method of  claim 11  wherein the human immunodeficiency virus antigen is a recombinant human immunodeficiency virus gp160 envelope protein obtained from HIV 213  (ATCC 2247).  
     
     
         13 . A method for screening a biological sample for HIV from a patient with Idiopathic Chronic Lymphopenia comprising: 
 obtaining a biological sample from a patient having or suspected of having idiopathic chronic lymphophenia;    exposing said sample to a nondenatured human immunodeficiency virus antigen under conditions that will allow immunocomplex formation between any antibody in the sample and the recombinant protein to provide an incubation mixture; and    identifying the presence of immunocomplex formation in the incubation mixture with the labeled human immunodeficiency virus antigen by anti-antibody secondary reagents,    wherein the presence of immunocomplex formation with antibody in the sample provides a screen for HIV in idiopathic chronic lymphophenia.    
     
     
         14 . An assay plate comprising a multiplicity of microtiter wells comprising a substantially nondenatured and purified human immunodeficiency virus envelope protein capable of binding an early anti-human immunodeficiency virus antibody.  
     
     
         15 . The assay plate of  claim 14 , wherein the microtiter wells are coated with the substantially nondenatured and purified recombinant human immunodeficiency virus envelope protein.  
     
     
         16 . The assay plate of  claim 14 , wherein the substantially nondenatured and purified recombinant human immunodeficiency virus envelope protein is further defined as a recombinant gp160 envelope protein.  
     
     
         17 . A kit for screening a sample for early anti-human immunodeficiency virus comprising: 
 an assay plate as defined in  claim 14;     a container means comprising labeled secondary antibody having binding affinity for anti-HIV early antibody.    
     
     
         18 . A target antigen preparation capable of immunologically binding an early anti-HIV antibody comprising a recombinant HIV envelope protein gp160 obtained from a purified eukaryotic cell lysate, said eukaryotic cell lysate comprising a lysate from HIV infected mammalian cells, said cells being infected with HIV AC-1 , a mammalian cell infected with HIV C  and a mammalian cell infected with HIV 213 .  
     
     
         19 . The target antigen of  claim 18  wherein the mammalian cell lysate is prepared by a process of solubilizing the HIV expressing mammalian cells with about 0.1% to an about 10% digitonin.  
     
     
         20 . The target antigen of  claim 18 , wherein the mammalian cells are CEM cells.  
     
     
         21 . The target antigen of  claim 18 , wherein the mammalian cells are Mu-1-Lu cells.  
     
     
         22 . An assay plate for use in detecting early anti-HIV antibody comprising cells adherent to the assay plate expressing recombinant gp160 from a viral isolate selected from the group consisting of HIV 213 , HIV- AC-1 , and HIV C .  
     
     
         23 . The assay plate of  claim 22  wherein the cells are Mu-1-Lu cells.