proteins considered as antigens in western blotting

• The proteins considered to represent HIV antigens in Western Blotting are obtained from mitogenically stimulated cultures in which tissues from AIDS patients are co-cultured with cells derived from non-AIDS patients-usually established leukaemic cell lines.
• Following the detection of the enzyme reverse transcriptase (RT) in the cultures, the supernatant, and more often the cell lysates, are spun in density gradients.

• Material which bands at 1.16 gm/ml is considered to represent "pure HIV" and consequently the proteins found at that density are considered to be HIV antigens.

• The immunogenic HIV proteins are thought to be coded by three genes, namely gag, pol and env.

• The gag gene codes for a precursor p53/55, which is then cleaved to p24/25 and p17/18. the pol gene codes for p31/32, and the env gene codes the precursor proteins p160 which cleaved to p120 and p41/45. 
• So all the basic Proteins that considered to be antigens in western Blotting are as follows;

p 120 protein
This is the mostly found proteins as antigens in Western Blotting for HIV test,

•The generally accepted view is that p 120 and p41 are cleavage products of p 160 , which is found only in infected cells and not in the virus. However, p 120 is a component only of the knobs (spikes) on the surface of HIV particles; The knobs are found only in the budding (immature) particles; and not in cell free (mature) particles; immature particles are "very rarely observed".

•Despite these findings, when "purified HIV" is tested against AIDS sera, strong bands corresponding to p 120 and p 160 develop. The solution to these contradictions was found when it was shown that p80 (vide infra) and "the components visualized in the 120-160 -kDa region do not correspond to gp 120 or its precursor but rather represent oligomers of gp41".

p41 protein

•p41 is one of the proteins detected by both Gallo's and Montagnier's groups in the first HIV isolates and protiens considered as antigens in western blotting . However, Montagnier and his colleagues observed that AIDS sera reacted with a p41 protein both in HIV and HTLV-I infected as well as non-infected cells, and concluded that the p41 band "may be due to contamination of the virus by cellular actin which was present in immunoprecipitates of all the cell extracts".

•Although Gallo's group did not find such reaction with p41 in non-infected cells, they did find a p80 protein and concluded that the reaction was "non-specific“.

•Actin is an ubiquitous protein which is found in all cells as well as bacteria and several viruses. Well known retroviruses such as the mouse mammary tumour virus and Rous sarcoma virus have also been shown to contain actin of cellular origin and it has been postulated that this protein plays a key role in both retroviral assembly and budding.

•It is also known that oxidation of cellular sulphydryl groups, as is the case in AIDS patients, is correlated with assembly of polymerised actin, and that the level of actin antibody binding to cells is determined by the physiological state of the cells.

•For this reason actin antibody binding to cells has been proposed "as a sensitive marker for activated lymphocytes.

•Platelets from healthy individuals also contain a p 41/45 protein which reacts with sera from homosexual men with AIDS and immune thrombocytopenic purpura (ITP) and which "represents non-specific binding of IgG to actin in the platelet preparation“.

p32 protein

•In 1987 Henderson isolated the p 30-32 and p 34-36 of "HIV purified by double banding" in sucrose density gradients and this protiens also considered as antigens in western blotting. By comparing the amino-acid sequences of these proteins with Class II histocompatibility DR proteins, they concluded that "the DR alpha and beta chains appeared to be identical to the p 34-36 and p 30-32 proteins respectively’’.

 p 24/25 protein

•Detection of p24 is currently believed to be synonymous with HIV isolation and viraemia protiens considered as antigens in western blotting.

•Apart from a joint publication with Montagnier where they claim that the HIV p24 is unique, Gallo and his colleagues have repeatedly stated that the p24s of HTLV-I and HIV immunologically cross-react.

•Genesca et al.(14) conducted WB assays in 100 ELISA negative samples of healthy blood donors; 20 were found to have HIV bands which did not fulfil the then ( 1989 ) criteria used by the blood banks for a positive WB.

•These were considered as indeterminate WB, (WBI), with p24 being the predominant band, (70% of cases). Among the recipients of WBI blood, 36% were WBI 6 months after transfusion, but so were 42% of individuals who received WB-negative samples.

•Both donors and recipients of blood remained healthy. They concluded that WBI patterns "are exceedingly common in randomly selected donors and recipients and such patterns do not correlate with the presence of HIV-1 or the transmission of HIV-1", "most such reactions represent false-positive results";

• Antibodies to p24 have been detected in 1 out of 150 healthy individuals, 13% of randomly selected otherwise healthy patients with generalised warts, 24% of patients with cutaneous T-cell lymphoma and prodrome and 41% of patients with multiple sclerosis.

•Ninety-seven percent of sera from homosexuals with ITP and 94% of sera from homosexuals with lymphandenopathy or AIDS contain an antibody that reacts with a 25Kd membrane antigen found in platelets from healthy donors and AIDS patients, as well as a 25 Kd antigen found in green-monkey kidney cells, human skin fibroblasts, and herpes simplex cultured in monkey kidney cells. This reaction was absent in sera obtained from non-homosexual patients with ITP or non-immune thrombocytopenic purpura.

•Conversely, the p24 antigen is not found in all HIV positive or even AIDS patients. In one study, the polymerase chain reaction (PCR) and p24 were used to detect HIV in patients at various CDC stages from asymptomatic to AIDS. p24 was detected in 24% patients and HIV RNA in 50%.

•In another study, "In half of the cases in which a subject had a positive p24 test, the subject later had a negative test without taking any medications that would be expected to affect p24 antigen levels...the test is clinically erratic and should be interpreted very cautiously".

                                                    

 p 17/18 protein

•In addition to the p24 band, the p 17/18 band is the most often detected band in WB of healthy blood donors protiens considered as antigens in western blotting.

•Sera from AIDS patients bind to a p18 protein in mitogenically stimulated HIV infected T-cells, but not to non-infected, unstimulated lymphocytes.

•However, when the lymphocytes are mitogenically stimulated, but non-infected, the AIDS sera bind to a p18 protein in these non-infected lymphocytes.

•A monoclonal antibody (MCA) to HIV p18, reacts with dendritic cells in the lymphatic tissues of a variety of patients with a number of non-AIDS related diseases; and the "same pattern of reactivity was present in normal tissue taken from uninfected individuals as in those taken from HIV positive subjects".

•AIDS patients and those at risk have high levels of antibodies to the ubiquitous protein-myosin,(22) which has two subunits of molecular weights 18, 000 and 25, 000 . In view of all the above evidence it is difficult to defend the view that the bands p41 (and thus p 160 and p 120 ), p32, p24 or p18 represent specific HIV proteins. Even if it could be shown that all these proteins are HIV specific, it cannot be automatically assumed that antibodies that react with each of these proteins are specific to HIV infection so these protiens are considered as antigens in western blotting.