Para identificar los anticuerpos como anticuerpos "reales" del VIH, se realiza una segunda prueba en la misma muestra de sangre mediante la técnica "Western Blot". Esta prueba visualiza la reacción de los anticuerpos frente a proteínas estructurales independientes del VIH.
- El Western Blot detecta anticuerpos a una serie de proteinas
NINGUNA DE LAS CUALES es específica del VIH.
Proteins Considered to be HIV Antigens
The proteins considered to represent HIV antigens 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. amowing the detection of the enzyme reverse trans.criptase (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 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 protein p160 which is cleaved to p120 and p41/p45. (1)
The p120 protein.
The generally accepted view is that p120 and p41 are cleavage products of p160, which is found only in infected cells and not in the bichito. However, p120 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".(2)
Despite these findings, when "purified HIV" is tested against AIDS sera, strong bands corresponding to p120 and p160 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 gp120 or its precursor but rather represent oligomers of gp41".(3)
The p41 protein.
p41 is one of the proteins detected by both Gallo's and Montagnier's groups in the first HIV isolates. 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 bichito by cellular actin which was present in immunoprecipitates of all the cell extracts".(4) 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"(5).
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 bichito and Rous sarcoma bichito 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.(6,7) It is also known that oxidation of cellular sulphydryl groups, as is the case in AIDS patients (8), is correlated with assembly of polymerised actin (9), 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"(10).
Platelets from healthy individuals also contain a p41/45 protein which reacts with sera from gays men with AIDS and immune thrombocytopenic purpura (ITP) and which "represents non-specific binding of IgG to actin in the platelet preparation"(11).
The p32 protein.
In 1987 Henderson isolated the p30-32 and p34-36 of "HIV purified by double banding" in sucrose density gradients. By comparing the amino-acid sequences of these proteins with Class II histocompatability DR proteins, they concluded that "the DR alpha and beta chains appeared to be identical to the p34-36 and p30-32 proteins respectively"(12).
The p24/25 protein.
Detection of p24 is currently believed to be synonymous with HIV isolation and viraemia. However, 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 (13);
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.(15)
Ninety-seven percent of sera from gayss with ITP and 94% of sera from gayss 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-gays patients with ITP or non-immune thrombocytopenic purpura.(11)
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%.(16)
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".(17)
The p17/18 protein.
In addition to the p24 band, the p17/18 band is the most often detected band in WB of healthy blood donors.(18)
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.(19)
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;(20) and the "same pattern of reactivity was present in normal tissue taken from uninfected individuals as in those taken from HIV positive subjects".(21)
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 p160 and p120), 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.
- Cada una de las bandas del Western Blot corresponde a una proteina diferente. El diagnóstico VIH-positivo o VIH-negativo se decide según criterio del número de bandas que se oscurecen y las proteinas que contienen.
- El criterio varía de pais a pais, por ejemplo, en Africa un paciente es seropositivo con solo DOS bandas, en Inglaterra se necesiten TRES bandas, en Australia tienes que dar un mínimo de CUATRO bandas.
- El Western Blot por tanto tampoco es un test específico, ni reproducible, ni científico, y conduce al absurdo de que un "infectado" africano se "cura" de su infección si toma el avión y se hace de nuevo la prueba en Australia.
Standarisation of HIV Antibody Tests
An antibody test becomes meaningful only when it is standardised, that is, when a given test result has the same meaning in all patients, in all laboratories, in all countries. From the first antigen-antibody reactions performed by Montagnier's (4) and Gallo's (23) groups (fig.1, 2) it was found that: not all of the "HIV proteins" react with all sera from AIDS patients or even sera from the same patients obtained at different times; and that sera from AIDS patients may react with proteins other than those considered to be HIV antigens. Because of these variable reactions, an essential requirement was to establish criteria as to what constitutes a positive WB.
Initially, Montagnier's group considered p24 sufficient to define a positive WB, whereas Gallo's group considered p41 sufficient. Most, if not all other laboratories, used the criteria recommended by the CDC, namely the presence of a band at either p24 or p41. By 1987 it became obvious that those bands were not HIV specific. Furthermore, till 1987 "there were as many WB procedures as there were laboratories doing the assay".(24) Since then, all major laboratories have changed their criteria for WB interpretation but in the United States there are still no nationally agreed criteria, even among the major laboratories:
In 1987 the Food and Drug Administration (FDA) licensed a WB kit manufactured by DuPont. The DuPont kit remains the only licensed WB kit and is used by a minority of laboratories. It specifies "extremely stringent" criteria for a positive result namely "specific bands representing three different gene products: p24 (gag), p31 (pol), and an env band, either gp41, gp120 or gp160" (24).
The American Red Cross defines a positive result as presence of antibodies to at least one gene product from each of the gag, pol and env genes, without specifying which bands.
The Association of State and Territorial Public Health Laboratory Directors/Department of Defence/CDC consider a WB positive if two out of p24, gp41 and gp120/160 are reactive.
The Consortium for Retrovirus Serology Standardization (CRSS) defines a positive WB as the presence of antibodies to at least p24 or p31/32, and gp41 or gp120/160 (25).
All the other major USA laboratories for HIV testing have their own criteria. For all laboratories, a negative result requires the absence of any and all bands including bands which do not represent "HIV proteins". All other patterns which do not satisfy a given laboratory's criteria for a positive or negative test are regarded as WBI by that laboratory.
Thus, in the scientific literature, no strips have been published of a standard positive WB. Patient samples may show varying degrees of reactivity with different proteins, thus showing different band development patterns...Each laboratory performing Western Blot testing should develop its own criteria for band interpretation. Alternatively, band interpretation may be left to the clinician".
In addition to the obvious problems associated with the lack of standardisation, all of the above interpretations possess major problems:
When the FDA criteria are used to interpret the WB, only a minimal number (less than 50%) of AIDS patients have a positive WB, that is, are infected with HIV. If the criteria of the CRSS are used, the percentage of AIDS patients testing positive increases to 79%.
As already mentioned, Henderson and his colleagues have shown that p31/32 is a non-HIV protein. Pinter and his colleagues have shown that p160 and p120 are oligomers of gp41. They have also shown that the WB pattern obtained is dependent on many factors including temperature and the concentration of sodium dodecyl sulphate used to disrupt the "pure bichito", and concluded:
"Confusion over the identification of these bands has resulted in incorrect conclusions in experimental studies. Similarly, some clinical specimens may have been identified erroneously as seropositive, on the assumption that these bands reflected specific reactivity against two distinct viral components and fulfilled a criterion for true or probable positivity. The correct identification of these bands will affect the standards to be established for Western Blot positivity: it may necessitate the reinterpretation of published results"(26).
The finding that the p31/32 band represents a cellular protein, and that p120 and p160 are oligomers of p41, reduces the criteria of the CRSS and that of the American Red Cross to two bands, p24 and p41, which according to Colonel Donald Burke are "less than perfectly specific",(27). The above findings reduce the criteria of the Association of State and Territory Public Health Laboratory Directors/Department of Defence/CDC to p24 or p41, generally accepted as being non-specific.
Despite the above evidence, even at present, the p160, p120 and the p41 bands are considered to represent distinct viral envelope glycoproteins. In fact, the current WHO guidelines consider a serum positive for HIV-1 antibodies if "two envelope glycoprotein bands (with or without) other viral specific bands are present on the strip".
Las instrucciones de las pruebas para administrar el Western blot advierten: "
No use esta prueba como la única base para el diagnóstico de la infección por VIH-1" (Epitope Organon Teknika).
En forma similar, las instrucciones que acompañan a los reactivos de una prueba frecuentemente usada para la PCR o "Carga Viral" advierte:
"la prueba de ampliación genética para monitorizar al VIH-1 no está prevista para ser usada como una prueba rastreadora del VIH ni como prueba diagnóstica para confirmar la presencia de infección por VIH" (Roche 2003).
Por tanto, las compañías farmacéuticas fabricantes de los reactivos para estas pruebas reconocen el hecho de que ni la prueba de ELISA, ni la de Western blot, ni la de "Carga Viral" para VIH
son específicas para diagnosticar la infección por VIH.
El sindrome de inmunodeficiencia puede deberse a varios motivos y el vih puede ser uno de ellos.
No puede ser causa de inmunodeficiencia un supuesto agente que falla todos los postulados de Koch.
Para ver si es vih o no, es necesario una segunda prueba para detectar las proteinas del bichito y confirmar la existencia del patógeno.
Ya hemos visto que ninguna prueba es específica, como reconocen los propios laboratorios. Por tanto ninguna prueba sirve para "confirmar" a otra porque todas padecen del mismo problema. Hoy en dia NO HAY UN ESTÁNDAR frente al que confirmar una infección por VIH.
Hay fotografías del patógeno infectando linfocitos. Fotos que vosotros eruditos llamáis "manchas".
Sin concentrar, aislar y caracterizar esas partículas nadie puede deciur qué son. Vosotors afirmáis que es VIH porque queréis vender antivirales.
Por si fuera poco vais mucho más allá y afirmais que dichas partículas son patógenas... pasándoos por el forro de los narices los cinco postulados de Koch.
Sois vendedores de crecepelo modernos, armados de pruebas pseudocientíficas que no detectan lo que dicen detectar, que atribuís causas virales a enfermedades que no lo son con el fin de vender un producto que no tiene salida, INCLUSO A GENTE SANA a la que acabáis asesinado por administrarles quimioterapia de enfermo terminal (AZT).
