The Western Blot Test

Dec 22 2008

The Western Blot test places multiple proteins (antigens) believed to be from HIV in a line on a test strip (according to molecular weight). Serum is added to see which antibody/antigen reactions occur. This test allows not only positive or negative results, but also ‘indeterminate’, where at least one protein reactions, but not enough to be considered positive. There are many different interpretations of this test, so it is possible to be positive under one interpretation and indeterminate in another, used by a different organization or in a different country. Since the Western Blot is an antibody test, like the initial ELISA test, it is not fully independent, and the very common use of this as a confirmatory test for ELISA is thus questionable.

“The clinical and public health implication of HIV Western blot (WB) indeterminate results is yet to be appraised in sub-Saharan Africa, including Nigeria. Using HIV Tri Line Test enzyme-linked immunosorbent assay (ELISA), 1286 patients (600 males and 686 females; age range, 5-60 years) with symptoms suggestive of HIV infection were screened. A total of 1020 (79.3%) of the patients…were HIV seropositive…Western blot analysis of sera from the 1020 HIV-seropositive individuals using the BIO-RAD NEW LAV-BLOT I specifying World Health Organization (WHO) interpretive criteria, confirmed the HIV serostatus of 815 (79.9%) of them with 205 (20.1%) individuals having indeterminate results…Patients aged 11-20 years old recorded the highest percentage of indeterminate results (31.7%) while those aged 21-30 years recorded the least (14.2%)…Result confirmed the limitation of Western blot assays in HIV confirmatory serodiagnosis. After obtaining HIV indeterminate Western blot result, clinicians should consider the total profile for the patient, reassess risk factors for HIV infection, perform a HIV retesting at 3-month intervals for 6 months or use an alternate HIV antibody confirmatory assay and running antibody tests for other human retroviruses.”
Uneke CJ et al. Western blot-indeterminate results in Nigerian patients HIV serodiagnosis: the clinical and public health implication. AIDS Patient Care STDS. 2007 Mar;21(3):169-76.

“Specimens that have bands present [on Western Blot] but do not fulfill the criteria for positivity are called Western blot indeterminate, and a follow-up specimen should be requested, usually collected three to four weeks after the initial specimen. In follow-up, patients will either show a definitive pattern indicating that they have seroconverted or will demonstrate the same banding pattern as previously observed. In the latter circumstance, the vast majority of these patients are HIV-negative and have nonspecific antibody. In these cases, if the patient is considered to be at risk or is particularly anxious, a qualitative PCR may be recommended to confirm that the patient is truly HIV-negative. Because these indeterminate banding patterns may be seen in patients who are not infected, the Western blot does not make a good screening test for HIV.”
Fearon M. The laboratory diagnosis of HIV infections. Can J Infect Dis Med Microbiol. 2005 Jan;16(1):26-30.

“[According to Australian guidelines] WB [Western Blot] indeterminate group 1 [occurs with] Reactivity to viral proteins, but not to p18, p24 or any envelope glycoproteins. WB indeterminate group 2 [occurs with] Reactivity to viral proteins including p18, but not to p24 or any envelope glycoproteins. WB indeterminate group 3: Reactivity to viral proteins including p24 but not to any envelope glycoproteins. WB indeterminate group 4: Reactivity to envelope glycoproteins but to less than three other viral proteins…all WB indeterminate reactivity should be followed up for 12 weeks…”
Guidelines for interpreting HIV testing results. Australian National Reference Laboratory. 2004

“C135 is the longest infected recipient [via blood transfusion] (over 20 years)…[and] has maintained an indeterminate HIV-1 Western blot since infection and triple-nested PCR was required to detect HIV-1 DNA”
Birch MR et al. An examination of signs of disease progression in survivors of the Sydney Blood Bank Cohort (SBBC). J Clin Virol. 2001 Oct;22(3):263-70.

“Persons demonstrating antibodies to HIV-1 should be referred for medical evaluation, which may include testing by other techniques…Accurate diagnosis of HIV-1 infection is important in determining an individual’s risk for developing AIDS. Accuracy is complicated by false-positive and false-negative (EIA) results…Slight ambiguities exist in the designation of the molecular weights of the HIV-1 antigens [used in this test]…Although a [Western] blot POSITIVE for antibodies to HIV-1 indicates infection with the virus, a diagnosis of Acquired Immundeficiency Syndrome or AIDS can only be made clinically if a person meets the case definition of AIDS established by the CDC. POSITIVE blot results using any specimen type (serum, plasma, or urine) should be followed with additional testing [even though the Western Blot test is usually claimed to be a ‘confirmatory’ test]…The clinical implications of antibodies to HIV-1 in an asymptomatic person are not known. However, a larger proportion of such persons have virus detectable in their peripheral blood and some will develop immunodeficiency. INDETERMINATE blots should not be used as the basis for diagnosis of HIV-1 infection…A negative blot does not exclude the possibility of infection with HIV-1.”
Human Immundeficiency Virus type 1 (HIV-1) Western Blot kit. Cambridge Biotech. 1998 Jun 2…dgeHIVUrine.pdf

“Confirmatory western blot analyses were performed in urine samples of all the ESNs [exposed to HIV, but sero-negative], their HIV-seroopositive sexual partners, and low risk controls…a gp41 monoband and other HIV bands (p68, p55, p52, p34, p25, p18), but not gp120 and/or gp160, were detected in urine samples from most of the ESNs [how could uninfected people have antibodies to HIV proteins?]”
Mazzoli S et al. HIV-specific mucosal and cellular immunity in HIV-seronegative partners of HIV-seropositive individuals. Nat Med. 1997 Nov;3(11):1250-7.

“HIV serology was determined using enzyme-linked immunosorbent assay (ELISA) and confirmed by Western blot. Subjects were categorized as positive in accordance with current [CDC] criteria [for Western Blot] if positive for p24 or p31 plus gp41 or gp120/160 until 1 November 1989, or if positive for two or more of p24, p41 and gp120/160 after 1 November 1989.”
Moss AR et al. HIV seroconversion in intravenous drug users in San Francisco, 1985-1990. AIDS. 1994 Feb;8(2):223-31.

“The specificity of immunoassays for detecting HIV antibodies has major shortcomings. Almost all reactions, especially in low risk populations, represent false-positive results. This has been observed with Western blot (WB), which is widely used as a confirmatory test…A serum panel of 21 blood-bank donors and two serum samples from patients with primary Sjögrem’s syndrome were analysed…All sera were positive on two commercial WB assays, as well as on home-made blots using recombinant p24 proteins. All sera appeared to be HIV-negative on a third-generation ELISA. Specimens of these sera were also HIV-1-antigen negative and [by] PCR. Infection could not be detected after cocultivation of donor-derived lymphocytes with permissive cells. Furthermore, in a retrospective study of blood donations, seroconversion was not observed in any of the recipients of this blood. We performed a radioimmunoprecipitation assay (RIPA) using recombinant p24 proteins…No reaction was observed…herpes simplex virus type 1 (HSV-1), strain 17, may be the agent that caused the cross-reactivity in…two cases.”
Langedijk JP et al. Identification of cross-reactive epitopes recognized by HIV-1 false-positive sera. AIDS. 1992 Dec;6(12):1547-8.

“HIV-1 antibodies were detected in blood and semen of all 28 sample pairs. Blood and semen samples of three known seronegative controls were consistently negative…Western blot analysis of 13 paired samples showed different blood and semen IgG antibody patterns…Antibody reactivity against the gp160 band was almost as intense in seminal plasma fractions as in blood plasma, whereas antibody reactivity against p55, p24, and p17 bands was undetectable when seminal plasma was run at dilutions comparable to those of blood plasma.”
Wolff H et al. A comparison of HIV-1 antibody classes, titers, and specificities in paired semen and blood samples from HIV-1 seropositive men. J Acquir Immune Defic Syndr. 1992;5(1):65-9.

“The initial study group consisted of 1,129 addicts (949 men and 180 women) consecutively admitted to the National Institute of Mental Health’s former Clinical Research Center at Lexington, KY, between May 15, 1971, and May 14, 1972…The WB results from the earlier study, which had employed a technique enhanced by the use of an avidin-biotin system, were reanalyzed. The Centers for Disease Control issued diagnostic criteria in 1985 recommending that a WB be considered positive if either band p24 or band gp4l was present alone or in combination with other bands. On rereading, blots with isolated p24 bands were considered to be indeterminate. One 1985 WB, with bands at both the 24 and 55 kilodalton regions, was included among the positives, since the interpretation of this pattern had previously been unclear. The 1971-72 serum specimens were not available for retesting… The two former patients whose 1971-72 WB results were most strongly reactive had current ELISA and WB assays that were negative. Their immune function parameters were inconsistent with immune suppression… The results of the ELISA and WB assays performed on the 1971-72 specimens remain an enigma. Some were interpretable as positive, although only weakly reactive. One explanation is that the results observed in 1985 were true positives, that PDAs in the early 1970s had antibodies to an HIV-like agent that was nonpathogenic, and that the WBs subsequently converted from positive to negative. Loss of HIV antibodies in asymptomatic homosexual men has been reported. It is possible that antibodies to a nonpathogenic virus would have disappeared during the 17 to 18 years between admission to Lexington and the 1989 followup. Although this potential cannot be ruled out, it is more likely that the earlier results were false positives. The reasons for false positivity are unclear, but cross reactivity with related retroviruses may be one possibility. The HIV Western blot assay may cross-react with antibodies to HTLV-I in the p24 and p55 antigen regions, but not the gp4l region. These serum specimens were tested for the presence of HTLV-I/HTLV-II antibodies by other investigators, and a 6.3 percent seropositivity rate for the entire cohort was observed. It is not known if these 10 persons were seropositive for this related retrovirus; however, it is unlikely that this type of cross reactivity accounted for the previous results, given the distribution of the bands and the fact that reactivity was not detected during selected 1989 followup WB screening. The earlier false positivity could be the consequence of either the state of the serum specimens or the test kit or assay employed. It has been suggested that artifactual findings may occur as a consequence of frequent thawing and refreezing of serum aliquots, and that frequent refreezing might affect the physical properties and serologic characteristics of the serum protein moieties. The available evidence would suggest that long-term storage and repeated thawing and refreezing does not affect subsequent testing for serum constituents”
Lange WR et al. Followup study of possible HIV seropositivity among abusers of parenteral drugs in 1971-72. Public Health Rep. 1991 Jul-Aug;106(4):451-5.

“Indeterminate patterns on Western blot are common in non-HIV_infected people…Western blot testing should not be done routinely as a screening test for HIV-1 infection because…it would result in a large increase in the number of indeterminate specimens, nearly all of which would not be from persons with HIV-1 infection…Possible explanations for the indeterminate reactions include exposure to an unidentified immunogen [immune system stimulator], such as another retrovirus, the presence of nonspecific comigrating antigens in the viral lysate used to prepare the Western blot strips and the presence of epitopes, posttranscriptionally modified antigens, or new antigenic sites in the viral antigen that are not present on the recombinant p24 antigen.”
Povolotsky J et al. Differences in human immunodeficiency virus type 1 (HIV-1) anti-p24 reactivities in serum of HIV-1-infected and uninfected subjects: analysis of indeterminate western blot reactions. J Infect Dis. 1991 Feb;163(2):247-51.

“[In a study of healthy persons with repeatedly positive ELISA antibody tests but ‘indeterminate’ Western Blot ‘confirmatory’ tests]…After a median of 14 months (range, 1 to 30) from the time of the initial test, 65 subjects (66%) were still repeatedly reactive for HIV-1 antibody on at least one immunoassay. In 91 subjects (92%) the Western blot results were still indeterminate, whereas in 8 they were negative. No donor met the criteria for a positive Western blot assay for HIV-1, and none had evidence of HIV-1 or HIV-2 infection on culture or by any other test. We conclude that persons at low risk for HIV infection who have persistent indeterminate HIV-1 Western blots are rarely if ever infected with HIV-1 or HIV-2…we were unable to discern an association between any measure studied and indeterminate status with respect to Western blot results or a specific band.”
Jackson JB et al. Absence of HIV infection in blood donors with indeterminate western blot tests for antibody to HIV-1. N Engl J Med. 1990 Jan 25;322(4):217-22.

“The criterion for a Western blot assay with positive results was a speciment that exhibited at least two of three bands at p24, gp41 and gp120/160.”
Garland FC et al. Incidence of human immunodeficiency virus seroconversion in US Navy and Marine Corps personnel, 1986 through 1988. JAMA. 1989 Dec 8;262(22):3161-5.

“The currently licensed Du Pont Western blot test specifies that the test result should be interpreted as positive only when the detected bands include p24 and p31, and gp41 or gp120/160. Conversely, a negative Du Pont Western blot test result requires the absence of any and all bands–not just viral-bands…[Three] Alternative criteria have been proposed by various groups. ASTPHLD has proposed that a positive test result be defined by the presence of any two of the following bands: p24, gp41, and gp120/160 (13). The Consortium for Retrovirus Serology Standardization (CRSS) has defined a positive test result as the presence of either p24 or p31, plus a diffuse envelope band (i.e., gp41 or gp120/160) (14). The American Red Cross has defined a positive test result as greater than or equal to 1 band from each of the GAG, POL, and ENV gene-product groups (15). These three groups and DuPont all agree that an indeterminate result is the presence of any other band or bands that fail to meet the positive criteria, and that a negative result is the absence of all bands. The criteria for a negative Western blot interpretation specify “no bands.” This interpretation is essential because some observed bands may reflect the presence of antibodies to HIV regulatory proteins or may indicate partially processed or degraded viral structural proteins. Furthermore, different Western blots (commercial, as well as “in-house” preparations) and different virus-antigen preparations used to prepare Western blots may contain different numbers and concentrations of both viral-specific and contaminating cellular proteins that may have unpredictable molecular weights…the ASTPHLD definition gives the highest percentage of positive and the lowest percentage of indeterminate results…On the basis of the results described above, CDC concurred with the ASTPHLD criteria and recommends their use in public health and clinical practice.”
CDC. Interpretation and Use Of Western Blot Assay for Serodiagnosis of Human Immunodeficiency Virus Type 1 Infections. MMWR. 1989;38(S-7):1-7.

“To interpret human immunodeficiency virus (HIV) Western blots, the specific nature of each band must be understood. Bands near the top of each strip, for example, were thought to reflect the reactivity of antibodies with HIV gp160 envelope precursor and the gp120 envelope glycoprotein. However, we have unexpectedly found that viral antigens, migrating with mobilities of 160 and 120 kd [molecular weight] on commercial Western blot strips, are primarily multimers of the HIV transmembrane glycoprotein gp41 that react with antibodies to gp41. 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.”
Zolla-Pazner S et al. Reinterpretation of human immunodeficiency virus western blot patterns. N Engl J Med. 1989 May 11;320(19):1280-1.

“The pattern of bands on protein or Western immunoblot (IB) for antibodies to antigens of human immunodeficiency virus (HIV) is not always reproducible…positivity may be simulated by the reactivity of serum with nonviral proteins of molecular sizes similar to viral components. Finally, technical vagaries may result in apparent rather than real loss of antibody to HIV capsid antigen (p24)…Routine Quality Control with Two Positive QC specimens…Only 101 (56%) of the 179 assays showed the presence of p24, p32, and gp41 and/or gp120, a pattern that would be reported as positive by all sets of criteria in use at reference laboratories…Routine Quality Control with Two Negative QC Specimens…An absence of all bands…was reported for only 67 assays (67%) [i.e. 33% of tests on negative western blot controls showed one or two positive bands]…Physicians should remember that any given IB result can be erroneous and seek further confirmation by a later sample if applicability of the result to the patient seems questionable. The criteria for positivity when only some bands are seen continues to be a problem. Reactivity at either p24 or gp41, without p32 or gp120 has sometimes been considered adequate for confirmation of anti-HIV positivity. This pattern was seen 10 times with one of the positive samples inn two laboratories but a combined total of 19 times in three laboratories with the two negative samples. It is obvious that p24 alone or gp41 alone, without p32 or gp120, must be reported as indeterminate and the IB repeated on the same or a subsequent specimen. On the other hand, requiring three bands among p24, p32, and gp41 or gp120 would have permitted a positive interpretation of only 101 or 179 assays (56%) of QC(+) [positive quality control] specimens. Requiring two of the three would result in a correct interpretation for 166 of 179 (93%) assays of QC(+) samples and 99 of 101 (98%) assays of QC(-) specimens. The criteria for positivity and negativity for any given IB assay, however, must minimize false positivity and false negativity, even if reassay of specimens is necessary for a moderate number of sera. An indeterminate pattern (i.e.,, not interpretable as necessarily positive or negative) tended to occur in clusters on a given day of testing or on successive dates, particularly for the negative QC samples. This seems attributable to a systematic technical problem at that particular time. Whenever we observed this occurrence, we resubmitted to the same facility after discussion of the problem or checked reproducibility in another laboratory.”
Edwards VM, Mosley JW. Reproducibility in quality control of protein (Western) immunoblot assay for antibodies to human immunodeficiency virus. Am J Clin Pathol. 1989 Jan;91(1):75-8.

“Applicants for United States military service, October 1985 to March 1986…were screened with a commercial enzyme-linked immunosorbent assay (ELISA), and positive samples were immediately retested in duplicate with the ELISA. All samples positive on repeat ELISA were tested with the Western blot method for reactivity with HIV proteins. Specimens were classified as positive if antibodies to HIV encoded protein gp41 or proteins p24 and p55 were detected.”
Burke DS et al. Human immunodeficiency virus infections among civilian applicants for United States military service, October 1985 to March 1986. Demographic factors associated with seropositivity. N Engl J Med. 1987 Jul 16;317(3):131-6.

“In the absence of a 41-kd band, a blot must show both a 24-kd and a 55 kd band to be deemed positive…A second sample must be drawn and confirmed to be blot positive before a diagnosis is considered established.”
Burke DS, Redfield RR. False-positive western blot tests for antibodies to HTLV-III. JAMA. 1986 Jul 18;256(3):347.

“Of 1,027,786 units of donated blood tested, 10,385 (1.0%) were reactive on the initial EIA test for HTLV-III [HIV] antibody. Of these, 1723 (0.17%) were repeatably reactive, designated ‘EIA-positive’ (EIA+), and not released for transfusion. Of 1455 EIA-positive blood units, representing approximately 868,000 donors, that were tested by Western blot analysis, 333 were positive (WB+) [i.e. 83% of initial positive EIA’s were false positive based on a second EIA and only 3.8% of initial EIAs were confirmed by both a second EIA and Western Blot, the other 96% were false positives]”
Schorr JB et al. Prevalence of HTLV-III antibody in American blood donors. N Engl J Med. 1985 Aug 8;313(6):384-5.

(Posted: Apr 1 2009 at The Unhived Mind II)

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