Use of a 24-kilodalton Trypanosoma cruzi recombinant protein to monitor cure of human Chagas' disease

A 24-kDa recombinant protein from Trypanosoma cruzi (rTc24) was evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot (immunoblot) tests to identify treated chagasic patients considered parasitologically cured on the basis of persistently negative tests of hemocultures and lytic antibodies. Some of these patients were termed dissociated because their sera, although negative by the complement-mediated lysis test, were positive by conventional serology. The negative lysis test indicates the absence of active infection after specific treatment, but this assay requires live and infectious parasites and cannot be used easily in a laboratory routine. Here we tested rTc24 by ELISA and Western blotting as an alternative for the complement-mediated lysis test. For the group of patients with active infection despite the treatment (uncured patients), all the sera tested recognized rTc24 in both tests. For the dissociated patients, approximately 80% of the sera did not react with rTc24 in the ELISA or in Western blots, in agreement with the negative complement-mediated lysis tests. Thus, the 24-kDa T. cruzi recombinant antigen, when used for initial trials to evaluate cure of chagasic patients submitted to specific treatment, will allow the identification of most, but not all, cases.     

Validation of a new immunoblot assay (LiaTek HIV III) for confirmation of human immunodeficiency virus infection

BACKGROUND: Used as a supplemental assay, new anti-human immunodeficiency virus (HIV) immunoblots, employing recombinant and synthetic antigens, appeared to resolve the majority of samples with false-reactive Western blot results. Would it be possible to completely replace the Western blot by an immunoblot for confirmation and exclusion of HIV infection? STUDY DESIGN AND METHODS: The sensitivity of the new LiaTek HIV III immunoblot assay (Organon Teknika, Turnhout, Belgium) was tested on 416 Western-blot positive samples (386 HIV-1, 22 HIV-2, 1 HIV-1/2, and 7 HIV-O) and on 45 HIV-1 seroconversion samples. The specificity was tested on 146 samples from noninfected donors with false-positive results on a HIV screening test. RESULTS: All Western-blot-positive samples tested positive in the immunoblot (sensitivity: 100%). The immunoblot could not discriminate between HIV-1 and HIV-2 infection in 22 of 416 (5%) samples. The LiaTek assay showed reactivity in 28 of 45 seroconversion samples, whereas the Western blot reacted in 30 of 45 seroconversion samples. With false-positive donor samples, the immunoblot was indeterminate in 10 of 146 samples (specificity: 93%), and the Western blot was indeterminate in 44 of 146 samples (specificity: 70%). CONCLUSION: Like the Western blot, the immunoblot runs the risk of missing samples that are reactive by enzyme immunoassay during the early stage of HIV infection. Nevertheless, considering its superior specificity on false-positive donor samples, it appears that the immunoblot offers a cost-effective alternative to the Western blot assay for confirmation and exclusion of HIV infection.     

Evaluation of a dipstick method for the detection of human immunodeficiency virus infection

Serology has been a fundamental tool to prevent post-transfusional infection with human immunodeficiency virus (HIV) and for epidemiological surveys, the first step to attempt control of the pandemia. Enzyme immunoassay is in widespread use. Nevertheless, simpler methods are needed in many countries, where laboratory facilities and trained personnel are limited, and HIV prevalence is high. The evaluation of a simple and noninstrumented HIV antibody test is presented here. The test employs synthetic antigens of HIV-1 and HIV-2 attached to the teeth of a polystyrene comb, which fit into the wells of standard microtiter plates where samples are diluted. Captured antibodies are developed with colloidal gold-labeled Protein A. Three seroconversion panels plus 662 samples were tested, including HIV-1 and HIV-2-infected individuals, normal blood donors, and a noninfected baby born to a seroreactive mother. When compared with enzyme-linked immunosorbent assay (ELISA) and Western blot, the dipstick showed 100% sensitivity and 98.7% specificity. The simplicity of result evaluations and excellent reagent stability make the dipstick suitable for small blood banks and for epidemiological surveys.     

Tick-borne encephalitis diagnosis in patients with inflammatory changes in the cerebrospinal fluid in a region with very low prevalence

Tick-borne encephalitis (TBE)-IgG antibodies are used for the serologic detection of antigen contact caused by TBE infection or immunization. In the present study, enzyme-linked immune sorbent assay (ELISA) results from a group of patients with inflammatory changes in the cerebrospinal fluid (CSF) were re-examined using Western blot technology. The result of the TBE-IgG-ELISA was positive in 47 of the 904 sera samples tested. Retesting the sera with a Western blot confirmed this result in only 31.8% of the positive cases. In 134 of the 904 sera, the ELISA result was borderline. In 5.5% of these sera, the Western blot reacted specifically. The remaining 723 sera samples tested negative with the ELISA. Of these sera, 15 were selected randomly and retested with the Western blot; none of them tested positive. The high number of false positive ELISA results can be explained by the highly selected group of patients and the low prevalence of TBE in the region studied. In patients with meningitis or encephalitis with positive ELISA results and uncharacteristic clinical symptoms, the treating physician should consider the possibility of nonspecific reactions involving inflammatory mediators or cross-reactivity with other flaviviruses. The ELISA-mediated diagnosis of TBE should therefore be verified by means of the patient's history and clinical symptoms, as well as further serologic tests including the Western blot, the hemagglutination test and the neutralization test.     

Absence of pericentromeric heterochromatin (9qh-) in a patient with bilateral retinoblastoma

The diagnosis of HIV infection is based on screening of HIV antibodies and confirmed by a more specific supplementary test. The most common confirmation test is Western blot, which is expensive, time consuming and subject to technical skill. The present study was carried out to evaluate whether the anti-HIV-1 antibody titer is valid as a supplementary test for diagnosis of HIV-1 infection. Anti-HIV-1 antibody titers of 2,414 anti-HIV-1 positive sera determined by the particle agglutination (PA) method were analysed in comparison with the Western blot analysis. The Western blot negative result was found in 11 of 2,414 (0.46%) anti-HIV-1 positive sera, these sera also gave negative anti-HIV by ELISA. The PA titers of these sera were found in the range of 16 to 64. Seventeen samples (0.70%) with anti-HIV-1 in the titer range of 16 to 256 showed indeterminate Western blot analysis. The rest, 2,386 of these 2,414 sera (98.84%), were shown to be positive by Western blot. However, all of the 2,356 sera with antibody titers > or = 512 (97.6%) demonstrated positive Western blot results. Five cases among the 17 (29.4%) indeterminate sera were examples of early seroconversion of HIV infection, which were confirmed in follow up specimens. The results suggest that only the samples with antibody titers < 512 are required to be confirmed for HIV infection by Western blot. It is possible that early seroconversion may be inferred from anti-HIV titers. Therefore, in order to reduce time and cost, the PA anti-HIV titer can be used as an alternative supplementary test for diagnosis of HIV-1 infection in most positive screened anti-HIV samples. Western blot is needed for testing in only a few cases.     

Cognitive performance, mood and experimental pain before and during morphine-induced analgesia in patients with chronic non-malignant pain

OBJECTIVE: To determine the ability of simple, rapid tests to identify HIV-1 antibody-positive specimens in field settings using the World Health Organization's (WHO) alternative testing strategies. DESIGN: Three-phase evaluation of simple, rapid assays using banked specimens and prospectively collected serum specimens at regional hospitals and rural clinics. METHODS: Seven test (Retrocell, Genie, HIVCHEK, SUDS HIV-1, Testpack, Serodia HIV-1, and HIV-1/2 RTD) were evaluated and results compared with standard enzyme immunoassay (EIA) and Western blot results (phase 1). Further evaluation consisted of prospective testing of routine specimens at regional (phase 2; n = 900) and rural, peripheral laboratories (phase 3; n = 1266) throughout Honduras with selected assays. RESULTS: Sensitivity and specificity were calculated for each assay and combination of assays for each phase to evaluate the effectiveness of the WHO alternative testing strategies. All tests in all phases were > 99% sensitive after correcting for technical errors, with two exceptions (SUDS, phase 1; HIVCHEK, phase 3). In phase 3, where the testing algorithm was diagnostic, several combinations of assays were 100% sensitive and specific using WHO strategy II or III. For the Honduras Ministry of Health, the combination of Retrocell and Genie was found to be equally sensitive, more specific (no indeterminate results), and less expensive than EIA/Western blot. CONCLUSION: Combinations of rapid, simple HIV antibody assays provide sensitivity and specificity performance comparable to EIA/Western blot. Application of these combinations in the WHO alternative testing strategies provides an inexpensive and effective method of determining HIV status. Assay combinations using these strategies can be easily performed in small, rural laboratories and have been implemented in routine HIV screening in Honduras.     

Cesium uptake studies on human erythrocytes

We compared the performance of second and third generation ELISA assays to detect antibodies to HIV-1 virus with conventional Western blotting (WB) and radioimmune Western blotting (RIWB). Both sera from commercial seroconversion panels and serial dilutions of a serum for HIV-1 antibodies were tested with Murex HIV Recombinant, Vidas bioMérieux HIV 1/2 (2nd generation ELISA) Murex HIV 1-2 (3rd generation ELISA), as well as with WB and RIWB. In seroconversion panels all ELISA assays were positive for the same serum with the exception of the first serum of Panel D which was negative with both sample Murex assays and borderline with Vidas assay. This serum was negative with WB but evidenced antibodies to gp160 p66, p51, p24 HIV-1 proteins when assayed by RIWB. In only two cases did WB reveal antibodies to HIV-1 proteins before ELISA assays (Panel A and E); not only did RIWB show the same sensitivity as WB in the two last panels, but it also detected antibodies to HIV-1 proteins earlier than WB, ranging from a few days (Panel C) to approximately 12 weeks (Panel D). The results obtained by testing the dilutions of the serum positive for anti HIV-1 antibodies showed the following degrees of sensitivity: Murex HIV 1-2 (the most sensitive), Murex HIV Recombinant and Vidas bioMérieux HIV 1/2. Although WB was more sensitive than the ELISA assays and picked out antibodies to gp160, gp120 and p24 HIV proteins at 1/4000 serum dilution, the most sensitive test was RIWB which at 1/20,000 serum dilution enabled detection of antibodies to gp160, p66 and p24 HIV proteins.     

Lower respiratory illness in infants and young children with cystic fibrosis: evaluation of treatment with intravenous hydrocortisone

The enzyme-linked immunosorbent assay (ELISA) using HLA class I molecules purified from pooled platelets has the potential to detect HLA antibodies with increased efficiency without sacrificing sensitivity or specificity. This test, which was originally developed in our institution, has been independently validated by recent studies and is now commercially available. We now present evidence of its usefulness as a routine HLA antibody screening test for renal transplant patients. A total of 515 patients were tested monthly by ELISA (13.9 tests/patient) and by antiglobulin-enhanced panel reactivity (6.3 tests/patient). In patients found to be unsensitized, the incidence of false-positive results was less for ELISA than for the panel studies. In patients who were highly sensitized, both tests performed equally well, whereas discordant results were registered mainly in cases of mild sensitization. Because 66% of our patients were not sensitized, the ELISA was effective in reducing the number of more involved tests aimed at characterizing the antibodies. These results provide a foundation to use the pooled platelet HLA ELISA on a routine basis for HLA antibody screening.     

Detection of Vibrio anguillarum by a sandwich enzyme-linked immunosorbent assay performed with monoclonal antibodies

Monoclonal antibodies (Mabs) against V. anguillarum were produced and characterized by Western blotting analysis, competitive binding assays and cross-reactivity tests. Their ability to detect V. anguillarum in a liquid culture was tested in a sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of these Mabs used as capture or tracer antibodies. One combination was selected as the most suitable for diagnostic applications, showing the highest sensitivity and specificity.     

New systems of cardiac mapping or life to the electrophysiologist may become complicated

We report the case of an infant with progressive human immunodeficiency virus (HIV) infection and persistent seronegativity. The child had Pneumocystis carinii pneumonia at 4 months of age and was documented to be HIV-infected by HIV-1 deoxyribonucleic acid (DNA) polymerase chain reaction (PCR), but enzyme-linked immunosorbent assay (ELISA) and Western blot tests for HIV-1 and HIV-2 specific antibodies remained negative until the infant was 10 months old. This case should increase awareness about the possibility of seronegative HIV infection in infants and stress the fact that in questionable cases, even if the screening serology is negative, additional methods of diagnosis (ie, PCR, viral culture, and p24 antigen) should be considered.     

I-123 uptake in nonfunctional struma ovarii

Sera from 211 ostriches were tested for the presence of Newcastle disease virus (NDV)-specific antibodies by the virus neutralisation test, the haemagglutination inhibition (HI) test and a recently developed avian paramyxovirus serotype 1 (PMV-1) specific monoclonal antibody blocking ELISA (b-ELISA). The virus neutralisation test was used as the reference for the estimation of the sensitivity and specificity of the b-ELISA and HI tests. Of the 211 sera, 140 contained NDV-specific neutralising antibodies, 130 were positive by the HI test and 122 by the b-ELISA. The sensitivity, specificity and predictive accuracy of the HI and b-ELISA tests relative to the virus neutralisation test were similar. The good agreement between the HI and b-ELISA test (kappa = 0.85) suggested that the two methods are interchangeable.     

Induction of cyclooxygenase-2 in alveolar macrophages after acid aspiration: selective cyclooxygenase-2 blockade reduces interleukin-6 production

Sera from HIV-1-infected haemophiliacs were examined for human immunodeficiency virus (HIV) specific antibodies and for platelet crossreactive antibodies. Using HIV sepharose 4B affinity columns for serum absorption, antibodies against various HIV antigens, including HIV lysate. HIV-p24 and HIV-gp120, were eluted either by low or by high pH buffer. The eluates were examined by ELISA for HIV specificity and by flow cytometry for platelet crossreactivity. Two types of HIV antibodies could be eluted, i.e. acid-sensitive and alkaline-sensitive antibodies. HIV antibodies were obtained in 26/29 acid eluates and in 25/29 of the alkaline eluates from HIV-lysate columns; 96% (25/26) of the acid-eluted antibodies were HIV-specific but 48% (12/ 25) of the alkaline-eluted antibodies also showed crossreactivity to platelets. Of the 20 alkaline-eluted HIV-p24 antibodies, 40% (8/20) reacted specifically with HIV-p24 and 60% (12/20) were platelet crossreactive. In contrast, of the alkaline-eluted HIV-gp120 antibodies (n=17), 88% (15/17) were HIV gp120-specific and only 12% (2/17) were platelet crossreactive. Western blot analysis of platelets demonstrated that the anti-p24 antibodies recognized three bands with approximate molecular weights of 72000 to 95000. 69% of the serum antiplatelet antibodies showed platelet glycoprotein IIbIIIa specificity. Anti-HIV antibodies could be eluted from platelets. Hence, platelet crossreactive antibodies in HIV infection are primarily alkaline-sensitive and are associated predominantly with HIV p24 antibody; these antibodies may play a role in the immune thrombocytopenia of HIV-infected haemophiliacs.     

Pain due to epidural tumor in cancer patients. Report of two cases and differential diagnosis

INTRODUCTION: Blood is generally used for detection of antibodies associated with infections. However, removal of blood samples can be problematic and is painful for the patient. It requires suitable equipment and skilled staff, both of which may be expensive. Some patients refuse to allow removal of blood samples because they find it painful and traumatic. Removal of blood samples from children, newborns, immunocompromised or overweight subjects is often particularly difficult. In addition, some religions forbid the taking of blood samples. Thus, it is necessary to develop alternative, simple, painless methods of sampling body fluids that give results as accurate as those obtained with blood samples. Saliva has been suggested as a possible alternative. Epidemiological studies and other reports have shown that saliva may be of value for the detection of HIV antibodies. AIM: To evaluate the potential of saliva for detection of antibodies and seroconversion. MATERIALS AND METHODS: Section I: Saliva and serum from 1,023 subjects, including 150 AIDS patients, 251 TB patients of known HIV status and 622 subjects from at-risk groups were tested. Sera from subjects with unknown HIV status were systematically tested by Abbott recombinant HIV1/HIV2 EIA. Section II: Saliva and sera were obtained from a population at risk of HIV infection. Two hundred and fifty consenting adults were found to be HIV-negative. Saliva was collected from each subject once per week and tested the same day with the Abbott test pack and Wellcozyme GACELISA. A spot of blood was also collected, dried and tested using the Wellcozyme GACELISA protocol. Whenever a positive result was obtained for any test, a blood sample was taken the same day or as soon as possible, for Western, blot analysis. Saliva was collected with an OMNISAL device (SDS, Vancouver, USA) placed under the tongue. The indicator turned blue when enough fluid had been collected. The device was then removed and the saliva placed in a tube with stabilized product. It was then transferred to a casting tube with a filter. RESULTS: Section I: Saliva from 96 of 150 AIDS patients (64%) tested positive. Serum from these patients also tested positive for HIV. Thirty-two subjects of the 622 from at-risk groups tested positive for HIV in the Abbott recombinant HIV1/HIV2 EIA with saliva (5.14%). Serum from these subjects also tested positive for HIV in Western blot. However, saliva EIA gave 5 false negative results for patients who tested positive in rapid tests and Western blot. We optimized the procedure and obtained 98.69% sensitivity and 100% specificity. Section II: One individual tested positive in saliva and dried blood spot tests in the fourth week of the study. Western blot using serum samples obtained on the next and following days showed a progressive increase in the intensity of the bands, beginning with P24 and GP160 (Fig. 2). CONCLUSION: This study shows that testing saliva is effective for determining HIV status early in seroconversion.     

Diagnosis of contagious bovine pleuropneumonia: problems and recent developments

While it is easy to diagnose contagious bovine pleuropneumonia (CBPP) in an animal in the acute clinical stage, subacute and chronic forms are more difficult to diagnose. Recourse to laboratory tests is essential to confirm any suspicion of CBPP. As standard diagnostic procedures (isolation, culture, biochemical tests, serological tests) are lacking in specificity and sensitivity, improvements are needed. Progress in molecular biology techniques has led to new tests, among which are the polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) using monoclonal antibodies. Application of these techniques to CBPP offers a number of advantages, and has considerably enhanced the specificity and sensitivity of diagnosis.     

Expression of aquaporins in the rat ocular tissue

The current HIV pandemic is complicated by the spread of distinct types and subtypes of HIV. The currently used conventional diagnostic tests have shown limitations in the detection of antibodies against all HIV-1 subtypes, as demonstrated by recent identification of HIV-1 subtype O. To evaluate quantitatively the diagnostic potential of a double ELISA strategy for the detection and partial differentiation of HIV-1, HIV-1 subtype O and HIV-2 infections blood samples were examined at five different test centers: Blantyre, Malawi; Abidjan and Daloa, Ivory Coast; Yaoundé, Cameroon; Munich, Germany. All tests results, including ELISA extinction values and Western blot profiles, were forwarded to Munich for final interpretation. An indirect anti-HIV-1/2 ELISA and a competitive anti-HIV-1 ELISA were used in combination for the initial screening of blood specimens. All anti-HIV positive and anti-HIV negative samples were subjected to immunoblot analysis. Independent of the diversity of the extinction profiles, and of the test manufacturer, the quantitative evaluation of the ELISA extinction values could define two extinction areas with a 100% predictive value for HIV-1 seropositivity and HIV seronegativity; extinction values > 2 by the indirect ELISA and < 0.2 by the competitive ELISA for an anti-HIV-1 subtype A to I positive result; extinction values < 0.2 by the indirect ELISA and > 1.0 by the competitive ELISA for an anti-HIV negative result. Additionally, the quantitative evaluation of the extinction profile provides partial information on the HIV-1 subtype as far as the distinction in group M and group O is concerned. In conclusion, the quantitative evaluation of this double ELISA strategy can reduce the number of blood specimens that require additional confirmatory testing in developing countries and can be superior to the immunoblot method during early seroconversion.     

An enzyme-linked immunosorbent assay (ELISA) for monitoring guineapigs and rabbits for Bordetella bronchiseptica antibodies

An enzyme-linked immunosorbent assay (ELISA) for monitoring antibodies specific to Bordetella bronchiseptica in guineapigs and rabbits was developed. In conventional and SPF colonies of guineapigs and rabbits, the ELISA was equally successful in detecting infected animals when compared to selective cultivation from the respiratory tract. The ELISA showed a sensitivity of 100% and a specificity of 90% in guineapigs. In rabbits the sensitivity and specificity of the ELISA were 97% and 91% respectively. In rabbit sera from infected colonies, ELISA activity showed a statistically significant correlation with titres obtained in the micro-agglutination test. Since serologically unrelated strains of the bacterium exist, the monitoring of animals for B. bronchiseptica infection by ELISA should be performed with various antigens.     

Effect of angiotensin ii on Na+/H+ exchangers of the renal tubule

A commercial enzyme-linked immunosorbent assay (ELISA) designed to detect allergen-specific immunoglobulin (Ig)E antibodies were evaluated in 36 atopic dogs and in 12 normal dogs. The test showed a sensitivity of 72.23% and a specificity of 41.6%. Positive and negative predictive values were 76.47 and 35.71% respectively. Correlation between the ELISA kit results and intradermal skin testing varied depending on the allergen and ranged from 47.1 to 80.4%, although positive correlation (i.e. allergens positive in both tests) ranged rom 2.7 to 19.4%. In conclusion, this serological test gave both false positive and false negative results. Sensitivity, specificity and predictive values indicate that this ELISA may not be useful in canine atopy. Although correlation studies were hampered by the impossibility of using the same allergenic extracts, the correlation observed between intradermal and serological testing indicates that results from both tests are not interchangeable.     

Comparison of PF4/heparin ELISA assay with the 14C-serotonin release assay in the diagnosis of heparin-induced thrombocytopenia

The diagnosis of heparin-induced thrombocytopenia (HIT) may be affirmed by demonstrating heparin-dependent anti-platelet antibodies using the 14C-serotonin release assay (SRA). In this study, results of the SRA was compared with the recently described platelet factor 4 (PF4)/heparin enzyme-linked immunosorbent assay (ELISA). Compared with the SRA, the sensitivity and specificity of a PF4/heparin ELISA was 87% and 92%, respectively, using an assay developed in our laboratory; and 90% and 98%, respectively, using a commercially developed kit (Diagnostica Stago, Asnieres, France). However, antibodies to PF4/heparin were also detected in up to 8% of patients whose plasma was negative by SRA, and 23% of patients receiving heparin who were not thrombocytopenic. These data indicate that results obtained with the PF4/heparin ELISA and the SRA are generally in accord in patients with a clinical diagnosis of HIT. However, discrepant results occur in approximately 20% of cases because of the greater sensitivity of ELISA and the possible involvement of other heparin-binding proteins. The fact that each assay contributes independent information in some cases must be considered in the sequence of test performance and in providing consultation to the practicing hematologist.     

Sarcoplasmic reticulum Ca2+ loading in rabbits 8 and 15 weeks after coronary artery ligation

Antibodies against rabbit hemorrhagic disease virus (RHDV) from 352 red fox (Vulpes vulpes) sera collected in Germany (Mecklenburg-Vorpommern) in 1993 were tested by a blocking enzyme-linked immunosorbent assay (ELISA) test kit. Ninety samples with positive or suspected results also were analyzed by the hemagglutination inhibition test (HIT). Eighteen serum samples (5%) were positive with the blocking ELISA and eight of these also were positive with HIT. The 18 positive sera also were tested by blocking ELISA for antibodies against European brown hare syndrome virus (EBHSV) and by sandwich ELISA to detect for antibodies against RHDV and EBHSV antigen. Antibodies were not found against EBHSV using the blocking ELISA. With the sandwich ELISA, six samples were positive against RHDV and also against EBHSV, indicating cross-reactivity between determinants of both viruses. However, antibody titers against RHDV were higher than against EBHSV in five samples, and in one animal titers were similar. In addition, two positive samples were investigated by Western blot immunoassay which showed clear positive reactions with the two main peptide bands of EBHSV and RHDV. Comparison of the areas below the peaks of the bands after immunoblotting indicated that there was a stronger reaction with the two main polypeptides of RHDV than with the two main peptides of EBHSV. This is the first report of calicivirus antibodies in free-ranging red foxes. Based on the specificity of the tests, the antibodies detected against caliciviruses may be induced by RHDV. There is a potential link for RHDV between free-ranging rabbits and foxes.     

New laboratory guidelines for serologic diagnosis of Lyme disease: evaluation of the two-test protocol

Recent guidelines established by the Association of State and Territorial Public Health Laboratory Directors (ASTPHLD) and the U.S. Centers for Disease Control and Prevention (CDC) recommend the use of a two-test protocol for the serologic diagnosis of Lyme disease (LD). The two-test protocol relies on a sensitive screening test, which is followed by specific immunoglobulin M (IgM) and/or IgG immunoblotting (IB), depending on the date of disease onset, of all samples with equivocal and positive screening test results. We evaluated a commercially available IgM-IgG enzyme-linked immunosorbent assay (ELISA) and separate IB tests for IgM and IgG antibodies to Borrelia burgdorferi as candidate assays for the two-test protocol. Serum samples obtained from healthy controls (n = 29), from patients with diagnoses or laboratory findings associated with serologic cross-reactivity to LD (n = 24), and from patients with well-documented early- and late-stage LD provided by the CDC and the College of American Pathologists (n = 53) were examined to determine each assay's individual sensitivity and specificity. No false-positive results were detected among the healthy controls by either ELISA or IB, whereas four false-positive ELISA results were recorded within the cross-reactive group. None of these sera, however, were positive for either IgM or IgG reactivity according to IB band criteria. With regard to the patients with LD, we determined the sensitivity and specificity of the ELISA to be 96 and 100%, respectively, compared with the reference data provided for these specimens. When we compared our IB results with data from CDC, the assay sensitivity and specificity were 80 and 96.2%, respectively, for IgM and 81.8 and 95.8%, respectively, for IgG. Pursuant to this evaluation we assessed the suitability of the two-test protocol by performing a retrospective analysis using clinical history to define samples as positive or negative for LD. We determined clinical sensitivity and specificity for all study subjects (n = 112) to be 50 and 100%, respectively. A reduction in the clinical sensitivity of the two-test protocol was associated with a lack of antibody response or seroconversion in LD patients treated with antibiotics. We conclude that the CDC-ASTPHLD guidelines provide useful criteria for test performance and interpretation aimed at standardizing the serologic diagnosis of LD.