Causes of para-allergy to tuberculin

Experiments on 56 rabbits infected with microorganisms of the genera Mycobacterium, Nocardia and Rhodococcus revealed that in response to the action of different antigens used T and B systems of immunity induced synthesis of antibodies reactive with nonspecific antigens (mycobacterial antigens, tuberculin). In some cases the statistically significant correlation between the dynamics of blast transformation and the specific lysis of lymphocytes in animals with Nocardia and Rhodococcus infections (in comparison with the controls) was determined when P.P.D. tuberculin was used as specific antigen. In the rabbits sera infected by Nocardia and Rhodococcus complement-fixation and hemagglutination antibodies to mycobacterial antigens were detected. These rabbits also exhibited skin reaction to P.P.D. tuberculin. The presence of common group-specific antigens in Nocardia and Rhodococcus, as well as in mycobacteria, determined the capacity of the former to sensitize experimental animals to tuberculin, with should be taken into consideration in making the allergic test to tuberculosis.     

Partial suppression of cell mediated immunity in chromoblastomycosis

The cellular immune response of 8 patients from the Brazilian Amazon region with chromoblastomycosis was analyzed. Primary immunological responses of patients were tested by contact sensitization to 2,4-dinitro-chlorobenzene (DNCB), or rejection of first set skin allografts. 2 of 8 patients were reactive to DNCB after sensitization, and skin allograft rejection occured in an average of 14 days. Capacity of patients to mount recall immunological responses was measured by skin testing with two fungal antigens and three bacterial antigens. Delayed skin reaction to trichophytin and Candida antigens was negative in the majority of the patients. However, reactivity to mycobacterial (tuberculin), and bacterial (staphylococcal, streptococcal) antigen was high, or only slightly diminished respectively. The data suggest that patients with chromoblastomycosis have suppressed nonspecific, cell mediated immunity for some antigens (skin allografts, DNCB, fungal antigens), while reactivity to bacterial and mycobacterial antigens is not impaired.     

Immunodiagnosis of nocardiosis.

A specific immunodominant 54-kDa antigen was purified from a culture filtrate of Nocardia asteroides by immunoaffinity chromatography. The chromatography column was prepared with immunoglobulin G obtained from sera from patients with lepromatous leprosy. Unbound solutes consisted of specific, partially purified N. asteroides antigens, primarily a 54-kDa band, accompanied by two others of 31 and 62 kDa. The Western blot (immunoblot) technique was applied to detecting the immunologic response to nocardiae in the serum of nocardiosis patients. Each of the serum samples from immunosuppressed or immunocompetent patients infected with N. asteroides reacted with the 54-kDa band, and two reacted with the 31- and 62-kDa bands. There was no reaction to either the 54- or the 31-kDa antigen with all serum samples obtained from patients with tuberculosis, except for one, with all serum samples obtained from patients with leprosy, or with all sera obtained from healthy controls. The partially purified 54-kDa antigen, specific for N. asteroides, was used as the immunogen to generate monoclonal antibodies (mAbs) and two mAbs were selected. As determined by Western blot, both mAbs reacted with the 54-kDa band. Using indirect immunofluorescence or enzyme immunoassay with whole N. asteroides micro-organisms, the mAbs did not react with N. asteroides cells. No cross-reactivity with mycobacterial antigens, either culture-filtrate antigens or tuberculin, was exhibited with any of the two mAbs. These mAbs are candidates to be used for the development of a sensitive and specific diagnostic test for nocardiosis.     

Enzyme-linked immunosorbent assay and Western blot antibody determination in sera from patients diagnosed with different helminthic infections with Anisakis simplex antigen purified by affinity chromatography

An evaluation of the sensitivity and the specificity of the Anisakis simplex antigens purified by affinity chromatography was performed using sera from patients diagnosed with Anisakis sensitisation and sera from patients previously diagnosed with different helminthic infections. Only the sera of the patients diagnosed with Schistosoma mansoni or Onchocerca volvulus parasitic infections were negative against the A. simplex antigen and its purified fractions (PAK antigen: A. simplex antigen purified using columns prepared with anti-A. simplex rabbit IgG and PAS antigen: PAK antigen purified using columns prepared with anti-Ascaris suum rabbit IgG). However all the sera were positive against the A. suum antigen. In all the sera from the patients diagnosed with Anisakis sensitisation, the antibody levels detected using the purified antigens (PAK and PAS antigens) were lower than the observed using the A. simplex crude extract with the highest diminution in the case of the IgG. When these same sera were tested against the A. simplex crude extract by Western blot, several bands of high molecular masses were observed as well as, intense bands at 60 and/or 40 kDa. A concentration of these last proteins was observed in the PAK and the PAS antigens. When the sensitivity and the specificity determinations were performed, only seven of the 38 patients diagnosed of Anisakis sensitisation were positive, as well as, the sera from the patients diagnosed with parasitisms by Echinococcus granulosus or Fasciola hepatica.     

Characterization of serologic and cell-mediated reactivity of a 38-kDa antigen isolated from Mycobacterium tuberculosis

An antigen of Mycobacterium tuberculosis with an m.w. of 38,000 has been isolated by affinity chromatography using a monoclonal antibody. This antibody bound only to an antigen found in M. tuberculosis and Mycobacterium bovis BCG. The specificity of the antigen was tested in a vertical study by immunodetection on western blots reacted with hyperimmune sera against M. tuberculosis, M. bovis, and 10 other Mycobacterium species. The antigen was detected only by antisera to M. tuberculosis and M. bovis. Specificity in cell-mediated immunity was tested by skin tests in guinea pigs sensitized with M. tuberculosis, Mycobacterium intracellulare, and Mycobacterium kansasii and by lymphocyte proliferation tests. The 38-kDa antigen induced positive skin test reactions regardless of the Mycobacterium species used to sensitize the animal. The ability of the 38-kDa antigen to sensitize for cell-mediated immunity was tested by injecting mice with the 38-kDa antigen and challenging their lymphocytes in vitro with various mycobacterial antigens. Lymphocyte proliferation was observed in the presence of 38-kDa antigen, M. tuberculosis sonicate antigen, and tuberculin purified protein derivative and to M. kansasii and M. intracellulare. The 38-kDa antigen may contain a specific epitope detected by serology, but also contains epitopes that are cross-reactive for cellular immunity.     

Immune reactions in patients with superficial bladder cancer after intradermal and intravesical treatment with bacillus Calmette-Guérin

Summary The immune reactivity of patients with strongly recurrent superficial bladder cancer was followed after combined intravesical and intradermal bacillus Calmette-Guérin (BCG) immunotherapy. All patients in this study were previously treated without success with intravesical chemotherapy. The BCG treatment regimen consisted of weekly administrations with BCG (RIVM) for six consecutive weeks, both intravesically and intradermally. In this study, sera and peripheral blood leukocytes (PBL) of patients were tested serially. Besides BCG-antigen-specific reactions, e.g. skin reactivity to purified protein derivatives of Mycobacterium tuberculosis (PPD), antibody formation and antigen stimulation of PBL in vitro, non-antigen-specific immune reactivities were also measured, e.g. mitogen response and spontaneous cytotoxic activity of PBL. In addition the antibody response to bladder carcinoma antigens and the cytotoxic activity of PBL for the bladder carcinoma cell line T24 and the natural-killer-sensitive K562 cell line were investigated. The results obtained from the various assays were evaluated for their prognostic value in relation to the length of the tumor-free interval after the BCG treatment. Because sera and PBL were only obtained during the first 6 months after the BCG treatment, the immune reactivity was compared to the clinical results at that same time. At 6 months after therapy 12 out of 40 BCG-treated patients were tumor-free whereas 28 out of 40 showed a recurrence. Skin reactivity to tuberculin PPD was measured in 40 patients during a period of 3–6 months after therapy. Of patients who showed a recurrence of the tumor within 6 months, 48% of them showed a transient response or developed no response at all to PPD. In the group of patients with a longer tumor-free period (n=10), only one patient lost the response to tuberculin PPD. Although PBL of a limited number of patients were tested, it was observed that the cytotoxicity to the bladder carcinoma cell line T24, and the natural-killer-sensitive K562 cell line increased in a number of the patients (7 out of 14, and 9 out of 14 respectively). Reactivity of PBL to mitogens and subset distribution (ratio T-helper: T-suppressor/cytotoxic) were not influenced by the BCG treatment. Antibody response to mycobacterial antigen was detected in 9 out of 23 patients investigated. Of these 9 patients, 8 belonged to the group with a recurrence of the tumor within 6 months (n=17). There was no correlation between the skin reactivity and the antibody response to tuberculin PPD. Furthermore, none of the 25 patients showed an antibody response to bladder carcinoma antigens. Sera of bladder carcinoma patients (n=19) reduced the mitogen-induced proliferation of lymphocytes, compared to sera of healthy controls (n=13), indicating the presence of circulating suppressor factor(s). Our results indicate that the absence of a Mantoux conversion or the presence of transient reaction to tuberculin PPD were highly related (91%) to a relapse of the disease. On the other hand, the cytotoxic activity of PBL to T24 and K562 cell lines, or their reactivity to tuberculin PPD or mitogens, gives no predictive information about the clinical results (tumor-free interval) of the BCG therapy. Abbreviations used: BCG, bacillus Calmette-Guérin; NK, natural killer; PBL, peripheral blood leukocytes; PPD, purified protein derivative of Mycobacterium tuberculosis; ELISA, enzyme-linked immunosorbent assay     

Thymic dependence of cell-mediated immunity to avian sarcomas in chickens. Immunological characterization of a nonvirion antigen in virus-infected cells.

Chickens bearing tumors which have been induced by avian retroviruses express cellmediated immune responsiveness against antigens which are associated with these neoplasms. We have employed a peripheral lymphocyte stimulation test to characterize antigens which are found in the supernatant fluids of avian retrovirus-infected chicken embryo fibroblast (CEF) cells and in the plasma of birds which have been inoculated with avian myeloblastosis virus (AMV). The results indicated that the antigenic activity being measured was virus group specific, cell transformation independent, and nonvirion in nature. Paradoxically, expression of such antigen(s) was restricted to cells which were actively synthesizing progeny avian retrovirus particles, and was absent in mammalian nonproducer cells which had been transformed by avian sarcoma viruses. Ability to respond immunologically to such antigen(s) was present in animals which had been inoculated with either leukosis or sarcoma viruses. Thymectomy, but not bursectomy, was stimulatory to tumor growth and abolished sensitized lymphocyte immune responsiveness in this system.     

Development of multiplex serological assay for the detection of human African trypanosomiasis

Human African trypanosomiasis (HAT) is a disease caused by Kinetoplastid infection. Serological tests are useful for epidemiological surveillance. The aim of this study was to develop a multiplex serological assay for HAT to assess the diagnostic value of selected HAT antigens for sero-epidemiological surveillance.We cloned loci encoding eight antigens from Trypanosoma brucei gambiense, expressed the genes in bacterial systems, and purified the resulting proteins. Antigens were subjected to Luminex multiplex assays using sera from HAT and VL patients to assess the antigens' immunodiagnostic potential. Among T. b. gambiense antigens, the 64-kDa and 65-kDa invariant surface glycoproteins (ISGs) and flagellar calcium binding protein (FCaBP) had high sensitivity for sera from T. b. gambiense patients, yielding AUC values of 0.871, 0.737 and 0.858 respectively in receiver operating characteristics (ROC) analysis. The ISG64, ISG65, and FCaBP antigens were partially cross-reactive to sera from Trypanosoma brucei rhodesiense patients. The GM6 antigen was cross-reactive to sera from T. b. rhodesiense patients as well as to sera from VL patients. Furthermore, heterogeneous antibody responses to each individual HAT antigen were observed. Testing for multiple HAT antigens in the same panel allowed specific and sensitive detection. Our results demonstrate the utility of applying multiplex assays for development and evaluation of HAT antigens for use in sero-epidemiological surveillance.     

Isolation of a New Soluble Antigen from the Yeast Phase of Histoplasma capsulatum

A method is described by which a soluble antigen was prepared from the yeast phase of Histoplasma capsulatum. This soluble preparation had a specificity greater than that of whole-cell yeast-phase antigens. In complement fixation tests with sera from human cases of histoplasmosis, blastomycosis, and coccidioidomycosis, the soluble antigen reacted in 12.1% of 141 tests with heterologous sera, whereas conventional whole-cell yeast antigens reacted in 47.3% of 91 tests with heterologous sera. The reactivities of the two types of antigens with homologous sera were essentially the same.     

Serological detection of B-cell ("Ia-like") antigens in serum of normal donor and in some sera of leukaemic patients

Some sera from normal donors (1/18) and from leukaemic patients (2/7 with acute myeloid leukaemia [AML], 1/4 with chronic lymphatic leukaemia [CLL], 0/3 with acute lymphoblastic leukaemia [ALL]; with high numbers of leukaemic cells expressing Ia-like p28,33 antigen on the leukaemic cell surface) inhibited the complement mediated cytotoxic activity of highly specific xenogenous anti Ia-like sera (which were prepared by immunization of rabbits with insoluble membrane fractions of B-type lymphoid lines) at a titre 1:4 or less. This effect was not observed with antisera directed against other membrane marker determinants (e.g. T lymphocyte specific antigens). These results suggest that at least a small proportion of membrane bound Ia-like antigens can be released from cell surfaces and in some patients these Ia-like moieties are detectable (by sensitive inhibition assays) in the serum.     

Comparison of antigens recognized by xenogeneic and allogeneic anti-Ia antibodies: Evidence for two classes of Ia antigens

Previously published data suggest that both xenogeneic and allogeneic anti-Ia sera can recognize carbohydrate-defined antigenic determinants on the surface of lymphocytes. There is also evidence, based on studies with allogeneic anti-Ia sera, that protein-defined Ia antigens exist. In this paper the relationship between these two types of Ia antigen was examined. It was found that in capping studies, the allogeneic anti-Ia serum could cap off the antigens recognized by the xenogeneic antiserum, whereas the xenogeneic antibodies could, at least partially, clear the surface of lymphocytes of Ia antigens detected by the allogeneic antibodies. On the other hand, when immunoprecipitates of radioiodinated cell-surface antigens were examined by SDS-polyacrylamide-gel electrophoresis, it was found that the xenogeneic anti-Ia serum did not immunoprecipitate any labeled material. In contrast, the allogeneic antiserum immunoprecipitated a labeled molecule which corresponded to the protein-defined Ia antigen described by others. Finally, it was shown that serum Ia antigens could be bound by either mouse or rabbit anti-Ia antibody, and this binding blocked any further reactivity with either serum. These results were interpreted as suggesting that two separate classes of Ia antigen molecule appear on the lymphocyte surface-one class has carbohydrate-defined antigenic specificities and the other has protein-defined determinants. Allogeneic anti-Ia sera contain antibodies against both these antigenic systems, whereas xenogeneic sera recognize only the carbohydratedefined series. The genetic implications of this interpretation are discussed.     

Identification and characterization of mitochondria autoantigens in progressive systemic sclerosis: identity with the 72,000 dalton autoantigen in primary biliary cirrhosis

Sera from eight out of 62 (14.5%) patients with progressive systemic sclerosis (PSS) reacted by immunoblotting with a 72,000 dalton antigen and one, a patient with concomitant primary biliary cirrhosis (PBC), reacted with the 72,000 dalton and a 47,000 dalton antigen. Reactivity with these antigens was not seen with any of 111 control sera. The antigens with minor variations in m.w. were present in a variety of cultured cells and tissue homogenates from different species. Subcellular fractionation studies localized the antigens to the mitochondria. Of 19 sera from patients with other diseases selected for immunofluorescence staining for anti-mitochondria autoantibody, nine reacted with the 72,000 dalton antigen, seven reacted with both the 72,000 and 47,000 dalton antigens, and three reacted with the 47,000 dalton antigen. These results show that serum reactivity with the 72,000 dalton and 47,000 dalton mitochondria autoantigens is found with some patients with PSS. Because mitochondria autoantibodies that are reactive with the 72,000 dalton and 47,000 dalton polypeptides are also found in patients with PBC, the present finding provides additional support for the association of PSS with PBC. Prior absorption of rat liver homogenate with PBC sera removed PSS serum reactivity with a 63,000 dalton antigen, the equivalent 72,000 dalton antigen in rodents, and vice versa, showing that both PBC and PSS sera recognize the same antigen.     

Cellular immunity in Q fever: modulation of responsiveness by a suppressor T cell-monocyte circuit

Human infection with the rickettsia Coxiella burnetii presents as an acute flulike primary Q fever, as a subacute granulomatous hepatitis, or, rarely, as chronic endocarditis. We have previously described lymphocyte unresponsiveness to Coxiella antigen in patients with Q fever endocarditis. This unresponsiveness was antigen specific and was mediated in part by adherent suppressor cells. In this report we show that the adherent suppressor cells work via prostaglandin E2 (PGE2)4 production. Addition of the cyclooxygenase inhibitor indomethacin to cultures of PBMC from patients with endocarditis or chronic laboratory exposure resulted in consistent increases in Coxiella-specific lymphocyte proliferation. The degree of increase in proliferation induced by indomethacin correlated strongly with the amount of PGE2 produced in a 4-hr culture stimulated by Coxiella antigen, but it also correlated with the sensitivity to inhibition of mitogenesis by PGE2. The suppressor mechanism was antigen nonspecific, because induction of suppression in vitro by Coxiella antigen also suppressed Candida-induced proliferation when both antigens were present in the same culture. Addition of indomethacin to these antigen cocultures totally reversed the Coxiella-induced suppression, confirming the evidence above that the nonspecific effector mechanism of suppression was prostaglandin (PG)-mediated. Elicitation of suppression, however, was antigen specific and involved a T cell-monocyte suppressor circuit. Supernatants from Coxiella-stimulated immune T cells and from the suppressor subset (OKT8+-enriched) of those T cells, but not unstimulated immune cells, induced augmented PGE2 production by unrelated nonimmune PBMC. We conclude that the lymphocyte unresponsiveness characterizing patients with Q fever endocarditis is modulated in part by an antigen-specific T suppressor cell which secretes a lymphokine to stimulate PGE2 production by adherent cells.     

In vitro assay for responsiveness of lymphocytes to transfer factor by a new leukocyte migration inhibitory test.

Transfer factor (TF) causes nonimmune lymphocytes to produce leukocyte migration inhibitory factor (LMIF) in the presence of purified protein derivative (PPD). The activity of TF was measured by leukocyte migration inhibitory test (LMIT). The LMIT was a modification of the conventional agarose droplet method. To express the activity of LMIF quantitatively and simply, LMIF titer was introduced. The LMIF titer was obtained from the combination of two factors, LMIF dilution and cell migration diameter, and therefore this made the LMIT much more sensitive as compared to the conventional LMIT. The responsiveness of lymphocytes from acute lymphoblastic leukemia (ALL) and from cell-mediated immunodeficiency in children to TF was assayed by LMIT. In ALL, the lymphocyte responsiveness was poor in relapse but improved with remission. The responsiveness was remarkably well in 3 patients with cell-mediated immunodeficiency. This method appears useful for the in vitro evaluation of responsiveness of lymphocytes to TF.     

Antibodies to bacterial and tumor-derived antigens in sera from normal guinea pigs.

Antibodies that react with radiolabeled antigens derived from guinea pig line-10 tumor cells and Mycobacterium bovis (BCG) were detected in sera from normal tumor-free strain-2 guinea pigs (NGPS). Binding by NGPS to the two antigens was inhibited by extracts of either line-10 cells or BCG. Binding by NGPS to the line-10 antigen was inhibited by a number of other bacterial extracts. NGPS was tested after absorption with a variety of cells including line-10, line-1, normal guinea pig spleen, normal adult and fetal liver cells. Results indicated that some of the antibodies in NGPS were directed to line-10-specific determinants. The specific stimulating antigen for these antibodies was not identified but because of the antigenic relationship between BCG, line-10 cells and other bacteria, antibodies to line-10-associated antigens might have been induced by exposure to environmental microorganisms.     

Enzyme-linked immunosorbent assay (ELISA) for IgM, IgG and IgA class antibodies against Candida albicans antigens: development and comparison with other methods

An extract of Candida albicans was used as an antigen on microtitre plates in the enzyme-linked immunosorbent assay (ELISA) to measure IgM, IgG and IgA class antibodies in the sera of hospitalized patients. It was found that of these patient sera that reacted positively in Ouchterlony immunodiffusion (ID) when undiluted, 58% were also positive in the ELISA against the same antigen preparation. However, all the sera with an ID titre of 1:2 or higher were ELISA-positive, demonstrating especially IgG and IgA. Of the sera positive by counterimmunoelectrophoresis against somatic and metabolic antigens of C. albicans, 86% were positive by ELISA. Reactions in precipitin-negative sera, if they occurred, usually demonstrated IgM or IgA. The sera with high passive haemagglutination or indirect immunofluorescence titres against surface antigens of C. albicans were positive in the IgG and IgA assays, while approximately one third were positive in the IgM assay.     

Reactivities of 4 murine coronavirus antigens with immunized or naturally infected rat sera by enzyme linked immunosorbent assay

Four murine coronavirus antigens, sialodacryoadenitis virus (SDAV) strain TG, Parker's rat coronavirus (PCV) strain 8190, mouse hepatitis virus (MHV) strains S and NuU, were examined for their reactivities to hyperimmunized and naturally infected rat sera by ELISA. With the immunized sera, SDAV and PCV antigens reacted best with respective homologous sera. MHV antigens reacted with all antisera, anti-SDAV, anti-PCV, and anti-MHV-S at approximately the same level, and MHV-S showed a slightly higher reactivity than MHV-NuU. The reactivities of the sera from various colonies to these antigens were in the order--from high to low--of SDAV, MHV-S, MHV-NuU, and PCV. None of sera negative for SDAV antigen reacted positively to the other antigens. Within the sera positive for SDAV, the positivities were in the order of MHV-S, MHV-NuU, and PCV. These results suggested that, although homologous antigens are best to detect SDAV or PCV infection by ELISA, MHV antigen can be used if highly cross-reactive viral strain is selected.     

Functional heterogeneity among the T-derived lymphocytes of the mouse. VII. Conversion of T1 cells to T2 cells by antigen.

The T1 subpopulation of peripheral T cells was defined in mice by its short half life, insensitivity to anti-thymocyte sera (ATS) in vivo, and slow kinetics of response to antigen. The T2 subpopulation was defined by its long life time, elimination by ATS in vivo, and rapid response to antigen. Mice containing only T1-type T cells were constructed by adult thymectomy (ATx) followed immediately by the elimination of T2 cells by ATS treatment. Immunization of these mice with SRBC led to the production of memory helper cells in the T2 subpopulation. This process depended on the presence of T1 cells and for the most part required SRBC immunization, although a few SRBC-specific T2 cells reappeared in the mice in the absence of antigen. We conclude that T1 cells can give rise to T2 cells in an antigen-driven step and that the two populations correspond to virgin and memory T cells, respectively.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Western blot diagnosis of vivax malaria with multiple stage-specific antigens of the parasite.

Western blot analysis was performed to diagnose vivax malaria using stage-specific recombinant antigens. Genomic DNA from the whole blood of a malaria patient was used as templates to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1) of Plasmodium vivax. Each amplified DNA fragment was inserted into a pGEX-4T plasmid to induce the expression of GST fusion protein in Escherichia coli by IPTG. The bacterial cell extracts were separated on 10% SDS-PAGE followed by western blot analysis with patient sera which was confirmed by blood smear examination. When applied with patient sera, 147 (91.9%) out of 160 vivax malaria, 12 (92.3%) out of 13 falciparum malaria, and all 9 vivax/falciparum mixed malaria reacted with at least one antigen, while no reactions occurred with 20 normal uninfected sera. In the case of vivax malaria, CSP-1 reacted with 128 (80.0%) sera, MSP-1 with 102 (63.8%), AMA-1 with 128 (80.0%), SERA with 115 (71.9%), and EXP-1 with 89 (55.6%), respectively. We obtained higher detection rates when using 5 antigens (91.9%) rather than using each antigen solely (55.6-80%), a combination of 2 (76.3-87.5%), 3 (85.6-90.6%), or 4 antigens (89.4-91.3%). This method can be applied to serological diagnosis, mass screening in endemic regions, or safety test in transfusion of prevalent vivax malaria.