Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.

We describe a new approach for retrieval of antigens from formalin-fixed, paraffin-embedded tissues and their subsequent staining by immunohistochemical techniques. This method of antigen retrieval is based on microwave heating of tissue sections attached to microscope slides to temperatures up to 100 degrees C in the presence of metal solutions. Among 52 monoclonal and polyclonal antibodies tested by this method, 39 antibodies demonstrated a significant increase in immunostaining, nine antibodies showed no change, and four antibodies showed reduced immunostaining. In particular, excellent immunostaining results were obtained with a monoclonal antibody to vimentin as well as several different keratin antibodies on routine formalin-fixed tissue sections after pre-treatment of the slides with this method. These results showed that after antigen retrieval: (a) enzyme predigestion of tissues could be omitted; (b) incubation times of primary antibodies could be significantly reduced, or dilutions of primary antibodies could be increased; (c) adequate staining could be achieved in long-term formalin-fixed tissues that failed to stain by conventional methods; and (d) certain antibodies which were typically unreactive with formalin-fixed tissues gave excellent staining.     

Differential distribution of RNA polymerase B and nonhistone chromosomal proteins in polytene chromosomes of Drosophila melanogaster

The distribution patterns of nonhistone chromosomal proteins (NHCP) associated with pulse-labeled RNA were determined by indirect immunofluorescence on salivary gland chromosomes of Drosophila melanogaster using monoclonal antibodies. By staining for two different antigens simultaneously, using antibodies tagged with different fluorescent probes, it became possible to position RNA-associated antigens as well as RNA polymerase B in relation to each other. Three separate staining patterns could be observed with anti-NHCP antibodies, none of which showed a pattern which was identical with that of RNA polymerase B. Furthermore, no correlation with the synthesis of the primary trancript, as monitored by the RNA polymerase B content of chromosomal sites, could be found by following the fluorescence patterns during inactivation of intermolt puffs or activation of early ecdysone-induced puffs. Finally, no strict correlation was observed between puffing activity and the accumulation of a certain antigen in these selected chromosomal sites.     

Commercial polyclonal and monoclonal histostaining PAP kits

Summary Universal, polyclonal and monoclonal immunoperoxidase staining kits from BioGenex, Dako and Ortho were employed for the localization of antigens such as gastrin, prostate specific antigen, IgA, IgG, AFP and CEA in histological sections from formaldehyde fixed and paraffin embedded human specimens. The kit components were controlled by immunohistological and serological assays and were also compared with self-prepared reagents. In connection with specific primary antibodies, universal/basic kits gave reliable localization of defined antigens. The optimal concentration of the primary antibodies had to be established by dilution experiments. In the case of polyclonal kits, typical antigen localization was obtained in selected tissue sections with all the respective kits. CEA kits also stained strongly NCA molecules present in organs such as colon, stomach and liver. BioGenex polyclonal kits gave almost stronger stainings than kits from Dako and Ortho. Irrespective of which kit from different commercial sources is used, development of peroxidase activity with AEC/H₂O₂ often had to be stopped far below the recommended incubation time of 40 min or overstaining with color change from reddish to muddy green occurred. The latter was attributed to insufficiently balanced kit reagents, an interpretation wich was supported by quantitative serological studies. Sensitivity of immunohistological reactivity was much enhanced by pretreatment of tissue sections with Pronase. Thus, stronger immunostainings and larger numbers of positive cells were detected than in conventionally rehydrated sections. Incubation of sections with self-prepared primary antibodies, linking antibodies and PAP complexes gave essentially the same antigen localization as with commercial kits, but antibodies isolated by our affinity chromatography led to a better staining contrast with absence of nonspecific background. The advantage of monoclonal over polyclonal kits was the background-free staining of sections. Other-wise, antigens were localized in the same cell types, although cellular reactivity was usually less intense than with polyclonal antibodies. This, however, could be overcome by Pronase treatment of the sections prior to incubation.     

The sequential appearance of components of the synaptonemal complex during meiosis of the female rat.

This paper describes the light microscopy (LM) and electron microscopy (EM) localization of synaptonemal complex (SC) antigens in oocytes of rats. For this purpose, we used monoclonal antibodies (Mabs) that recognize components of 30 + 33, 125, and 190 kDa antigens of SCs of rat spermatocytes. The LM localization was performed by immunofluorescence and the EM localization by immunogold staining. The reaction of the Mabs with oocytes was similar to the reaction with spermatocytes, but weaker. The 30 + 33 kDa as well as the 190 kDa antigens could always be demonstrated if axial elements of the SC were present, irrespective of whether these were paired or unpaired. Thus, these antigens could be detected from leptotene--early zygotene until diplotene. The 190-kDa antigen appeared in a diffuse manner just before the appearance of the 30 + 33 kDa antigens. The 30 + 33 kDa antigens were not only detected in the axial elements of SCs but also in characteristic aggregates, which appeared in zygotene and persisted until after the SCs had disappeared. Such aggregates had rarely been observed in spermatocytes. The 125 kDa antigen was only present in the tripartite segments of SCs, at the inner edge of the lateral elements. Thus, the reaction of the Mab against the 125 kDa antigen was detectable in zygotene, pachytene, and very early diplotene. It appeared later than 30 + 33 kDa and 190 kDa antigens and it disappeared earlier. We found that several steps of the immunostaining procedure could cause variation in the intensity of the Mab reaction.     

Nonlabeled secondary antibodies augment/maintain the binding of primary, specific antibodies to cell membrane antigens.

BACKGROUND: In studies on surface membrane antigen expression using immunofluorescence techniques, it is commonly observed that direct staining gives weaker signals than the signals following indirect staining with fluorochrome-conjugated secondary antibodies. This is most marked when cells have also been permeabilized in order to stain intracellular protein. The commonly accepted explanation for this observation is that fluorochrome-conjugated secondary antibodies bind to a higher number of binding sites on the primary antibody, as compared to the binding of conjugated primary antibodies to the membrane antigens. Another hypothesis might be that the antibody/antibody complexes formed on the membranes when using the indirect technique may have an augmented ability to bind the membrane epitopes. The present study was performed in order to check this hypothesis. MATERIALS AND METHODS: Peripheral blood mononuclear cells were stained with fluorochrome-conjugated anti-CD antibodies directly without or with a second-step application of nonconjugated goat anti-mouse IgG antibodies, followed by different fixation and permeabilization methods. The cells were analyzed by flow cytometry. RESULTS: A second-step application of nonconjugated goat anti-mouse IgG antibodies following direct staining with fluorochrome-conjugated anti-CD antibodies gave a significant increase in membrane antigen expression on permeabilized cells as compared to direct staining alone. The secondary antibody must be bivalent, since whole IgG or F(ab')(2) fragments of the goat anti-mouse antibodies showed effects, while Fab fragments did not. CONCLUSIONS: Nonlabeled secondary antibodies are able to influence the binding of primary, specific antibodies to cell membrane antigens on cells treated with permeabilizing agents necessary for staining intracellular proteins. The improved membrane antigen expression seems to be due to the formation of a network of primary and secondary antibodies on the cell surface, with increased ability for maintaining binding to CD antigens.     

Monoclonal antibody immunocytochemistry: novel method extending usefulness of monoclonal antibodies for antigen visualization

We describe a novel procedure combining the multiple-site reactivity of polyclonal antibodies with the defined single epitope-specificity of monoclonal antibodies. The method is based on previous findings that IgG molecules often only react with tissue-bound antigens with one of their two antigen-combining sites; thus, the remaining site is free to bind subsequently added antigen. In the procedure devised, such (undenatured) antigen is subsequently detected by a specific monoclonal antibody and the reaction is finally revealed by immunogold-silver staining. Antibody subpopulations to contaminating antigens may well be present in the polyclonal antiserum and may well bind first to tissue and then to the corresponding contaminants in the crude antigen preparation applied as second layer. Such contaminants will, however, not react with the monoclonal antibody and will therefore not be immunocytochemically detected. The method has been evaluated with one antigen which cannot be detected by monoclonal antibodies in paraffin sections (glial fibrillar acidic protein) and with another antigen (human chorionic gonadotropin) which can only be detected by the monoclonal antibody when occurring in high concentrations. In both cases the procedure resulted in strong specific staining of the antigens with no background.     

Optimization of immunohistochemical techniques to detect extracellular matrix proteins in fixed skin specimens

Complete antigen visualization in the context of well-preserved tissue architecture is the goal of all immunohistochemical techniques. Frozen tissue section techniques achieve optimal antigen visualization but preserve tissue architecture poorly. On the other hand, formalin-fixed tissue section techniques preserve tissue architecture very well but result in antigen masking. Enzymatic digestion or salt extraction of formalin-fixed sections has been used to reestablish antigen expression. Recently acid-alcohol-fixed tissue has been used as a successful compromise between tissue architecture preservation and the visualization of cytoskeletal antigens. In an attempt to find an improved immunohistochemical process for non-cytoskeletal antigens, we compared avidin-biotin immunofluorescence staining in frozen, formalin-fixed, and acid-alcohol-fixed tissues. The fixed tissues were either untreated or treated with enzyme digestion or salt extraction. For this study, we examined healing cutaneous wounds in Yorkshire pigs with antibodies to fibronectin, laminin, von Willebrand factor VIII, and keratin. Although tissue architecture was poor, frozen sections provided the best antigen visualization and were therefore used as the standard for complete antigen expression. Formalin-fixed tissues had excellent tissue architecture, but most antigens were completely masked. Pre-treatment technique only partially overcame the antigen masking caused by formalin. In contrast, acid-alcohol fixation preserved tissue architecture almost as well as formalin and sometimes allowed complete antigen visualization; however, laminin and fibronectin were partially masked. Total recovery of the expression of these antigens could be obtained by pre-treating the acid-alcohol-fixed tissue with either hyaluronidase or 1 M NaCl. Therefore, acid-alcohol-fixed tissue appears best for extracellular matrix (ECM) protein immunostaining as well as for cytoskeletal staining. However, certain ECM antigens require hyaluronidase or 1 M NaCl treatment for optimal visualization.     

Cell-surface associated proteins of corneal fibroblasts: dissection with monoclonal antibodies

It is now generally accepted that the cell surface is involved in the interaction of the cells with the extracellular matrix. To identify and characterize cell-surface-associated components of corneal fibroblasts, several monoclonal antibodies were developed. Hybridomas were developed by fusing mouse myeloma cells SP2/OAg14 with spleen cells from mice immunized with membrane fractions of corneal fibroblasts grown in culture. Twenty-five hybridomas secreting monoclonal antibodies to cell-surface components were selected by an enzyme-linked immunosorbent assay using corneal fibroblasts grown in microtiter plates as the substrate. Immunohistochemical staining demonstrated that the antigenic determinants recognized by these antibodies were not present on corneal epithelial cells, but were present on skin fibroblasts. The antigenic determinants recognized by two of these antibodies, designated 10D2 and 716, were matrix components of the corneal stroma. Immunochemical characterization of the antigens was carried out by indirect precipitation of the radioactively labeled cellular proteins with the monoclonal antibodies and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the precipitates. Four antibodies were able to precipitate antigens from cell extract in detectable amounts. Antibodies designated 5E2, 9G2, and 10D2 recognized antigens consisting of polypeptides of approximate molecular weights 105K and 110K, while antibody 716 recognized an antigen of 100K molecular weight. However, based on the tissue distribution and cell-surface distribution, these antibodies reacted with different antigenic determinants. The antigen recognized by 716 was also secreted by cells in culture but consisted of 220K and 200K polypeptide chains. It was tentatively identified as cellular fibronectin, based on the reaction of this antigen with polyclonal antibodies to plasma fibronectin.     

Immunogold labeling of blood-group antigens in human salivary glands using monoclonal antibodies and the streptavidin-biotin technique

Summary We investigated the localization of blood-group antigens A, B, and H in human labial salivary and submandibular glands by applying a postembedding immunogold method using monoclonal antibodies in combination with the streptavidin-biotin bridge technique. The H, A, and B antigens were only detected in mature secretory granules (SGs), which were mainly found in cells in the late phase of the maturation cycle. In immature SGs, which were present in cells in the early or middle phases of the maturation cycle, these antigens were not detected. All other cytoplasmic organelles were not labeled by the monoclonal antibodies used. In blood-group-O secretors, H antigen was present in almost all of the mature SGs. In blood-group-A secretors, the labelling for H antigen exhibited a mosaic-like pattern, i.e. only some of the mature SGs contained H antigen. With respect to the A and B antigens, a similar mosaic-like pattern of staining was observed in blood-group-A and-B secretors, respectively. To the best of our knowledge, this is the first time that the distribution of blood-group antigens A, B, and H in human tissues has been demonstrated at the electron-microscope-level using monoclonal antibodies.     

Incompatible A antigen expressed in tumors of blood group O individuals: immunochemical, immunohistologic, and enzymatic characterization

Monofucosyl type 1 chain A (type 1 Aa) and difucosyl type 1 chain A (ALeb), but not other types of A antigens, have been detected by application of carrier type-specific monoclonal anti-A antibodies (AH21 and HH3) in colonic tumors of blood group O individuals. An A-transferase activity (UDP-Gal-NAc:H-alpha-GalNAc transferase) was demonstrated in the extract of one of the O tumors expressing A antigen. The incidence of A antigen expression in O tumors was found to be two out of 15 cases, based on TLC immunostaining of glycolipid extracts, and five out of 50 cases, based on immunofluorescent staining of tumors with AH21 and HH3 antibodies.     

免疫磁珠技术分离唾液A/B血型抗原并用于吸附血清A/B抗体

目的:应用免疫磁珠分离技术获得具有良好抗原性的A/B血型抗原,并探究其作为ABO血型抗体吸附剂去除A/B抗体的可行性。方法:将含有血型物质的唾液进行预处理,再与包被了抗体的磁珠混合,分离出纯度较高的A/B抗原,运用酶联免疫及凝集抑制试验验证所得抗原的抗原性及是否存在交叉反应。用未纯化A/B抗原和纯化A/B抗原包被磁珠,对含有抗A/B IgM、IgG的血清进行抗体吸附,用纯化A/B抗原对100份来自O型血孕妇的临床血清样本进行抗体吸附,分别评价其吸附效果。结果:纯化抗原与对应抗体反应后,其吸光度显著高于对照组(A抗原与A抗体0.85±0.12 vs.0.27±0.03,P0.01;B抗原与B抗体0.86±0.09 vs.0.24±0.06,P0.01),与其它类型抗体反应后的吸光度值与对照组比较差异无统计学意义(P0.05)。进行红细胞凝集抑制试验时,纯化抗原可显著抑制相应抗体与红细胞的凝集反应,对其它类型抗体与红细胞的凝集没有抑制作用。血清抗体吸附实验表明纯化抗原的吸附效率比未纯化抗原的高(97.00%vs.88.00%,P0.001)。临床样本抗体吸附实验显示,纯化A抗原对抗A IgM/IgG的吸附效率分别为96.88%、98.44%;纯化B抗原对抗B IgM/IgG的吸附效率分别为96.88%、98.44%。结论:磁珠纯化抗原能特异性地与对应抗体结合,有效吸附血清中的血型抗体,有望作为合成A/B抗原的替代品。     

Immunogold labeling of blood-group antigens in human salivary glands using monoclonal antibodies and the streptavidin-biotin technique

We investigated the localization of blood-group antigens A, B, and H in human labial salivary and submandibular glands by applying a postembedding immunogold method using monoclonal antibodies in combination with the streptavidin-biotin bridge technique. The H, A, and B antigens were only detected in mature secretory granules (SGs), which were mainly found in cells in the late phase of the maturation cycle. In immature SGs, which were present in cells in the early or middle phases of the maturation cycle, these antigens were not detected. All other cytoplasmic organelles were not labeled by the monoclonal antibodies used. In blood-group-O secretors, H antigen was present in almost all of the mature SGs. In blood-group-A secretors, the labeling for H antigen exhibited a mosaic-like pattern, i.e. only some of the mature SGs contained H antigen. With respect to the A and B antigens, a similar mosaic-like pattern of staining was observed in blood-group-A and -B secretors, respectively. To the best of our knowledge, this is the first time that the distribution of blood-group antigens A, B, and H in human tissues has been demonstrated at the electron-microscope-level using monoclonal antibodies.     

Antigenic similarity between the coccidian parasites Toxoplasma gondii and Hammondia hammondi

The antigens that are present in the coccidian parasites Toxoplasma gondii and Hammondia hammondi were demonstrated and defined by using SDS-PAGE and immunoenzymatic techniques with 125I-labeled and unlabeled antigens of T. gondii and sera of mice infected orally or intraperitoneally with H. hammondi . All cell surface antigens of T. gondii that were labeled with 125I were recognized by antibodies in the sera of the mice infected with H. hammondi except the antigen of approximate molecular weight of 21.5 Kd. This suggests that this antigen is specific for T. gondii. Various antigens in the T. gondii-lysed antigen preparations were recognized by antibodies to H. hammondi . The number of recognized antigens increased as the infection of the mice with H. hammondi progressed. Oral infection with H. hammondi appeared to induce the formation of antibodies that recognized more T. gondii antigens than infection by intraperitoneal inoculation.     

Ultrastructural localization of human kidney antigens using monoclonal antibodies.

A highly sensitive method of ultrastructural-immunoperoxidase staining was developed for use with monoclonal antibodies which have been raised in this laboratory to a variety of antigens of the human kidney. Because of the susceptibility of the antigens to fixation and processing, a four layer, pre-embedding method of staining was used. Results confirmed and clarified previously reported light microscopy results, indicating that an antigen recognized by the PHM5 antibody was found on the podocyte cell membrane within the glomerulus and was not present within the glomerular basement membrane. The antigen was also present on the extraglomerular endothelial cell membrane. The study also demonstrated the presence of an antigen specific to endothelial cells throughout the renal cortex, and gave further insight into the precise localization of glomerular basement membrane components including fibronectin. The method of staining is now being used together with detailed ultrastructural studies to identify the cells produced from isolated glomeruli in tissue culture.     

Monoclonal antibody analysis of simian virus 40 small t-antigen expression in infected and transformed cells.

The monoclonal antibody PAb280 binds to small t antigen but not to large T antigen. Its binding site within the unique region of small t antigen was localized by studying its reaction with simian virus 40 mutants, other papovaviruses, and bacterial expression vectors coding for fragments of small t antigen. The antibody was used to define the cellular location of small t antigen by immunocytochemistry and by immunoprecipitation of subcellular extracts of infected cells. PAb280 reacts strongly with a cytoplasmic form of small t antigen that appears to be associated with the cytoskeleton and is not detected by antibodies directed to the common N terminus of small t and large T antigens. Immunoperoxidase staining of cells infected by the simian virus 40 defective strain SV402 with PAb280 and other anti-T antibodies demonstrated that this virus produced an N-terminal fragment of large T antigen as well as small t antigen. In cells infected by the virus, this fragment was located in the cell nucleus but was very unstable. These results suggest that the activity of the SV402 virus in transformation assays may not be entirely due to the action of small t antigen alone.     

Characterization and quantitation of membrane antigens of New World Leishmania species by using monoclonal antibodies in Western blot and flow microfluorometric assays

Membrane-specific monoclonal antibodies generated against promastigotes of New World Leishmania species were used in Western blot, ELISA, and flow microfluorometric assays to characterize their antigen specificity and to determine the external surface distribution of the reactive epitopes. Three major membrane antigens of molecular weight 72 KD, 55 KD, and 42 KD were identified as well as a dominant antigen that migrated as a broad band on SDS-PAGE, corresponding to a molecular weight of 10-15 KD. By dot-ELISA this antigen was also found to be excreted by promastigotes into their culture medium. One minor membrane antigen of 25 KD and a triplet component of 66, 58, and 56 KD were also identified. While assays performed on air-dried promastigotes revealed the almost ubiquitous presence of the 72 KD and 55 KD antigens, indirect immunofluorescent staining of live promastigotes followed by flow cytometric analysis revealed that these antigens had no external exposure. Antibodies binding the 55 KD component were also reactive toward purified mammalian tubulin. The remaining antigens had a variable distribution on the eight isolates utilized, and these quantitative differences could be used to distinguish isolates of the Leishmania mexicana complex from those belonging to the Leishmania braziliensis complex.     

Potential of immunogold-silver staining for the study of leukocyte subpopulations as defined by monoclonal antibodies

The potential of immunogold-silver staining for study of leukocyte subpopulations, as defined by monoclonal antibodies in cell suspensions, was examined. The cells were labeled in suspension as described for immunogold staining. Cytocentrifuge preparations of the suspensions were then immersed in a physical developer. By light microscopy, cells reacting with the monoclonal antibodies showed dark granules on their surface membrane. The morphology of the cells, as revealed by a panoptic counterstain, was comparable with that seen in routine cell smears for differential counts. The numbers of T-cells, T-helper/inducer cells, and T-cytotoxic/suppressor cells counted by this method in normal peripheral blood were nearly identical to those identified by immunogold staining and immunofluorescence microscopy in the same cell suspensions. The good morphological delineation also made possible rapid and accurate identification of particular leukocyte subsets in complex cell suspensions. Atypical lymphocytes from patients with infectious mononucleosis displayed the surface phenotype of activated T-cytotoxic/suppressor cells. Different maturation stages of neoplastic cells in patients with acute myeloid leukemia showed differences in surface antigen expression. Immunological detection of cell surface antigens could be combined with cytochemical staining of intracellular enzymatic activities. Finally, the labeling could be performed on cells prefixed on glass slides.     

Chromogen-based immunohistochemical method for elucidation of the coexpression of two antigens using antibodies from the same species

The authors established a chromogen-based, double immunolabeling method using antibodies from the same species without any unwanted cross-reactivity. In addition, time-consuming staining steps were shortened by using polymer-based secondary antibodies. Taking advantage of the nature of the chromogen 3-amino-9-ethylcarbazole (AEC), which is used as a horseradish peroxidase substrate for antibody detection, the AEC-derived signals in the first color development were easily eliminated by alcohol treatment. Therefore, the signals from the first staining did not interfere with those from the subsequent second staining, which used the chromogen 3,3'-diaminobenzidine. The co-localization of antigens within the same cell could be confirmed using this method, because cell images of the individual dye staining steps could be obtained and developed. The images from each step could be expressed in pseudo-colors in a dark field by using a computer. As a result, merged images could be constructed that resembled the images acquired by the fluorescent immunolabeling technique. The resolution of this method enabled analysis of the coexpression of two antigens in the same cell in the same section. The authors have named this staining technique the elucidation of the coexpression of two antigens in a cell using antibodies from the same species (ECSS).     

Sensitive detection of immunogold-silver staining with darkfield and epi-polarization microscopy

We evaluated the contribution of darkfield and epi-polarization microscopy to the detection of leukocyte cell surface antigens with immunogold-silver staining (IGSS). Lymphocyte cell surface differentiation antigens were labeled with monoclonal antibodies and IGSS as described for brightfield microscopy. In darkfield and epi-polarization microscopy the labeling appeared as bright spots on a dark background. The sensitivity of detection was much higher than that of brightfield microscopy. Sixteenfold higher dilutions of the monoclonal antibody could be used to detect all cells expressing the antigen in the cell suspension. However, non-specific staining was also better visualized. The latter could be reduced to a level comparable to that of brightfield microscopy only by use of weaker labeling conditions. A 25% reduction of the silver enhancement time was necessary for this purpose. However, these weaker labeling conditions also reduced the intensity of the specific staining. Therefore, the efficiency of IGSS, as detected with darkfield and epi-polarization microscopy, was only fourfold greater than that found with brightfield microscopy or that of an immunofluorescence procedure. Especially in combination with transmitted light, to improve cell identification, epi-polarization microscopy is a reliable and sensitive method for detection of immunogold-silver-labeled cell surface antigens for diagnostic and research purposes.     

Detection of leishmania antigen in kala azar patients using monoclonal antibodies.

Twenty-one monoclonal antibodies were produced against promastigote antigens of Leishmania donovani. Five monoclonal antibodies (Hyb.17, 6, 5, 4 and 2) identifying molecules associated with various L. donovani antigenic determinants ranging from 42-116 kDa were selected as 'capture antibodies' and compared with specific anti-leishmania antisera for detection of circulating leishmania antigens in kala azar patients' sera in a competitive-enzyme-linked immunosorbent assay system (ELISA). The anti-leishmania antisera could detect circulating antigen in 30% of kala azar cases while out of the five monoclonals, Hyb.17 could effectively detect circulating leishmania antigen in 85.4%. The efficacy of Hyb.6 was however low (31.7%). The antigens recognized by these monoclonal antibodies in the western blot assay could possibly represent the ones circulating in sera of patients suffering from kala azar. A cocktail of these monoclonal antibodies may be more useful than the conventional polyclonal antisera in detection of circulating antigen for clinical diagnosis of kala azar.