Enzyme-linked Immunoelectrotransfer Blot Technique Research Articles

Improved Dot-ELISA Assay Using Purified Sheep Coenurus cerebralis Antigenic Fractions for the Diagnosis of Zoonotic Coenurosis

Clinicians face significant problems in the diagnosis of zoonotic coenurosis. The current study aimed to develop an improved dot-Enzyme-linked-immunosorbent assay (dot-ELISA) for the diagnosis of zoonotic coenurosis using sheep Coenurus cerebralis scolices purified antigen (CcS-Ag) and to compare the obtained results with those of indirect ELISA and Enzyme-linked immunoelectrotransfer blot technique (EITB). Sera were collected from humans and sheep infected or suspected of infection with Coenurus cerebralis, control cases, and cases infected with other parasites. The CcS-Ag was proved to be the most specific antigen. This antigen was fractionated, and its specific polypeptides against anti-C. cerebralis antibodies (ACc-Ab) were identified using EITB. Fractions at the molecular weight (MW) of 48 and 58 kDa were proved as the only specific ones, eluted from the gel and concentrated, then dotted on the NC sheet as pooled antigen before its evaluation in the diagnosis of infection using dot-ELISA. Dot-ELISA demonstrated absolute 100% sensitivity and 100% specificity as recorded by EITB, compared to both fractions on a nitrocellulose (NC) sheet using surgically proved infected human or sheep sera as a gold standard. Diagnosis by ELISA using crude CcS-Ag revealed similar sensitivity but lower specificity (75%). The diagnostic accuracy of dot-ELISA was proved by comparing its results with postmortem data obtained post slaughtering of 20 suspected sheep and patients investigated by computed tomography (CT) and magnetic resonance imaging (MRI). In conclusion, the selection of specific fractions after EITB to be used in dot-ELISA improved the diagnostic value of the test as a diagnostic tool gathering the benefits of ELISA and EITB.

Cysticercosis in Madagascar.

Cysticercosis (CC) is the most common parasitic disease of the central nervous system. It is endemic in most developing countries where pigs are raised and consumed. An overview of all available data of this parasite in Madagascar is lacking. We conducted a literature review, collecting information on published and available literature about cysticercosis in Madagascar between January 1st, 1990 and June 30th, 2020. Out of 858 publications; 61 were included, issued from peer-review indexed journals, non-indexed journals, books, Ministry reports and press releases. In Madagascar, porcine cysticercosis has been reported since 1901; human cysticercosis is highly prevalent with an overall estimated seroprevalence between 7 and 21%. Serological analysis is based on Enzyme-Linked Immunosorbent Assay (ELISA) and Enzyme-linked immunoelectrotransfer blot techniques (EITB) for confirmative testing. Neurocysticercosis (NCC) is the most common pattern of cysticercosis in Madagascar and it is reponsible for pediatric morbidity causing more than 50% of epilepsy cases. Though CT-Scan is now available and tends to be considered the gold standard for NCC diagnosis, it remains unaffordable for most Malagasy patients and implies the proposal of a diagnostic algorithm for physicians. Our review has revealed that human taeniasis and bovine cysticercosis is a considerable burden in Madagascar. A national control program has been developed aiming to decrease the seroprevalence rate from 16 to 10% in 2015. The aim of the country is now to implement a CC control and elimination program. Meanwhile, some massive cysticercosis screenings have been conducted in the capital Antananarivo to drive people's attention on this widespread infection.

Production of monoclonal antibodies against target schistosomal antigen secreted in the urine of Schistosoma mansoni-infected patients

This study was undertaken to develop an immunodiagnostic test of active human schistosomiasis mansoni using a monoclonal antibody which targets urinary schistosomal antigen. Polyclonal antisera raised in rabbits against the processed urine of Schistosoma mansoni-infected patients showed very high and significant reactivity with ES product of ova compared with other different S. mansoni antigens. The monoclonal antibody (4.23) was reactive with repetitive epitopes of S. mansoni soluble egg antigen and ES product of ova with molecular mass range of 65–23 kDa and 80–23 kDa, respectively. It recognised different stages of the parasite life-cycle, with no cross reaction with Fasciola or hydatid antigen. MAbs were characterised by isotyping, immunoelectrophoresis, SDS–PAGE and the enzyme-linked immunoelectrotransfer blot technique, ELISA, and their recognition of carbohydrate or protein antigenic epitopes by periodate oxidation and trichloroacetic acid treatment of the antigen. It was used for detection of circulating schistosomal antigen in an antigen capture antibody sandwich ELISA on sera and urines of 58 S. mansoni-infected patients, 17 S. haematobium-infected patients, 15 parasite-free negative healthy controls and sera from 13 schistosomiasis-free patients harbouring Fasciola or hydatid infections. The percentage sensitivity of the assay in the serum of S. mansoni-infected patients was 98.4% and in urine 94.8%. A positive correlation was found between the number of faecal S. mansoni eggs and the circulating antigen, both in serum and in urine. Antigen circulating in urine correlated with that in the sera of S. mansoni patients. These data provide a sensitive and non-invasive method almost comparable with the use of sera for immunodiagnosis of schistosomiasis and an indirect way to reflect the intensity of infection. Australian Society for Parasitology.

Production of monoclonal antibodies for the identification of the eggs of Taenia solium.

Two antibodies, one monoclonal and one polyclonal, were produced and used to identify Taenia solium eggs, using the enzyme-linked immuno-electrotransfer blot technique (EITB). Different life-stages of Taenia solium and T. saginata, including eggs from gravid proglottids recovered, post-treatment, from patients infected with the tapeworms, and eggs of Diphyllobothrium pacificum and Hymenolepis nana from other patients were tested with these antibodies. The monoclonal antibody only recognized the eggs and immature proglottids of T. solium. The polyclonal antibody, however, not only reacted with the eggs, cysticerci and immature proglottids of T. solium but also with the eggs and immature proglottids of T. saginata. The sensitivity and specificity of the EITB were both 100% using the monoclonal antibody but only 78% and 60%, respectively, using the polyclonal antibody. Diagnostic bands for T. solium eggs corresponded to proteins of 22.5 kDa using the polyclonal antibody and 22.5-37 kDa using the monoclonal antibody. Species-specific fluorescence was obtained with an anti-T. solium monoclonal antibody which bound to egg-derived oncospheres of T. solium but not to those of T. saginata.

Identification of surface antigen of Trichomonas vaginalis.

Plasma membrane proteins of a Korean isolate of Trichomonas vaginalis HY-1 were fractionated for antigen analysis. Homogenates of T. vaginalis were fractionated by the differential centrifugation using sucrose step-gradient method. The interface layer from the 25%/45% sucrose was collected as a plasma membrane fraction and its purity was examined by transmission electron microscopy. The antigenicity of plasma membrane fraction was analysed by enzyme-linked immunoelectrotransfer blot technique with immune rabbit serum and compared with surface antigen labelled with N-hydroxysuccinimide-biotin. The fluffy fraction of 25%/45% sucrose interface was homogeneous and membrane particles were present as extended sheet and concentric vesicles showing typical trilamellar appearance under transmission electron microscope. Seven fractions at 40, 50, 60, 110, 130, 140 and 150 kDa were identified as the antigenic membrane proteins in EITB with anti HY-1 rabbit serum. The common band at 60 kDa was detected both in antigenic fractions of plasma membrane and surface protein labelled with NHS-biotin. This result indicates that this protein is considered as a major surface antigen of T. vaginalis. The role of this surface antigen at 60 kDa should be studied further.

Comparative antigen analysis of Trichomonas vaginalis by enzyme-linked immunoelectrotransfer blot technique

Analysis of six isolates of Trichomonas vaginalis was carried out with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunoelectrotransfer blot (EITB). Trichloroacetic acid-treated antigens of the 6 isolates revealed 25 protein profiles ranging 12-170 kDa of molecular weight in SDS-PAGE. In EITB, the specific immunogenic bands were visualized at 51 kDa and 96 kDa when HY-1 antigen was probed with different mice sera immunized with 6 isolates of T. vaginalis. The banding patterns with different sera showed isolate-to-isolate variability. In EITB, homologous antigen (HY-1) did not show any enhanced response in reacting to homologous antiserum (HY-1) when 6 isolates of T. vaginalis were probed with a single serum (HY-1). It is assumed that the different banding patterns of six isolates show isolate-to-isolate variability and immunogenic common bands in 41, 47, 74 and 94 kDa on EITB may connote the important significance on immune response in T. vaginalis infection.

Immunodiagnosis of Dracunculiasis by Falcon Assay Screening Test-Enzyme-Linked Immunosorbent Assay (Fast-Elisa) and by Enzyme-Linked Immunoelectrotransfer Blot (EITB) Technique

The Falcon assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) technique were used to test human sera with Dracunculus medinensis adult worm antigen in order to assess their potential value in the immunodiagnosis of dracunculiasis. The human sera used were from patients with prepatent and patent D. medinensis infections or from patients infected with other nematodes (Onchocerca volvulus and Loa loa) or trematodes (Schistosoma mansoni and S. haematobium), as well as uninfected Nigerian and Puerto Rican normal controls. In the FAST-ELISA, the sera from prepatent and patent dracunculiasis patients gave the highest absorbance values relative to normal human sera. The highest cross-reactivity was observed with onchocerciasis sera; no cross-reactivity was seen with sera from individuals with loiasis or schistosomiasis mansoni or haematobia. By the EITB, sera from dracunculiasis patients specifically recognized a 16 kDa protein (Dm 16) and antibodies to Dm 16 disappeared 2 months after worm extraction. Recognition of Dm 16 occurred from the late prepatent stage. A 17 kDa protein (Dm 17) was also recognized by dracunculiasis sera, but antibodies to Dm 17 disappeared more slowly and were present 1 year after recovery. The 16 kDa and 17 kDa antigens of D. medinensis may be useful in the immunodiagnosis of dracunculiasis.

Identification of a 17-kilodalton Fasciola hepatica immunodiagnostic antigen by the enzyme-linked immunoelectrotransfer blot technique.

Sera obtained from human patients, calves, sheep, and rabbits infected with Fasciola hepatica were tested by the Falcon assay screening test enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) techniques with Fasciola hepatica excretory-secretory antigens in order to evaluate their immunodiagnostic potential. The study included sera from 13 patients infected with F. hepatica or a history suggesting fascioliasis, 5 patients infected and treated with bithionol or praziquantel (3 were cured with bithionol), 10 patients infected with Schistosoma mansoni, 6 infected with Trichinella spiralis, and 13 controls and sera from calves, sheep, and rabbits with a primary F. hepatica infection. By FAST-ELISA with F. hepatica excretory-secretory antigens, the serum samples from fascioliasis patients gave the highest absorbance values, and the schistosomiasis patient sera gave intermediate values compared with a normal human serum control. Also by FAST-ELISA, the values for serum from patients with fascioliasis decreased steadily after cure, reaching normal levels 20 to 47 weeks postcure. In contrast, the serum from two patients who had been treated but were not yet cured had high levels of antibodies for up to 3 years of infection. By EITB, the serum samples from humans, rabbits, cattle, and sheep with fascioliasis recognized two antigenic polypeptides of 17 and 63 kilodaltons (kDa) in the form of sharp bands. For humans, this recognition lasted for at least 3 years of infection. Sera from individuals with schistosomiasis mansoni or trichinosis or from normal controls did not recognize the 17-kDa F. hepatica antigenic polypeptide. However, serum from one human with S. mansoni and one with T. spiralis infection has slight bands in the 63-kDa region, suggesting cross-reactivity. Reactivity to the 17-kDa polypeptide was absent in fascioliasis patients at 1 year postcure. Reactivity to the 63-kDa polypeptide was significantly diminished in fascioliasis patients at 1 year postcure. The sera from rabbits with a primary F. hepatica infection also recognized both the 17- and 63-kDa antigenic polypeptides by week 4 of infection. Reactivity to both antigens diminished significantly 6 weeks postcure and disappeared by 8 weeks postcure. The sera from infected cattle and sheep recognized these two antigenic polypeptides by week 8 of infection. These studies suggest that the 17-kDa F. hepatica excretory secretory antigen is an excellent candidate for the immunodiagnosis of acute and chronic fascioliasis. Purification of this antigen and its application to quantitative serologic tests will permit further analysis of its predictive value to evaluate cure.

Acquired immunity in schistosomiasis with purified fasciola hepatica cross-reactive antigens

We have previously demonstrated that a Fasciola hepatica-derived adult worm antigen, which is cross-reactive with Schistosoma mansoni and designated Fh SmIII(M), induce resistance to challenge infection with S. mansoni in mice. The current review concerns the methods developed to isolate and partially characterize a major component of Fh SmIII(M), a 12-kDa polypeptide, as well as immunity studies involving this antigen. Utilizing conventional gel filtration, followed by diethylaminoethyl (DEAE) Sephadex A-120 and monitoring the fractions by polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunoelectrotransfer blot techniques (EITB), we were able to isolate the 12-kDa antigenic polypeptide to homogeneity. Conventional gel filtration chromatography was followed by high-pressure, liquid anion, exchange chromatography, when highly purified material was needed, although the effective yields diminished drastically with the latter. Mice, rabbits and calves with a primary infection of F. hepatica developed antibodies (detectable in enzyme linked immunosorbent assay (ELISA) to the F. hepatica 12-kDa polypeptide within 2 weeks of infection. Mice with a primary infection of S. mansoni developed significant, but low, levels of anti-12-kDa antibodies by 7 weeks post-infection. Immunization of mice with microgram amounts of this 12-kDa polypeptide in Freunds' adjuvant resulted in the development of up to 77% less S. mansoni worms than the controls. Treatment with either endoglycosidase H, neuraminidase or dithiotreitol had no effect on the protein's mobility on sodium dodecyl sulfate (SDS)-PAGE or in its recognition by antibodies, suggesting the absence of carbohydrate moieties or disulphide bonds in relation to its antigenic determinants. Degradation by proteinase K further confirmed its polypeptide nature and points to recombinant DNA technology for the large-scale manufacture of this potential vaccine. Further use of this antigen in immunity studies should greatly contribute to the clarification of the mechanisms involved in cross-resistance against schistosomiasis.

Evaluation of Immunodiagnostic Antigens in the Excretory-Secretory Products of Fasciola hepatica

The metabolic antigens of F. hepatica have been shown to be a source of potential immunodiagnostic antigens. We have fractionated F. hepatica excretory-secretory (ES) antigens by conventional gel filtration and HPLC, analyzed these fractions in PAGE, and evaluated their immunogenicity by ELISA with sera from experimentally infected rabbits to identify potential serodiagnostic antigens for fascioliasis. A fraction enriched in high molecular weight components of ca. 150-160 kDa was found to be very reactive with sera from early fascioliasis. This fraction was successfully adapted to the DOT-ELISA, where titers up to 1:16,000 still appeared visually as positive. Both acute and chronic fascioliasis sera also recognized, in the enzyme-linked immunoelectrotransfer blot technique (EITB), prominent 25-30-kDa polypeptides that have previously been shown to be recognized by infected rabbits, cows, and sheep. We have therefore employed conventional gel filtration and HPLC gel exclusion chromatography as a 1-step procedure to obtain fractions enriched in antigens recognized in early fascioliasis. In addition, these antigens have been successfully applied to a sensitive, visual immunodiagnostic technique that can be easily employed in field studies.

Preparation and evaluation of the specificity of Parafilaria bovicola antigen for detection of specific antibodies by ELISA

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies in bovine sera against Parafilaria bovicola nematodes was developed and its sensitivity was compared with the immunodiffusion (ID) method. An exoantigen of P. bovicola which was shown to contain four major polypeptides was used in these procedures. The serological reactivity of the antigen polypeptides was defined by using the enzyme-linked immunoelectrotransfer blot technique (EITB) and whole-worm extract proteins. It identified only four serologically reactive polypeptides with sera from one experimentally infected calf and a verified field case. These two positive sera reacted mainly with four major antigens which coincided in molecular weights of the polypeptides of the exoantigenic preparation, namely, 43, 39, 28 and 25 KDa. Calves experimentally infected with P. bovicola showed a positive reaction with ELISA at 4 months after inoculation, and after this period a rapid increase in serum antibody response occured. In these cases the ID reaction was observed for the first time at 7 months after inoculation. The specificity of an ELISA method using crude exoantigen preparation of P. bovicola was tested for the diagnosis of bovine parafilariasis. No cross-reactivity was detected when the P. bovicola exoantigen preparation was tested against sera from calves experimentally infected with Onchocerca lienalis, as well as against the sera from cattle naturally infected with Dictyocaulus viviparus or from cattle chronically infected with Ostertagia ostertagi. In addition, testing of 740 field sera from cattle in areas non-endemic and endemic for P. bovicola indicated a specificity of the antigen preparation used. Forty sera from laboratory-confirmed field cases of P. bovicola infection were tested by ELISA and immunodiffusion. All of these sera were ELISA positive, whereas only 70% of these were positive in the ID test. Seven (2.1%) of 328 sera from 21 herds from non-endemic P. bovicola areas were ELISA positive, as opposed to none in the ID test. Of the 94 sera from six herds in areas endemic for P. bovicola infection, 51 (54%) were ELISA positive whereas only 24 (26%) were positive in the ID test. When 56 slaughtered cattle, with varying degrees of meat condemnations due to parafilariasis, were tested for P. bovicola specific antibody, 91% of the serum samples were positive by ELISA. These results suggest that the exoantigen of P. bovicola can be used in a sensitive and reliable serological detection of parafilariasis by ELISA.

Biochemical and immunological characterization of lipase during its secretion through cytoplasmic and outer membrane of acinetobacter calcoaceticus 69 v

The secretion of lipase through the cytoplasmic and outer membrane of Acinetobacter calcoaceticus 69 V was studied in relation to the growth phase. Lipase activity and its distribution in both membrane fractions was analysed by using membrane-dissolving and membrane-preserving assay conditions. The liberation of the enzyme from the cytoplasmic and outer membrane by means of deoxycholate was determined. With the help of antibodies the sidedness of the lipase of both membrane fractions in relation to the growth phases was analysed. With all these results, a model was assembled. In this model enzymatically active lipase first accumulates in the cytoplasmic membrane, showing properties of an intrinsic protein. Subsequently, the enzyme accumulates in the outer membrane with its attachment at the inner surface of this membrane being the next step. Out of the overloaded outer membrane the lipase is released into the surrounding medium without cell lysis or membrane-blebbing. The cytoplasmic and outer membrane-bound lipase were identical with the extracellular mature enzyme, as suggested by enzyme-linked immunoelectrotransfer blot technique.

Human Serologic Response to Subcellular Antigens of Trichomonas vaginalis

Trichomonas vaginalis organisms were processed by differential centrifugation to yield defined subcellular fractions. The various fractions were processed by methods to enhance solubility of membranes and other components. The soluble materials were analyzed for immunogenicity by a quantitative kinetic enzyme-linked immunosorbent assay (k-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) technique using human patient serum. The principal antibodies in the human serum were directed toward cytosol components, not against membrane components, although all fractions contained multiple antigenic moieties.

Analysis of antigen specificity using monoclonal and polyclonal antibodies to Cysticercus cellulosae by enzyme-linked immunoelectrotransfer blot technique.

To analyse the antigen specificity of patients sera from 24 confirmed neurocysticercosis and a monoclonal antibody, SDS-PAGE using 10-15% linear gradient gel and EITB were done. Cystic fluid, saline extracts of scolex and of whole worm of C. cellulosae, saline extracts of sparganum, hydatid cyst fluid, saline extracts of Fasciola, Clonorchis and Paragonimus were used as antigen. Of protein bands in cystic fluid of C. cellulosae, patient sera reacted frequently to bands of 152, 94, 64, 48, 24, 15, 10 and 7 kDa proteins. To saline extracts of scolex and whole worm of C. cellulosae, patients sera reacted frequently to 94, 64, 52, 39, 34, 15 and 10 kDa bands. Two bands in sparganum extract (130 and 64 kDa) and two bands in hydatid cyst fluid (52 and 27 kDa) were cross-reacting bands with sera from cysticercosis patient. Saline extracts of Fasciola, Clonorchis and Paragonimus did not exhibit cross-reacting bands. Monoclonal antibody to cystic fluid of C. cellulosae was found to react with low molecular weight proteins of 15, 10 and 7 kDa.

Characterization of protein and mannan polysaccharide antigens of yeasts, moulds, and actinomycetes.

Antigens in coccidioidin were compared with purified subfractions via tandem immunoelectrophoresis (IEP) and by a combination of advancing line and crossed IEP. Rocket IEP was suitable for titrating the reactions and showing the relationship between column fractions. These techniques required multicomponent antisera produced by hyperimmunization over many months and by the use of known standard migration pairs. The IEP variations were used to chart the development of antisera against coccidioidin factors, to monitor antigen purifications, and to test the immunochemical homogeneity of an isolated antigen. Mannose-based heteroglycans of Cryptococcus neoformans were recovered from the culture filtrate. After precipitation of the major viscous glucuronoxylomannan (GXM) with ethanol or cetyltrimethylammonium bromide, the supernate is reserved because it contains a galactoxylomannan (GalXM). After removal of glucuronic acid from the GXM, the resulting xylomannan of serotype A was amenable to 13C-nuclear magnetic resonance (NMR) spectrometry; it revealed nonreducing xylose, alpha-1,3-mannose, and alpha-1,2/1,3 disubstituted mannose, thus confirming by an independent means what was previously known. The characterization sequence of GalXM included: (1) gas-liquid chromatography (GLC) of neutral sugars as peracetylated aldononitriles; (2) methylation-fragmentation GLC mass spectrometry to determine the glycosidic linkages; and (3) 13C-NMR showing similarities to mannan of Saccharomyces cerevisiae. Affinity chromatography of the GalXM on concanavalin A separated the galactoxylo component from an adsorbed mannoprotein. Selection of monoclonal antibodies (MAbs) relies on presumptive enzyme immunoassays (EIAs) or radioimmunoassays for rapid screening of clones and for determination of isotypes; however, higher resolution confirmatory tests are needed to obtain MAbs of desired specificity. MAbs against Candida tropicalis mannan were labeled with horseradish peroxidase to use for detecting mannan in serum. MAbs against the partially purified "m" factor of histoplasmin were characterized by the enzyme-linked immunoelectro-transfer blot technique (EITB), revealing unsuspected complexity in the antigen. Secreted proteins of Nocardia asteroides were isoelectrically focused; three proteins, identified by EITB as promising to be specific for that actinomycete, were cut out of gels and used to immunize mice for production of MAbs. The fimbriae of Actinomyces viscosus and A. naeslundii that mediate lactose-reversible coagglutination with Streptococcus sanguis have been used to evoke MAbs.(ABSTRACT TRUNCATED AT 400 WORDS)

Calibration of prestained protein molecular weight standards for use in the “Western” or enzyme-linked immunoelectrotransfer blot techniques

Prestained protein molecular weight standards allow easy, direct visual location of electrophoretically transblotted lanes on nitrocellulose. They also provide a simple and accurate means for calibrating the molecular weights of resolved bands. Commercial prestained protein molecular weight standards, however, appear to have significantly different molecular weights from the original unstained proteins. We describe a calibration of these prestained molecular weight standards.

India ink staining of proteins on nitrocellulose paper

India ink staining of proteins that have been electrotransfer blotted onto nitrocellulose paper is described. This stain proved to be a useful adjunct to the enzyme-linked immunoelectrotransfer blot technique. It is more sensitive than Coomassie blue, amino black, and fast green stains and is simple to use.

29] Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis

This chapter describes the enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. The enzyme-linked immunoelectrotransfer blot technique (EITB) combines the high resolving power of gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and the high sensitivity of the enzyme-linked immunosorbent assay (ELISA) to produce an extremely powerful qualitative tool for studying antigen–antibody pairs. Using this procedure, antigens electrophoretically resolved on SDS–PAGE are transferred onto nitrocellulose or diazo sheets and identified by ELISA methods. The chapter discusses the general comments about the technique. The EITB is conducted in three stages: (a) the antigen mixture that can be extremely complex, such as solubilized whole cells, is first resolved by gel electrophoresis (two-dimensional gels can also be used); (b) the resolved gel is then electrophoretically blotted onto nitrocellulose sheets; and (c) the blotted nitrocellulose is then developed by ELISA.