A portable reflectometric photometer for quantitative enzyme immunoassay

A portable optoelectronic reflectometric photocolorimeter was designed. The photocolorimeter implements two operating modes (transmission and reflection), and its performance was tested in two systems of enzyme immunoassay for testosterone: microplate ELISA and membrane dot-ELISA. The detection threshold for microplate ELISA and membrane dot-ELISA was 0.1 and 0.6 ng/ml, respectively. The coefficient of correlation between the photocolorimeter readings and conventional photometric methods is r=0.999. Relative standard deviation of the results of photocolorimetric measurements (n = 4) within the optical density range of 0.03 to 1.00 ranged from 3.4 to 0.7%. The simple and versatile design of the photocolorimeter makes it appropriate for testing various substances both in laboratory settings and in the field.     

DOT-dye-immunoassay for the diagnosis of schistosomiasis mansoni.

A new serological assay dot-dye-immunoassay (dot-DIA) was evaluated for the diagnosis of schistosomiasis mansoni. This method consist of four steps: (a) biding of antigens to a nitrocellulose membrane (NC); (b) blocking of free sites of the NC; (c) incubation in specific primary antibody; (d) detection of primary antibody reactivity by color development using second antibody coupled to textile dyes. Sera from 82 individuals, 61 with Schistosoma mansoni eggs in the stool and 21 stool negative were tested by ELISA, dot-ELISA, and dot-DIA. A high level of agreement between the methods tested was observed for all sera tested: ELISA x dot-ELISA: 95.1%, ELISA x dot-DIA: 92.7% and dot-ELISA x dot-DIA: 97.6%. In this study, dot-DIA proved to be a feasible, sensitive, rapid and practical test for the diagnosis of schistosomiasis.     

Comparative evaluation of recombinant BP26 protein for serological diagnosis of Brucella melitensis infection in goats

An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of caprine brucellosis with purified recombinant BP26 (outer membrane protein 28) of Brucella melitensis 16M produced in Escherichia coli. Brucella protein named CP28, BP26, or Omp28 has been identified as an immunodominant antigen in infected cattle, sheep, goats, and humans. The recombinant BP26 (rBP6) ELISA performed with serum samples (n = 1738) taken from organized farms and field goats from Northern India and tested in two different batches of 778 and 960 animals for brucellosis. Positive results were compared with the traditional serum agglutination test (SAT), complement fixation test (CFT) and dot-ELISA. The diagnostic sensitivity of rBP26 ELISA, SAT, CFT and dot-ELISA was 87.5%, 56.25%, 62.5% and 75% respectively. The specificity of the rBP26 ELISA, SAT, CFT and dot-ELISA was 90%, 75%, 80%, and 85% respectively. The results of rBP26 ELISA positive animals were further compared and evaluated by tissue PCR using B. melitensis BP26 gene as target. This resulted in 100% positive correlation between rBP26 ELISA and BP26 PCR. Thus, these results confirmed the importance of BP26 as a suitable antigen and rBP26 ELISA, if well standardized, proved to be a good screening test for the serological diagnosis of caprine brucellosis.     

Comparative diagnostic potentiality of ELISA and dot-ELISA in prepatent diagnosis of experimental Fasciola gigantica infection in cattle

A glycoprotein (27 kDa) was isolated from crude somatic antigen of Fasciola gigantica by two steps affinity chromatography and was used in early detection of experimental fasciolosis in cattle by indirect ELISA and in dot-ELISA formats. Although, anti-27 kDa antibodies could be detected after 3 weeks post infection (WPI) by dot - ELISA which was one week later than indirect ELISA. The test, dot-ELISA, was more convenient in field application. By the test (dot-ELISA) the infection could be equally detected in animals infected with 100, 200 and 300 metacercariae of F. gigantica with high sensitivity. Further, the antigen (27 kDa) was not found to react with goat sera infected with Paramphistomum epiclitum, which are giving strong reaction to homologous immature and mature fluke antigens of P. epiclitum.     

A dot-ELISA mimicry western blot test for the detection of swine trichinellosis

A dot enzyme-linked immunosorbent assay (dot-ELISA) using antigens purified by monoclonal antibody affinity chromatography was developed for detecting Trichinella spiralis infection in swine. The test was as sensitive as an ELISA using excretory-secretory products as antigen and western blot analysis, and nearly as specific as the western blot. The dot-ELISA detected all of 20 low infections (0.08-4.74 larvae per gram of diaphragm), most of them by 5-6 wk postinfection. Sera from 1,960 farm-reared swine were tested by conventional ELISA, dot-ELISA, and western blot. Of the 1,960 sera, 262 (13.4%) were considered positive on conventional ELISA, 16 (0.82%) by dot-ELISA, and 15 (0.77%) by western blot. The improved specificity was achieved by employing species-specific denatured antigens. More importantly, the dot-ELISA was much simpler to perform than western blot analysis. The principles employed in this test can be adapted to other infectious diseases, such as AIDS.     

An evaluation of the dot-ELISA procedure as a diagnostic test in an area with a high prevalence of human Toxocara canis infection

The aim of this work was to evaluate a dot-enzyme-linked immunosorbent assay (dot-ELISA) using excretory-secretory antigens from the larval stages of Toxocara canis for the diagnosis of toxocariasis. A secondary aim was to establish the optimal conditions for its use in an area with a high prevalence of human T. canis infection. The dot-ELISA test was standardised using different concentrations of the antigen fixed on nitrocellulose paper strips and increasing dilutions of the serum and conjugate. Both the dot-ELISA and standard ELISA methods were tested in parallel with the same batch of sera from controls and from individuals living in the problem area. The best results were obtained with 1.33 μg/mL of antigen, dilutions of 1/80 for the samples and controls and a dilution of 1/5,000 for the anti-human IgG-peroxidase conjugate. All steps of the procedure were performed at room temperature. The coincidence between ELISA and dot-ELISA was 85% and the kappa index was 0.72. The dot-ELISA test described here is rapid, easy to perform and does not require expensive equipment. Thus, this test is suitable for the serological diagnosis of human T. canis infection in field surveys and in the primary health care centres of endemic regions.     

Cellulose acetate as solid phase in ELISA for plague

Antigen from Yersinia pestis was adsorbed on cellulose acetate discs (0.5 cm of diameter) which were obtained from dialysis membrane by using a paper punch. ELISA for human plague diagnosis was carried out employing this matrix and was capable to detect amount of 1.3 microg of antigen, 3,200 times diluted positive serum using human anti-IgG conjugate diluted 1:4,000. No relevant antigen lixiviation from the cellulose acetate was observed even after washing the discs 15 times. The discs were impregnated by the coloured products from the ELISA development allowing its use in dot-ELISA. Furthermore, cellulose acetate showed a better performance than the conventional PVC plates.     

Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

A dot enzyme-linked immunosorbent assay (dot-ELISA) was standardized using excretory-secretory antigens of Toxocara canis for the rapid immunodiagnosis of human toxocariasis. Thirty patients with clinical signs of toxocariasis, 20 cases with other parasitic diseases, and 40 healthy subjects were tested. A total of 0.2 ng of antigen per dot, serum dilution of 1:160 and dilution conjugate of 1:1000 were found optimal. The sensitivity and specificity of the assay were 100 and 95%, respectively. Comparable sensitivity of dot-ELISA and the standard ELISA was obtained, but only 3 cross-reactions occurred in the dot-ELISA, compared with 6 in the standard ELISA. Dot-ELISA is simple to perform, rapid, and low cost. Large-scale screening studies should be done to evaluate its usefulness under field conditions.     

单抗免疫斑点法和组织印迹法检测百合无症病毒

应用抗百合无症病毒（Lily symptomless virus,LSV）的单克隆抗体,建立了快速检测田间样品的免疫斑点法（Dot—ELISA）和组织印迹法（Tissue blot-ELISA）体系。LSV单抗稀释2,000倍时,Dot-ELISA中病叶粗汁液可被检出的最大稀释度为1：640。Tissue blot—ELISA中样品一次平切后第1次印迹与Dot—ELISA样品1：40稀释的结果相当,前4次印迹均可获得满意的显色效果。常规Tissue blot-ELISA的灵敏度低于Dot—ELISA,但用丙酮处理点过样的硝酸纤维素膜后,二者的灵敏度相当,且Tissue blot—ELISA操作更简便,适合田间大量样品的快速检测。     

Uniformity of protective antigens among isolates of the cattle tick, Boophilus microplus

Abstract. Gut membrane antigens were extracted from ten isolates of the cattle tick Boophilus microplus; the antigen extracts were probed with bovine antisera and three murine monoclonal antibodies (mAbs) in Western blots and dot-ELISA. The antisera had been obtained from cattle which were vaccinated with larval and gut extracts of B.microplus , and which were subsequently protected (84% and 94% respectively) against challenge with B.microplus. One of the mAbs (QU13) has been demonstrated to precipitate protective antigens from the midgut of B.microplus. Gut antigens from all ten isolates displayed similar reactivity profiles against bovine antisera and also against mAbs in Western blots. The end-point titres of antigens in dot-ELISA showed four-fold variation between isolates against bovine antisera, and also against mAb QUI 3. Larval membrane antigen extracted from N-strain B.microplus reacted with QU13 in dot-ELISA, indicating that protective antigens are common to both larval and adult stages of B.microplus. It was concluded that protective antigens recognized by QUI3 and antigens recognized by sera from protected cattle were conserved between the ten isolates examined, and between life-cycle stages.     

Dot-ELISA vs. PCR of fine needle aspirates of tuberculous lymphadenitis: a prospective study in India

OBJECTIVE: To evaluate an in-house dot-enzyme-linked immunosorbent assay (ELISA) for confirmation of clinically suspected cases of tuberculous lymphadenitis (TBLN). STUDY DESIGN: The study was performed at the postgraduate departments of microbiology and pathology of a tertiary-care teaching hospital in India. Suspected cases of TBLN were prospectively enrolled. Fine needle aspiration was done of enlarged lymph nodes in all patients, and 2 smears were prepared, 1 for acid-fast bacillus (AFB) demonstration and the other for cytologic examination. The remaining material was tested with in-house dot-ELISA and by IS6110 amplification with polymerase chain reaction (PCR), for diagnosis of TBLN. RESULTS: ELISA was more sensitive and detected 93.2% of cases. PCR and fine needle aspiration cytology (FNAC) detected 82.5% and 61.0% cases, respectively. AFB positivity was 33.1%. CONCLUSION: Application of dot-ELISA was more sensitive but less specific as compared to PCR. PCR, though expensive, should be used in problem cases because of its high specificity.     

Assessment of equine sperm mitochondrial function using JC-1

The fluorescent carbocyanine dye, JC-1, labels mitochondria with high membrane potential orange and mitochondria with low membrane potential green. Evaluation of mitochondrial membrane potential with JC-1 has been used in a variety of cell types, including bull spermatozoa; however, JC-1 staining has not yet been reported for equine spermatozoa. The aim of this study was to apply JC-1 staining and assessment by flow cytometry or a fluorescence microplate reader for evaluation of mitochondrial function of equine spermatozoa. Six ejaculates from four stallions were collected and centrifuged through a Percoll gradient (PERC). Spermatozoa were resuspended to 25 x 10(6) cells/mL, samples were split, and one sample was repeatedly flash frozen (FF) in LN2 and thawed. The following gradients of PERC:FF were prepared: 100:0 (100), 75:25(75), 50:50 (50), 25:75 (25) and 0:100 (0). Samples were stained with 2.0 microM JC-1 and assessed for staining by flow cytometry and by a fluorescence microplate reader. A total of 10,000 gated events was analyzed per sample with flow cytometry. The mean percentage of cells staining orange for the 100, 75, 50, 25 and 0 treatments was 92.5, 72.8, 53.4, 27.3 and 7.3, respectively. The expected percentage of spermatozoa forming JC-1 aggregates was correlated with the actual percentage of orange labeled sperm cells determined by flow cytometry (r2=0.98). Conversely, JC-1 monomer formation was negatively correlated with expected mitochondrial membrane potential (r2=-0.98). The blank corrected orange fluorescence, assessed by microplate assay, was significantly (P<0.0001) correlated with the expected (r2=0.49) and with the flow cytometric (r2=0.50) determination of percentage of spermatozoa with mitochondria of high membrane potential. Total orange and orange:green fluorescence was also correlated with mitochondrial function. These results indicate that JC-1 staining can accurately detect changes in mitochondrial membrane potential of equine spermatozoa. The relative fluorescence of JC-1 labeling patterns of equine spermatozoa can be accurately and objectively determined by flow cytometry and by a fluorescence microplate reader assay.     

Qualitative and quantitative detection of aflatoxin B1 in poultry sera by enzyme-linked immunosorbent assay

An indirect competitive inhibition type enzyme-linked immunosorbent assay (ELISA) has been developed for the detection of aflatoxin B₁, in poultry sera. Preincubation of aflatoxin B₁, samples with the antibody prior to competition yielded better results in terms of higher sensitivity. After competition, amount of antibody bound to solid phase was measured by incubation with anti-rabbit immunoglobulins coupled with horse raddish peroxidase. Intensity of colour decreased as the amount of free aflatoxin B₁, increased. Final detection of aflatoxin B₁, was made by (i) visual comparison with standard aflatoxin B₁ using dot-ELISA (qualitative) and (ii) by plate-ELISA, where optical density was measured at 492 nm (quantitative). Plate-ELISA was more sensitive than dot-ELISA, with sensitivity limits being 100 fg and 1 pg per 10 μl, respectively. However, due to ease and speed of performance, dot-ELISA has greater potential as a test for the diagnosis of mycotoxicosis at the field level.     

Differential expression of CMG peptide and crustacean hyperglycemic hormones (CHHs) in the eyestalk of the giant tiger prawn Penaeus monodon

Mouse antiserum against C-terminal amide of Pem-CMG (a peptide in the family of CHH/MIH/GIH) penta-deca peptide (RPRQRNQYRAALQRLamide=CMG-15) was generated and used for localization of the peptide in tissue and extract of the eyestalk of Penaeus monodon by means of immunohistochemistry and dot-ELISA in comparison with anti-T+ antiserum (T+=YANAVQTVamide : the putative C-terminal amide of crustacean hyperglycemic hormone (CHH) of Macrobrachium rosenbergii). The anti-CMG-15 antiserum did not show cross-reactivity to T+ peptide by dot-ELISA and vice versa for anti-T+ antiserum. In dot-ELISA of eyestalk extract of P. monodon after one step separation by RP-HPLC, anti-CMG-15 antiserum recognized different peptide fractions (F38-39) from those recognized by anti-T+ antiserum (F19, 40-41 and 47-51). Most of the T+ immunoreactive fractions (except F19) show higher hyperglycemic activity than the CMG immunoreactive fractions. In immunohistochemical localization, anti-CMG antiserum recognized only 2-3 neurons in medulla terminalis X-organ complex (MTXO) with long processes terminated in the sinus gland. The CMG-immunoreactive neurons were clearly distinct from CHH containing neurons situated in the same area. This evidence confirms the existing of CMG peptide which may play distinct roles from CHHs in hormonal regulation in P. monodon.     

A direct dot-enzyme immunoassay to detect human ovulation.

This paper describes an original dot-enzyme-linked immunosorbent assay (ELISA) for predicting ovulation in women, based on the detection of the pre-ovulatory estrogen peak in urine. A monoclonal anti-estrogen antibody is used which recognizes not only free estrogens but also some of their urinary metabolites (17-glucuro- and sulfo-conjugates) allowing a direct assay on early morning urines. Antigen is immobilized as a spot on a nitrocellulose membrane which is immersed in urine in the presence of this antibody. A peroxidase-labeled second antibody allows the detection of the first antibody bound to the membrane. Antigen and anti-estrogen antibody concentrations are chosen to obtain a maximal enzymatic coloration of spots corresponding to basal urinary estrogen levels and no coloration corresponding to the pre-ovulatory surge. Six menstrual cycles were studied, comparing dot-ELISA results with patterns of: (1) urinary estrogens measured by RIA either directly or after hydrolysis and extraction, and (2) basal body temperatures. The validity of the pre-ovulatory signal obtained and the requirements for an adaptation of this methodology to a reliable home kit are discussed.     

Hypersensitive immunoassay by using Escherichia coli outer membrane with autodisplayed Z-domains

In this work, the Z-domain of protein A was autodisplayed on the surface of the Escherichia coli outer membrane as a fusion protein of AIDA-1, and then layered on a microplate in order to develop a hypersensitive immunoassay. By using microplates with controlled hydrophilicity the formation of outer membrane layer was carried out, and the driving force for the layer formation was proven to be a hydrophobic interactions. Through the orientation control of the immobilized antibodies by using autodisplayed Z-domains, the limit of detection (LOD) and the sensitivity of this new immunoassay were determined to have improved as much as 30-fold compared to the conventional ELISA. The applicability of the new immunoassay for medical diagnosis was demonstrated by the detection of C-reactive protein (CRP) which is known to be a biomarker protein for inflammatory diseases.     

A quantitative fluorescence enzyme immunoassay for plant cytokinins

An enzyme-linked immunosorbent assay (ELISA) which used 4-methylumbelliferyl phosphate as an enzyme substrate was used to quantify two plant cytokinins. This assay detected as little as 0.03 pmol (approximately 10 pg) of cytokinin in microplate wells coated with a cytokinin-ovalbumin conjugate. The method measured competition between free cytokinin and the bound conjugate for reaction with monoclonal anticytokinin antibodies and used a standard curve prepared by use of known amounts of free cytokinin to quantify hormone levels in unknown samples. Standard curves which consisted of logit/log plots of fluorescence units versus picomoles of competing cytokinin measured from 0.03 to 256 pmol (approximately 10-85,000 picograms) of zeatin riboside (ZR) or isopentenyl adenosine. The fluorescence ELISA was compared with radioimmunoassay for the quantification of ZR in wheat (Triticum aestivum L., cultivar Stephens) seed samples. This fluorescence ELISA method is recommended for use in combination with a fractionation method, such as HPLC, to quantify cytokinins present in plant extracts.     

Application of monoclonal antibodies generated against Panton-Valentine Leukocidin (PVL-S) toxin for specific identification of community acquired methicillin resistance Staphylococcus aureus

Panton-Valentine Leukocidin (PVL) produced by community acquired methicillin Staphylococcus aureus (CA-MRSA) involved in skin and soft-tissue infections and necrotizing pneumonia comprised of two fractions, namely PVL S and PVL F. In the present study, three monoclonal antibodies designated as MAb1, MAb9 and MAb10 were generated against recombinant PVL-S (35 kDa) protein of S. aureus. All the three MAbs specifically reacted to confirm PVL-S positive strains of S. aureus recovered from clinical samples in Western blot analysis. Similarly all the three MAbs did not show any binding to other tested 14 different pathogenic bacteria belonging to other genera and species in Western blot analysis. Furthermore, a simple dot-ELISA method was standardized for the identification of PVL-S toxin containing S. aureus strains. Initially in dot-ELISA, Protein A (Spa) of S. aureus posed background noise problems due to the non-specific binding of antibodies resulting in false positive reactions. With the addition of 10 mM diethylpyrocarbonate (DEPC) along with 5% milk in PBS in the blocking step prevented this non-specific binding of Spa to mouse anti-PVL monoclonal antibodies in dot-ELISA. Once standardized, this simple dot-ELISA was evaluated with nine PVL positive strains recovered from food, environmental and clinical samples and the results were compared with PCR assay for the presence of PVL toxin genes and also with Western blot analysis. A 100% correlation was found between dot-ELISA, PCR assay and Western blot analysis. Collectively our results suggest the newly developed simple dot-ELISA system can be of immense help in providing, rapid detection of the PVL toxin containing S. aureus strains at a relatively low cost and will be a valuable tool for the reliable identification of CA-MRSA.     

Differentiation between thermophilic Campylobacter species by species-specific antibodies

P.L. GRIFFITHS. G.S. MORENO AND R.W. A. PARK. 1992. The four species of thermophilic camplyobacters, Campylobacter jejuni, C. coli, C. upsaliensis and C. lari , are difficult to distinguish from each other because of their lack of reactivity in many conventional biochemical and physiological tests. Those tests which do discriminate sometimes give discordant results. Species-specific antibody preparations (APs), capable of discriminating between the thermophilic campylobacter species by dot-ELISA, were raised by inoculation of mice with partially purified membrane protein. The APs produced were absorbed with cells of cross-reactive species and tested by dot-ELISA against reference and natural strains, the identities of which were confirmed by DNA/DNA hybridization. The results showed that such APs could be useful as an alternative to DNA/DNA hybridization for rapid species identification, for example in epidemiological surveys. Western blotting experiments with the APs showed that the specificity of the antibodies was not due to a single antigen.     

Dot enzyme-linked immunosorbent assay (dot ELISA) for antibodies to Encephalitozoon cuniculi

A dot-ELISA procedure was developed to detect antibodies against Encephalitozoon cuniculi. Sera from 84 rabbits, 22 dogs, 18 squirrel monkeys and 200 mice were tested by dot-ELISA and most also were tested by immunofluorescence. Comparison of the two tests showed that there was excellent agreement of the results (Kappa values greater than or equal to 0.74) in all four species. Dot-ELISA is a simple, quantitative, rapid alternative to immunofluorescence when large numbers of serum samples must be evaluated.