Las teorías neomarxistas

ELISA and immunoblot have confirmed efficacy and use in immunodiagnostic studies. They are relatively easy to set up in any laboratory but have the limitation of long assay-time especially in mass-screening community studies. A rapid

immunological diagnostic method that can be used in both the field and for initial differentiation of CE and AE at clinical level would be very useful (Craig et al., 2000; Bartholomat et al., 2000; Wang et al., 2004). Rapid serological test formats, such as dot-ELISA have been assessed for both human CE and AE, and although useful when associated with mass ultrasound screening, were temperamental and subjective in reading (Rogan et al., 1991).

Dot immuno-gold filtration assay (DIGFA) is a rapid immunodiagnostic test that uses colloidal gold conjugated antibody or antigen instead of enzyme or fluorescence conjugates (Valkirs and Barton, 1985; Beesley 1989; Chun and Chu, 1989; Xiao et al., 1995; Reddy 2006). Antibody or antigen are attached on a nitrocellulose membrane, heparinized blood or serum applied, and colloidal gold conjugated anti-human antibodies give a color change to show positive or negative. The procedure is similar to ELISA, but uses an infiltration system with colloidal gold to give a rapid result.

1.7.1 Colloidal gold preparation

Colloidal gold, also called "nanogold", is a suspension (colloid-like) of sub-micrometre-sized particles of gold in a fluid formation. The liquid shows usually an intense red colour for particles less than 100 nm (Wessling et al, 1996). The gold particles themselves can come in a variety of shapes, e.g. spheres, rods, cubes, and caps are some of those frequently observed.

Known since the ancient Roman times, colloidal gold was originally used as a method of staining glass as an intense red which the process was refined by Andreus Cassius and Johann Kunchel in the 17th century. In 1842, John Hershel found a method that used colloidal gold to record images on paper, called chrysotype (from the Greek word for gold). The first pure sample of colloidal gold was prepared in 1857 by Michael Faraday who used phosphorus to reduce a solution of gold chloride, which he called 'activated gold'. Faraday was also the first to recognize that the colour was due to the size of the gold particles. Modern scientific evaluation of colloidal gold did not begin until Michael Faraday's work of the 1850s(Mulvaney, 2003, Reddy, 2006). Colloidal gold has been widely applied in a wide variety scientific and technological fields, including electronics, nanotechnology due to its unique optical, electronic, and molecular-recognition properties, etc. (Rao et al, 1999, Mulvaney, 2003, Reddy 2006).

Generally, colloidal gold is produced in a liquid formular ("liquid chemical methods") by reduction (usually sodium citrate or sodium borohydride) of hydrogen tetrachloroaurate (HAuCl4). After dissolving HAuCl4 in distilled water,

the boiled solution is rapidly stirred while a reducing agent is added. This changes

Au3+ ions to reduce to un-ionized gold atoms. The solution becomes

supersaturated when most gold atoms reduced, and residual gold gradually starts to precipitate as sub-nanometer particles while other rest of the gold atoms stick to the existing particles. The similar size of particles could be obtained if the solution is stirred vigorously enough. To prevent the particles from aggregating, some sort of stabilizing agent that sticks to the nanoparticle surface is usually added. They can be functionalized with various organic ligands to create organic-inorganic hybrids with advanced functionality (Beesley, 1989; Reddy 2006).

Generally used sythenization methods for colloidal gold were sodium citrate reducing (Turkevich et al., 1951; Frens et al., 1970; Beesley 1989, Reddy 2006; Pong et al., 2007); organic reducing (like toluene) using tetraoctylammonium bromide (TOAB) (Brust et al. 1994-1998) and sonolysis (Zhang, et al., 2006)

1.7.2 Colloidal gold based immunodiagnostic assays

Colloidal gold conjugated antibody/antigen could be used in immunodiagnostic assays replacing enzyme or fluorescence dyes. Colloidal gold technique was first used in 1970s for immunological purpose in locating special antigen on the surface of cell through electric microscopy (Faulk and Taylor 1971; Horisberger et al., 1975).A dot immunogold infiltration assay (DIGFA) was used for detect HIV in 1989 (Beesley 1989; Chun and Chu, 1989). This technique is basically involves the antigen or antibody attached to a nitrocellulose membrane with a filter tissue underneath with the target antibody / antigen be combined on the membrane (Fig. 1.11). The procedure is similar to ELISA but using an infiltration system and colloidal gold provide a more rapid and reliable result.

Another related technique is the immunogold chromatography assay (IGCA) which has been widely used for example in HCG hormone detection for early diagnosis of pregnancy (May 1991). Gold conjugate and sample are allowed to flow to the capture (antigen or antibody) on the nitrocellulose membrane through chromatography, reaction in precipitation as a line (Millipore corp 1996). Other colloidal dyes such as Palami Red (UK), Samaron Red (Hoechst F.R.G) etc. were

used as an alternative to colloidal gold particles for antigen detection immunoassay with dipsticks (Snowden and Hommel, 1991).

Preparation of antibody-colloidal gold conjugate (Beesley, 1989) was using optimum volume of 1mg/ml antibody for conjugation with colloidal gold; and blocking with 1-10% BSA or gelatin or PEG 20,000 according different design; purification using centrifugation with different speed due to different size of colloidal gold; column chromatography to obtain the evenly size of gold particle. Dot Immuno-Gold Filtration Assay (DIGFA)

Fig. 1.16: The device for DIGFA. A: ready-to-use device; B: different

components of this device, including a plastic bottom for backup, an absorbent pad for collecting all the liquid, a piece of NC membrane as a reaction barrier, and a top cap with a middle well. C: reactions occurred in DIGFA, with specific antigen binded to NC membrane (solid phase), specific antibody in patient’s sample would combined with this antigen, and a colloidal gold conjugated anti-Ig could bind to above combination and showed color to judge the reaction.

DIGFA, using nitrocellulose (NC) membrane as a carrier which pre-coated antigen or antibody as a capture, is similar to immunoblot procedure excepted of a substrate step (Fig. 1.16). A sample (including antibody or antigen) dropped on the NC membrane followed by colloidal gold conjugate and washing buffer, antigen-antibody-conjugate would show red dot on the membrane and excessive reagents would filtrate through NC membrane to an absorbent tissue. Antigen or

Top cap NC membrane Absorbent pad Bottom C

antibody in sample / conjugate would combined to the antibody/antigen on NC membrane when the solution past through NC membrane and thus was more like a concentration with affinity chromatography. This made the procedure rapid (about 5 minutes), and washing step through filtration became more simple and rapid. DIGFA became one of the point of care test (POCT) due to rapid and simple (no special device needed).

Fig. 1.17: Immunogold chromatography assay strip

Immunogold chromatographic Assays, also called lateral flow assays or simply strip assays, have been developed for some time. This technique is based on an immunochromatographic procedure that utilizes antigen-antibody properties in a novel manner and provides rapid detection of analyte (Fig. 1.17) (Millipore corp 1996). They possess four benefits of user-friendly format, very short time to get test results, long-term stability over a wide range of climates and relatively inexpensive to make. These characteristics render it ideally suited for on site testing by untrained personnel, rapid point of care testing, and testing in the field for various environmental and agricultural analytes. In addition, they provide reliable testing that might not otherwise be available to third world countries. The principle behind the test is straightforward. Basically, any ligand that can be bound to a visually detectable solid support, such as dyed microspheres, can be tested for qualitatively, and in many cases even semi-quantitatively. Some of the more

common lateral flow tests currently on the market are tests for pregnancy (May 1991, Millipore corp 1996), strep throat , and chlamydia (Liu 2005; United States Patent 5393658; European Patent EP0258963; Shim et al., 2006). These are examples of conditions for which a quantitative assay is not necessary.

The main advantages of immunogold techniques are rapid and efficient. The procedure is simple, cheap, and easy to apply and does not require a reader device or machine and therefore cheap. Sensitivity could also be comparable to ELISA if good quality controls have been included. Such test have been used for diagnosis in some parasite diseases, such as antibody detection circulating antigen in toxoplasmasis (Wang et al., 2007; Li et al., 2004; Tang and Zhou, 2004), schistosomiasis (Zhu et al., 2002; Wu et al., 2005), cysticercosis (Liu et al., 2001, 2002), circulating antigen detection for Plasmodium falciparum (Moody, 2002; Li et

al., 2004; Grobusch et al., 2004) and Plasmodium vivax, Wuchereria bancrofti and Leishmania spp (Yang, 2003, Zhu, 2002; Garcia et al., 2000;).

Colloidal gold antibody conjugate based DIGFA or similar immuno- chromatographic assays have also become an acceptable rapid clinical bed-side detection method for drug screening and diagnosis of severe microbial and parasitic infections (Dar et al., 1994; Xiao et al., 1995; Dylan and Kevin, 1999; Feng et al., 2000; Garcia et al., 2000; Feng et al., 2002; Zhu et al., 2002; Sorell et al., 2002; Yang, 2003; Hujakka et al., 2003; Chen et al., 2005). Initial applications of DIGFA for human echinococcosis in China indicated good potential as a rapid test (Fu et al., 2000; Feng et al., 2002; Zhang et al., 2001).