As indicated in Table 1, the highest OD for positive control and the lowest OD for negative control and blank were at a dilution of 1/50

As indicated in Table 1, the highest OD for positive control and the lowest OD for negative control and blank were at a dilution of 1/50. against infection (12). Detection of serum anti-PRP antibodies (PRP-Ab), by a sensitive and specific assay, is therefore of great importance. The enzyme-linked PKR Inhibitor immunosorbent assay (ELISA) technique for the measurement of anti-Hib capsular polysaccharide antibodies was first successfully used in 1988 and, since then, it has been widely used all over the world (13-16). Rabbit polyclonal to ZNF101 The antibody titers obtained by this technique show acute, chronic and post-vaccination steps (17). A comparison among different assays, such as PCR, loop -mediated isothermal amplification (LAMP), radio immune assay (RIA) and ELISA cleared that Elisa has multiple advantages (18-21). The antibody levels against Hib capsular polysaccharide have been investigated in children in Iran, using ELISA kits (16). Although using commercial ready to use ELISA kits is easy and convenient, sometimes, homemade ELISA kits are required because of its affordability and also because of shortage and expensiveness of commercial kits (22). Moreover, using commercial ELISA kits to detect Hib antibody titer, especially for epidemiological studies, can cost much more than homemade kits. Homemade ELISA kits for several pathogens, such as (23) and (24) have been reported advantageous and cost-effective. Therefore, it was necessary to developed and optimize an PKR Inhibitor indirect-ELISA plate for the detection of Hib infection in children. 2. Objectives The is the most frequent causative agent of bacterial meningitis, in children aged 5 months to 5 years. The presence of anti-PRP antibody in the serum of non-vaccinated children 3-5 years old is common. Although there are different diagnostic methods to confirm the infection, the most used and preferred method is ELISA immuno-enzymatic method, as a screening test. It is necessary to prepare and develop antigen coated plates to study seroepidemiology of to evaluate its health impact. We designed and optimized anti-Hib enzyme immunoassay kit in our laboratory and compare it to vaccZymeHiBIgG (Binding site-UK). 3. Materials and Methods 3.1. Antigen Preparation The PRP was prepared from culture supernatants of Hib strains, which were obtained from the type bacteria collection of Pasteur Institute of Iran, Tehran, Iran (PTCC = 1623) PKR Inhibitor grown on culture media, including brain heart infusion broth (BHIB) (Difco, USA) and tripticase soy broth (TSB) (Difco, USA). In order to increase cell density and PRP titer, 60 liter fed batch fermentation was incorporated (Nova-palijas, contact-flow BV, the Netherlands) with 40 L working volume, at 37 1C (14). The PRP was prepared by precipitation with a mixture of alcohols, including ethanol 70%, methanol 99% and isopropanol 99%, with ratios of 60%, 20% and 20%, respectively. Then, the precipitate was centrifuged for one hour at 4000 rpm. The pellet was washed two times with pyrogen free water. After storing at 4oC for 24 hours, it was centrifuged for one hour at 4000 rpm. Resuspension of the precipitate was performed in 0.3 M sodium chloride. Orcinol was added to the pellet for assessing ribose (11, 14, 25, 26). The ribose concentration was determined by measuring the absorbance of the solution at 670 nm and comparing it to a standard curve prepared by assaying pure ribose. The PRP concentration was expressed in units of mg PRP per liter (14). After lyophilization, the purity of PRP was determined with nuclear magnetic resonance (NMR) and fourier transform infrared spectroscopy (FTIR). 3.2. Antigen Coatin An amount of 2 mg PRP antigen was dissolved in 1 mL of distilled water, after which 100 L of 0.1 M sodium periodate was added to this solution to the emerging aldehyde groups from vicinal hydroxyl groups of sugar moieties of PRP (27). The reaction mixture was stirred at room temperature for 20 minutes. The solution was dialyzed in 0.001 M sodium acetate buffer, with a pH of 4.4 and kept at 4oC for overnight. A two milliliters solution of 0.5 M bicarbonate containing 5 mg/mL BSA, with a pH of 9.6, was prepared. Dialyzed antigen was mixed with 2 mL of prepared BSA and stirred at room temperature for 2 hours. An amount of 400 L of 4 mg/mL sodium borohydride was gently added to the antigen solution and stirred for one hour. The antigen solution was dialyzed in carbonate buffer at 4oC for 24 hours. Micro plate wells (Greiner Bio-One GmbH, Germany) were coated with coating buffer (bicarbonate, pH = 9.6) containing prepared antigen solution (PRP-BSA) for 24 hours at room temperature (11). Then, the plates were washed two times with washing buffer (PBS 0.05% Tween 20, pH = 7.2), which contains no other proteins that might compete with the target antigen for attachment to the plastic.