DEVELOPMENT OF 17-a-HYDROXY PROGESTERONE IMMUNOASSAY USING DIFFERENT SPACERS IN IMMUNOGEN AND ENZYME CONJUGATE

17-a-hydroxy-progesterone is a clinically important steroid hormone which is mainly secreted by zona fasciculata cells of adrenal glands and is routinely measured to screen the patients of congenital adrenal hyperplasia (CAH), a disorder of the steroid biosynthetic pathway observed due to the malfunctioning of enzymes involved in the conversion of cholesterol to cortisol. The classical form of CAH is characterized by severe salt wasting and presence of pheno-typically altered genitalia by birth, whereas non-classical CAH manifests postnatally and mainly presents symptoms of hirsuitism and amenorrhoea. As 17-a-OH-P is an effective indicator for monitoring 21-hydoxylase functioning, its accurate and reliable measurement in serum is thus required in the diagnosis and management of CAH. Radioimmunoassays have been widely used for 17-a-OH-P estimation but they have been replaced by non-isotopic labels due to their inherent radiation hazards, disposable problems and license requirements. Among the non-isotopic assays enzyme immunoassays have been preferred over fluoroimmunoassay, chemiluminescent and bioluminescent immunoassays because of their simplicity and cost-effectiveness. The enzyme immunoassays reported so far for 17-a-OH-P estimation are either not very sensitive which is a requirement for post therapeutic management of CAH or are not very specific showing varying degree of crossreactions with other analogous steroids The present study has been taken up to develop a sensitive, specific and rapid ELISA for 17-a-OH-P by modifying the immunogen and enzyme conjugates using various spacer molecules. N-hydroxy succinimide-carbodiimide reaction has been used to prepare the immunogen and enzyme conjugates with various spacer molecules using 17-a-OH-P-3-CMO as hapten and HRP and BSA as enzyme and carrier protein respectively. In the first approach, immunogen and enzyme conjugate were prepared to develop a homologous immunoassay without having any spacer molecule in enzyme conjugate and immunogen. A very good immunoreactivity of the enzyme conjugate 17-a-OH-P-3-CMO-HRP was observed with anti-17-a-OH-P-3-CMO-BSA antibody. The immunoassay was highly specific with very good sensitivity of 0.18 ng/ml. This assay was further validated for analytical recovery, precision and its correlation with EQUAS scheme and well established RIA kit. In another approach, six homobifunctional spacers namely, urea(U), carbohydrazide (CH), adipic acid dihydrazide (ADH) , ethylene diamine (ED), gamaaminobutyricacid-urea (GU) and 6-aminocaproicacid-urea (AU) were used for preparing the enzyme conjugate with an aim to improve the sensitivity of the homologous assay without any significant compromise with the assay specificity. 17-a-OH-P-3-CMO-CH-HRP, 17-a-OH-P-3-CMO-ADH-HRP, 17-a-OH-P-3-CMO-U-HRP, 17-a-OH-P-3-CMO-ED-HRP, 17-a-OH-P-3-CMO-GU-HRP and 17-a-OH-P-3-CMO-6-ACA-HRP enzyme conjugates were characterized by Matrix Assisted Laser Desorption Ionisation—Time of Flight (MALDI-TOF) to know the steroid density and confirm the coupling of 17-a-OH-P-HRP. The immunoreactivity of anti 17-a-OH-P-3-CMO-BSA antibody was evaluated with these 17-a-OH-P-spacer-HRP conjugates. The use of spacer in enzyme conjugates improved the homologous assay sensitivity without any significant compromise on the specificity except in case of 17-a-OH-P-3-CMO-ADH-HRP where cross reaction was observed to be twice of the homologous assay. The assay using 17-a-OH-P-3-CMO-CH-HRP was found to have the best sensitivity of 0.008 ng/ml among the bridge heterologous assays by inserting spacer in the enzyme conjugate whereas with use of 17-a-OH-P-3-CMO-ADH-HRP the assay sensitivity was 0.06ng/ml which was least of all the bridge heterologous assays. The better assay sensitivity with the use of 17-a-OH-P-3-CMO-CH-HRP was mainly attributed to the efficiency of the conjugate in reducing the bridge binding and steric hindrance (in homologous assays) due to the presence of a five atom CH as a spacer molecule which is highly hydrophilic and rigid due to the presence of polar groups and mesomeric effect in the entire molecular chain length. But the presence of 4 hydrophobic methylene units in ADH renders the enzyme conjugate flexible enough so that steroid may easily fold to get buried either in the methylene units or in the hydrophobic pockets in the enzyme, resulting in decreased immunoreactivity and the ability to counter the steric hindrance posed by the enzyme. These methylene units in ADH also interact with the Ab due to hydrophobic interaction resulting in tighter binding of the enzyme conjugate thereby decreasing the sensitivity of the competitive assay. Moreover, the methylene units interact with the other steroids and solid phase by hydrophobic interactions to give higher cross reactions and non specific binding. All the other enzyme conjugates with hydrophilic spacers showed better assay sensitivity and specificity than with the use of 17-a-OH-P-3-CMO-ADH-HRP. The assay using 17-a-OH-P-3-CMO-CH-HRP was further validated for recovery, precision and correlation studies. ADH, ED and 6-ACA-GABA-ADH spacers were also used for preparing immunogens and resulted in generation of anti-l7-a-OH-P-3-CMO-ADH-BSA, anti-l7-a-OH-P-3-CMO-EDBSA and anti-l7-a-OH-P-3-CMO-6-ACA-GAGA-ADH-BSA BSA antibodies respectively which were evaluated for their tryptophan (Trp) fluorescence to analyze the effect of the spacers in the immunogens on the quality of the resulting antibodies. Anti-l7-a-OH-P-3CMO-BSA antibody which was generated against immunogen without any spacer showed highest immunoreactivity and Trp fluorescence. Though anti-l7-a-OH-P-3-CMO-ED-BSA Ab gave good binding with five out of the seven enzyme conjugates used and its Trp fluorescence was comparable to the anti-l7-a-OH-P-3-CMO-BSA antibody, it did not give displacement with the enzyme conjugate studied. Anti-l7-a-OH-P-3-CMO-6-ACA-GABAADH-BSA Ab gave binding and displacement with only two enzyme conjugates, while 17-a-OH-P-3-CMO-ADH-BSA did not give binding with any of the enzyme conjugates used and their Trp fluorescence was low compared to anti-l7-a-OH-P-3-CMO-ED-BSA Ab. Both the immunoassays thus developed using anti-l7-a-OH-P-3-CMO-6-ACA-GABA-ADH-BSA Ab were highly non specific showing almost five times cross reaction compared to the homologous assay having no spacer molecule in either of the immunoconjugates. It was concluded that spacers can be used for modifying and preparing the enzyme conjugates for improving the assay sensitivity instead of inserting them in the immunogens. A colloidal gold based rapid, immunochromatographic strip for 17-a-OH-P was developed by utilizing anti-l7-a-OH-P-3-CMO-BSA antibody and 17-a-OH-P-3-CMO-BSA—Gold conjugate. Gold nanoparticles were synthesized and characterized using various techniques. The size of NPs was 36nm with a zeta potential of -44.1mV as determined by Dynamic light scattering. AFM of 17-a-OH-P-3-CMO-BSA-Gold conjugate showed a distinct change in the surface topography as compared to the bare gold NPs. The immunochromatographic strip was prepared by coating anti-l7-a-OH-P-3-CMO-BSA antibody at the capture line, anti-BSA antibody at the control line and 17-a-OH-P-3-CMO-ADH-BSA-gold conjugate on reservoir matrix. Competitive inhibition assay was developed on this strip by using 17-a-OH-P standards of different concentrations. The lower detection limit of the developed assay was found to be 1.25 ng/ml by visual observation. The strip was semi-quantitative but took less than 20 min for result development as compared to 80mfin required in quantitative ELISA.