Production of a Human Recombinant Polyclonal Fab Antivenom against Iranian Viper Echis carinatus.

Venomous snakebite is a life-threatening injury in many tropical and subtropical areas including Iran. The gold standard treatment option for human envenomation is the use of antivenoms. Despite the unique effects of horse-derived antivenoms on the treatment of snakebite, they are not fully perfect and need improvements. In this study, human recombinant Fab fragment antivenom was produced in Rosetta-g bacterium using a gene library constructed in the previous study. The prepared Fab was purified in several steps, desalted, and lipopolysaccharide-depleted using ammonium sulfate solution and dialysis against phosphate buffer and Triton X-114 solution, respectively. Subsequently, the product was initially confirmed by the sodium dodecyl sulfate polyacrylamide gel electrophoresis and enzyme-linked immunosorbent assay (ELISA), respectively. Finally, the neutralization potency of the product was investigated in laboratory Syrian Mice. The obtained results showed corresponding reduced bands to Fab fragment with the molecular weight of about 28 kDa at a concentration of 3.1 mg/ml. There was a significant difference between the groups in terms of ELISA test (P<0.05). The neutralization potency of the product against the venom of Echis carinatus (E. carinatus) was about 7 LD50/ml (54.6 µg/ml) when tested on mice. Based on the results, the Fab fragment antivenom had the ability to neutralize the in vivo biological activity of the venom of Iranian E. carinatus. However, further studies are recommended to reach a suitable concentration of antivenom fragment.

[3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways].

Dikkoppf-1 (DKK1) is an antagonist of the canonical Wnt signaling pathway. The importance of DKK1 as a diagnostic and therapeutic agent in a wide range of diseases along with its significance in a variety of biological processes accentuate the necessity to decipher its 3D structure that would pave the way towards the development of relevant selective inhibitors. A DKK1 structure model predicted by the Robetta server with structural refinements including a 10 ns molecular dynamics run was subjected to functional and docking analyses. We hypothesize that the N-terminal region of the DKK1 molecule could be functionally important for both canonical and noncanonical Wnt pathways. Moreover, it seems that DKK1 could be involved in interactions with the Frizzled receptors, leading to the activation of the Planar Cell Polarity (PCP) pathway (activation of Jun N-terminal kinase (JNK) Pathway) and Wnt/Ca^(2+) pathway (activation of CamKII).

Daily timing of salivary cortisol responses and aerobic performance in lean and obese active females.

The main aim of the present study was to study the effects of morning and afternoon physical activities on cortisol responses in obese and lean women. Twenty women volunteered to participate in this study. Subjects were divided into an obese group (BMI =29.1 kg/m2) and a lean group (BMI =19 kg/m2). All subjects participated in an exercise program consisting of treadmill running at 65+/-2 % VO2max until exhaustion. In order to study effects of circadian rhythm, exercise was performed at a similar intensity and in similar environmental conditions at both 8:00 AM and 4:00 PM. Saliva specimens were collected at rest 20 minutes before activity and then immediately after the exercise in both morning and afternoon sessions. Morning and afternoon exercise resulted in a significant increase in salivary cortisol concentrations compared to basal levels in both lean and obese women (p<0.05), though the change in cortisol concentrations were higher in lean. The aerobic function of lean and obese women in the morning and afternoon showed a significant increase of 13.8 % and 5.9 %; respectively, with lean being consistently higher than obese. In conclusion, the stress response to exercise is related to circadian rhythm and individual's body weight. Based on the results of this study, it is suggested that overweight women perform exercises in the afternoon to minimize the stress response for the exercise volume performed (Tab. 1, Fig. 3, Ref. 39). Full Text in free PDF www.bmj.sk.

The effect of preseason training on mucosal immunity in male basketball players.

AIM: This study examined the effects of pre season training on restring level and acute response of mucosal immunity in male basketball players. METHODS: Twenty male basketball players performed 8 weeks progressive exercise training, consisting of interval and continuous parts. Five mL un-stimulated saliva was collected from each subject before, immediately and one hour after the end of one bout of exercise to exhaustion on treadmill at the beginning of the first week and end of 8 weeks to determine the acute responses. At the beginning of each 2 weeks (resting state) induced changes in basal mucosal immunity was evaluated. The concentration of sIgA and total protein was measured by the ELISA and Bradford methods respectively. RESULTS: One bout exercise training at beginning of first week decreased significantly sIgA level but not at the end of 8th week. Total protein did not change significantly at 1st week after exercise, but at eight week significantly increased and remained at high level until one hour after exercise. sIgA to total protein ratio at first week significantly decreased and remained constant one hour after exercise. At the eight week sIgA decreased significantly immediately after exercise and remained low until one hour after exercise. The comparison of sIgA and total protein levels indicates significant decrease after eight weeks training. CONCLUSION: These results suggest that repetition of single bout of exercise training have a cumulative effect on the mucosal immune system.

A new radiopharmaceutical compound (131I-PR81) for radioimmunotherapy of breast cancer: labeling of antibody and its quality control.

PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast and other tumors. The objective of this study was to evaluate the application of this antibody against MUC1 as a radioimmunotherapeutical agent. Monoclonal antibody (PR81) against MUC1 was prepared, characterized, purified, and labeled with 131I. The immunoreactivity of radiolabeled mAb PR81with MUC1 (the native protein), BSA-P20 (a 20 amino acid corresponding the tandem repeat of MUC1) and MCF7 cell line were performed by RIA. In vitro stability of radiolabeled mAb in human serum was determined by thin layer chromatography (TLC). Cell toxicity and in vitro internalization studies were performed with the MCF7 cell line, and the tissue biodistribution of the radioiodinated PR81 was evaluated in normal BALB/c mice at 4, 24 and 48 hrs. The tumor imaging was performed in BALB/c mice with breast xenograft tumors at 24 and 72 hr after the complex injection. The labeling efficiency was found to be 59.9% ± 7.9%. MAb-131I conjugates showed high immunoreactivity towards MUC1 protein, BSA-P20 and MCF7 cell line. In vitro stability of the labeled product in human serum was found to be more than %50 over 24 hr. Cell toxicity and in vitro internalization studies showed that the mAb-131I conjugate inhibited 80% growth of the MCF7 cultured cell lines in vitro in a high concentration and up to %60 of the conjugate internalized after 24 h. Biodistribution studies were performed in normal BALB/c mice at 4, 24 and 48 hrs post-injection and no important accumulation was observed in vital organs. The tumors were visualized with high sensitivity after 24 and 72 hr in radioimmunoscintographical studies. These results show that the new radiopharmaceutical may be considered as a promising candidate for therapy of breast cancer.

Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein.

A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance.

Production of chimeric recombinant single domain antibody-green fluorescent fusion protein in Chinese hamster ovary cells.

There is an increasing interest in the application of nanobodies such as VHH in the field of therapy and imaging. In the present study a stable genetically engineered cell line of Chinese hamster ovary (CHO) origin transfected using two sets of expression vectors was constructed in order to permit the cytoplasmic and extracellular expression of single domain antibody along with green fluorescent protein (GFP) as reporter gene. The quality of the constructs were examined both by the restriction map as well as sequence analysis. The gene transfection and protein expression was further examined by reverse transcription-polymerase chain reaction (RT-PCR). The transfected cells were grown in 200 microg/mL hygromycin containing media and the stable cell line obtained showed fluorescent activity for more than a period of 180 days. The production of fusion protein was also detected by fluorescent microscopy, fluorescent spectroscopy as well as by enzyme-linked immunosorbent assay (ELISA) analysis. This strategy allows a rapid production of recombinant fluobodies involving VHH, which can be used in various experiments such as imaging and detection in which a primary labeled antibody is required.

Expression and characterization of a recombinant single-domain monoclonal antibody against MUC1 mucin in tobacco plants.

A promising alternative to conventional antibodies is the single-domain antibody fragment of the Camelidae (V(HH)), which (because of features such as small length, high expression, solubility, and stability) is preferred to other antibody derivatives. In this report, a recombinant single-domain antibody (V(HH)) against MUC1 mucin in the tobacco plant, which may be considered as a suitable and economical alternative expression system, was produced. This antibody was expressed under the control of a strong constitutive promoter, CaMV35S, and NOS terminator. A plant high-expression sequence (Kozak sequence) was linked at the 5' end for overexpression of the V(HH) gene. The constructed cassette (pBIV(HH)) was transferred to agrobacterium, and the VHH gene was inserted into the plant genome by agrobacterium-mediated transformation. Transgenic lines were selected on kanamycin (100 mg/L) and maintained in soil, and subsequent generations were obtained. The presence and expression of the transgene was confirmed in the transformants by polymerase chain reaction (PCR), enzyme-linked immunosorbent assay (ELISA), and Western blot. Tobacco transgenic lines leave expressed V(HH) at levels varying from 1.12% to 1.63% of the total soluble protein. This report examines the transformation and expression of recombinant single-domain antibody (V(HH)) against antigen-associated tumor in tobacco plants.

Modified salting-out method: high-yield, high-quality genomic DNA extraction from whole blood using laundry detergent.

Different approaches have been used to extract DNA from whole blood. In most of these methods enzymes (such as proteinase K and RNAse A) or toxic organic solvents (such as phenol or guanidine isothiocyanate) are used. Since these enzymes are expensive, and most of the materials that are used routinely are toxic, it is desirable to apply an efficient DNA extraction procedure that does not require the use of such materials. In this study, genomic DNA was extracted by the salting-out method, but instead of using an analytical-grade enzyme and chemical detergents, as normally used for DNA isolation, a common laundry powder was used. Different concentrations of the powder were tested, and proteins were precipitated by NaCl-saturated distilled water. Finally, DNA precipitation was performed with the use of 96% ethanol. From the results, we conclude that the optimum concentration of laundry powder for the highest yield and purity of isolated DNA is 30 mg/mL. The procedure was optimized, and a final protocol is suggested. Following the same protocol, DNA was extracted from 100 blood samples, and their amounts were found to be >50 microg/mL of whole blood. The integrity of the DNA fragments was confirmed by agarose gel electrophoresis. Furthermore, the extracted DNA was used as a template for PCR reaction. The results obtained from PCR showed that the final solutions of extracted DNA did not contain any inhibitory material for the enzyme used in the PCR reaction, and indicated that the isolated DNA was of good quality. These results show that this method is simple, fast, safe, and cost-effective, and can be used in medical laboratories and research centers.

Preparation and characterization of a monoclonal antibody against mannoprotein of Candida albicans.

BALB/c mice were immunized via injection with whole cell of Candida albicans serotype A. The spleens were fused with myeloma cells of SP2/0 origin. A mannoprotein-reactive monoclonal antibody (MAb) was selected and characterized by ELISA technique. This MAb reacted with strains of Candida such as C. albicans, C. tropicalis, and C. albicans of the Persian Type Culture Collection (PTCC). However, our antibody did not react with other Candida species such as C. parapsilosis, C. glabrata, C. stellatoidae, C. lusitania, C. krusei, and S. cervisiae. These antibodies also did not recognize extracts of other fungal species such as Aspergillus fumigatus and Aspergillus flavus, and bacterial strains such as Staphylococcus aureus and Pseudomonas aeruginosa. Polyclonal antibody produced in this study could not differentiate the above species and was reactive towards all fungal species mentioned above except bacterial strains of S. aureus and P. aeruginosa. Western blot analysis of ligand affinity-purified mannoproteins of C. albicans wall protein using this MAb showed reactivity toward a single protein band in the region of 55-65 kDa molecular weight. The same antibody, when examined with unpurified C. albicans extract, reacted with a broad band in the region of 55-105 kDa, which we concluded was due to a possible different glycosylation pattern of mannoprotein in crude extract in which the higher molecular weight protein was eliminated by ligand-binding affinity purification.

Suppressive effects of caraway (Carum carvi) extracts on 2, 3, 7, 8-tetrachloro-dibenzo-p-dioxin-dependent gene expression of cytochrome P450 1A1 in the rat H4IIE cells.

Cytochrome P450 1A1 (CYP1A1) is among the cytochrome P450 classes known to convert xenobiotics and endogenous compounds to toxic and/or carcinogenic metabolites. Suppression of CYP1A1 over expression by certain compounds is implicated in prevention of cancer caused by chemical carcinogens. Chemopreventive agents containing high levels of flavonoids and steroids-like compounds are known to suppress CYP1A1. This study was carried out for assessment of the genomic and proteomic effects of caraway (Carum carvi) extracts containing high levels of both flavonoids and steroid-like substances on ethoxy resorufin dealkylation (EROD) activity and CYP1A1 at mRNA levels. Rat hepatoma cells co-treated with a CYP1A1 inducer i.e. TCDD (2, 3, 7, 8-tetrachlorodibenzo-p-dioxin) and different preparations of caraway extracts at concentrations of 0, 0.13, 1.3, and 13 microM in culture medium. After incubation (37 degrees C and 7% CO2 for 20 h), changes in EROD specific activity recorded and compared in cells under different treatments. The results show that caraway seed extract prepared in three different organic solvents suppressed the enzyme activity in hepatoma cells in a dose-dependent manner. The extracts added above 0.13 microM could significantly inhibit EROD activity and higher levels of each extract (1.3 and 13 microM) caused approximately 10-fold suppression in the enzyme activity. Accordingly, data obtained from the RT-PCR (TaqMan) clearly showed the suppressive effects of plant extract on CYP1A1-related mRNA expression. These data clearly show that substances in caraway seeds extractable in organic solvents can potentially reverse the TCDD-dependent induction in cytochrome P450 1A1.

Production and characterization of a new antibody specific for the mutant EGF receptor, EGFRvIII, in Camelus bactrianus.

EGFRvIII is the type III deletion mutant form of the epidermal growth factor receptor (EGFR) with transforming activity. This tumor-specific antigen is ligand independent, contains a constitutively active tyrosine kinase domain and has been shown to be present in a number of human malignancies. In this study, we report the production and characterization of camel antibodies that are directed against the external domain of the EGFRvIII. Antibodies developed in camels are smaller (i.e. IgG2 and IgG3 subclasses lack light chains) than any other conventional mammalian antibodies. This property of camel antibodies makes them ideal tools for basic research and other applications such as tumor imaging and cancer therapy. In the present study, camel antibodies were generated by immunization of camelids (Camelus bactrianus and Camelus dromedarius) with a synthetic 14-amino acid peptide corresponding to the mutated sequence of the EGFR, tissue homogenates of several patients with human glioblastoma, medulloblastoma and aggressive breast carcinoma, as well as EGFR-expressing cell lines. Three subclasses of camel IgG [conventional (IgG1, 160 kD) and heavy chain-only antibodies (IgG2 and IgG3, 90 kD)] were separated by their different binding properties to protein A and protein G affinity columns. The anti-EGFRvIII peptide antibodies from immunized camels were purified further using the EGFRvIII synthetic peptide affinity column. The purified anti-EGFRvIII peptide camel antibodies selectively bound to the EGFRvIII peptide and affinity-purified EGFRvIII from malignant tissues and detected a protein band of 140 kD from malignant tissues by Western blot. Affinity analysis showed that the antibodies from C. bactrianus and C. dromedarius reacted with peptide and antigen purified from a small cell lung cancer ascitic fluid with affinities of 2 x 10(8) and 5 x 10(7)M(-1) to the same extent, respectively. Since the functional antigen-binding domain of the anti-EGFRvIII antibodies in camels is much simpler and located only on the heavy chains of proteins, we are currently developing recombinant and smaller versions of the variable domain of these naturally occurring heavy-chain antibodies (V(HH)) for use in tumor imaging and cancer therapy.

Production of novel recombinant single-domain antibodies against tandem repeat region of MUC1 mucin.

Recently, the existence of "heavy-chain" antibody in Camelidae has been described. However, as yet there is no data on the binding of this type of antibody to peptides. In addition, there was not any report of production of single-domain antibodies in two-humped camels (Camelus bactrianus). In the present study, these questions are addressed. We showed the feasibility of immunizing old world camels, cloning the repertoire of the variable domain of their heavy-chain antibodies, panning and selection, leading to the successful identification of minimum-sized antigen binders. Antigen-specific fragments of the heavy-chain IgGs (V(HH)) are of great interest in biotechnology because they are very stable, highly soluble, and react specifically and with high affinity to the antigens. In this study, we immunized two camels (Camelus dromedarius and Camelus bactrianus) with homogenized cancerous tissues, synthetic peptide, and human milk fat globule membrane (HMFG), and generated two V(HH) libraries displayed on phage particles. Some single-domain antibody fragments have been isolated that specifically recognize the tandem repeat region of MUC1. The camels' single-domain V(HH) harbor the original, intact antigen binding site and reacted specifically and with high affinity to the tandem repeat region of MUC1. Indeed soluble, specific antigen binders and good affinities (in the range of 0.2 x 10(9) M(-1) to 0.6 x 10(9) M(-1)) were identified from these libraries. This is the first example of the isolation of camel anti-peptide V(HH) domains.

Production of a novel camel single-domain antibody specific for the type III mutant EGFR.

Camelids have a unique immune system capable of producing single-domain heavy-chain antibodies. The antigen-specific domain of these heavy-chain IgGs (VHH) are the smallest binding units produced by the immune system. In this study, we report the isolation and characterization of several binders against the epidermal growth factor receptor (EGFR) vIII retrieved from immune library of camels (Camelus bactrianus and Camelus dromedarius). The EGFRvIII is a ligand-independent, constitutively active, mutated form of the wild-type EGFR. The expression of EGFRvIII has been demonstrated in a wide range of human malignancies, including gliomas, and breast, prostate, ovarian and lung cancer. Camels were immunized with a synthetic peptide corresponding to a mutated sequence and tissue homogenates. Single-domain antibodies (VHH) were directly selected by panning a phage display library on successively decreasing amounts of synthetic peptide immobilized on magnetic beads. The anti-EGFRvIII camel single-domain antibodies selectively bound to the EGFRvIII peptide and reacted specifically with the immunoaffinity-purified antigen from a non-small cell lung cancer patient. These antibodies with affinities in the nanomolar range recognized the EGFRvIII peptide and affinity-purified mutated receptor. We concluded that using the phage display technique, antigen-specific VHH antibody fragments are readily accessible from the camelids. These antibodies may be good candidates for tumor-diagnostic and therapeutic applications.

Production of monoclonal antibody, PR81, recognizing the tandem repeat region of MUC1 mucin.

A monoclonal antibody (MAb) was generated by immunizing BALB/c mice with homogenized breast cancerous tissues. This antibody (PR81) was found to be of IgG(1) class and subclass, containing kappa light chain. PR81 reacted with either the membrane extracts of several breast cancerous tissues or the cell surface of some MUC1 positive cell lines (MCF-7, BT-20 and T-47D) tested by enzyme immunoassay and for MCF-7 by immunofluorescence method. PR81 also reacted with two synthetic 27 and 16-amino acid peptides, TSA-P1-24 and A-P1-15, respectively, which included the core tandem repeat sequence of MUC1. However, this antibody did not react with a synthetic 14 amino acid peptide that has no similarity with tandem repeat found in MUC1. The generated antibody had good and similar affinities (2.19 x 10(8) M(-1)) toward TSA-P1-24 and A-P1-15, which are mainly shared in the hydrophilic sequence of PDTRPAP. Through Western blot analysis of homogenized breast tissues, PR81 recognized only a major band of 250 kDa. This band is stronger in malignant tissue than benign and normal tissues.

Measurement of glutathione S-transferase and its class-pi in plasma and tissue biopsies obtained after laparoscopy and endoscopy from subjects with esophagus and gastric cancer.

OBJECTIVES: To develop an indirect enzyme-linked immunosorbent assay (ELISA) for measuring class-pi glutathione S-transferase (GST) in plasma, and tissue biopsies obtained from upper gastrointestinal cancer (UGI Ca) patients. METHODS: GST activity and GST-pi concentration were detected in normal human squamous esophageal epithelium, normal gastric cardia and their corresponding malignant tumor biopsies. RESULTS: Plasma GST was significantly higher (p < 0.05) in UGI Ca patients as compared to those obtained from normal individuals. Plasma GST-pi concentration in normal subjects was 6.6 +/- 1.9 ng/mg protein, whereas it was higher in UGI Ca patients (esophageal, 10.0 +/- 1.8; gastric, 10.7 +/- 1.7 ng/mL, p

Preparation and characterization of monoclonal antibody against digoxin.

Mouse-mouse hybridoma cell lines producing stable, highly specific and with good affinity monoclonal antibody (MAb) against the cardiac glycoside digoxin were established. Balb/c mice were immunized via injection of digoxin-3'-bovine serum albumin (BSA). The spleens of which were fused with myeloma cells of SP2/0 origin. Three clones designated as BBA, MBE, and BMG producing good antibodies displayed different patterns of fine specificity for digoxin and low cross-reaction with several digoxin analogues as elucidated by inhibition enzyme-linked immunosorbant assay (ELISA). All three MAbs were of the same class and subclass (IgG(1)). Affinity purification was performed for the selected clone BBA displaying the highest affinity and nearly no cross-reactivity with any of the structurally related molecules. Ultrafiltered concentrated hybrid cell supernatant was also purified by polyethylene glycol (PEG) 6000 precipitation for large-scale preparation and coated onto the wells of microtiter plates. The standard curve was constructed with a sensitivity of 10 pg/well covering up to 10 ng/well.

Characterization of a monoclonal antibody against neopterin using an enzyme-linked immunosorbent assay with penicillinase as label.

An active ester derivative of neopterin was prepared using 4-(N-maleimidomethyl) cyclohexan 4-carboxilic acid N-hydroxy succinimide ester (MCH-NHS), conjugated to bovine serum albumin (BSA) and injected for antibody production (for both monoclonal and polyclonal antibodies). High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. Neopterin was conjugated to the enzyme penicillinase by a one-step glutaraldehyde method, which was used as tracer. A novel enzyme immunoassay was developed using this conjugate to screen and characterize the monoclonal antibody (MAb) produced in these experiments. After limiting dilutions, it was found that antibody produced by one clone with a Ka value of 7.6 x 10-7 mol/L was specific for a number of structurally related molecules. This clone was found to be of IgG class and IgG2a subclass. The standard curvewas constructed with a sensitivity of 10 pg/well (100 pg/mL) covering up to 1 ng/mL.

Preparation and characterization of specific and high-affinity monoclonal antibodies against morphine.

A C6-hemisuccinate derivative of morphine was prepared and conjugated to bovine serum albumin. High titer antibody producing spleen cells were removed and fused with myeloma cells of Sp2/0 origin. A C3-hemisuccinate derivative of morphine was prepared and conjugated to enzyme penicillinase used as a tracer molecule. A novel enzyme-linked immunoadsorbent assay was developed using this conjugate to screen and characterize the monoclonal antibody produced in these experiments. After two successive limiting dilutions, antibodies produced by 5 clones with good affinities ranging from 10(8) to 10(12) M(-1) and less cross-reaction (least for codeine and other structurally related molecules) were selected. These clones were found to be of IgG class with kappa light chain. Subclass determination showed that two of the clones produced IgG2b and three of them produced IgG1 type of antibody. Affinity purifications were performed for the selected clone (MOR-I). Purified antibody was coated onto the wells of microtiter plate. The standard curve was constructed with a sensitivity of 100 pg/mL covering up to 10 ng/mL in buffer and urine. The slope of the standard curve for selected clone in buffer and urine was calculated to be -0.7 and -0.64, respectively.