Targeting the active site of the placental isozyme of alkaline phosphatase by phage-displayed scFv antibodies selected by a specific uncompetitive inhibitor

BACKGROUND: The isozymes of alkaline phosphatase, the tissue non-specific, intestinal and placental, have similar properties and a high degree of identity. The placental isozyme (PLAP) is an oncofetal antigen expressed in several malignancies including choriocarcinoma, seminoma and ovarian carcinoma. We had earlier attempted to isolate PLAP-specific scFv from a synthetic human immunoglobulin library but were unable to do so, presumably because of the similarity between the isozymes. In this work, we have employed a PLAP-specific uncompetitive inhibitor, L-Phe-Gly-Gly, to select isozyme specific scFvs. An uncompetitive inhibitor binds to the enzyme in the presence of substrate and stabilizes the enzyme-substrate complex. Several uncompetitive inhibitors have varying degrees of isozyme specificity for human alkaline phosphatase isozymes. A specific uncompetitive inhibitor would be able to unmask conformational differences between the otherwise very similar molecules. Also, such inhibitors would be directed to regions at/close to the active site of the enzyme. In this work, the library was first incubated with PLAP and the bound clones then eluted by incubation with L-Phe-Gly-Gly along with the substrate, para-nitro phenyl phosphate (pNPP). The scFvs were then studied with regard to the biochemical modulation of their binding, isozyme specificity and effect on enzyme activity. RESULTS: Of 13 clones studied initially, the binding of 9 was inhibited by L-Phe-Gly-Gly (with pNPP) and 2 clones were inhibited by pNPP alone. Two clones had absolute and 2 clones had partial specificity to PLAP. Two clones were cross-reactive with only one other isozyme. Three scFv clones, having an accessible His6-tag, were purified and studied for their modulation of enzyme activity. All the three scFvs inhibited PLAP activity with the kinetics of competitive inhibition. Cell ELISA could demonstrate binding of the specific scFvs to the cell surface expressed PLAP. CONCLUSION: The results demonstrate the biochemical modulation of scFv binding. Also, the scFvs bound to the active site and denied the access to the substrate. The selection strategy could generate specific anti-enzyme antibodies to PLAP that can potentially be used for targeting, for modulating enzyme activity in in vitro and in vivo and as probes for the active site. This strategy also has a general application in selecting antibodies from combinatorial libraries to closely related molecules and conformations

DEVELOPMENT AND VALIDATION OF STABILITY INDICATING HPTLC METHOD FOR DETERMINATION OF APIXABAN AS BULK DRUG

Objective: To develop and validate simple, sensitive stability indicating HPTLC (High performance thin layer chromatography) method for apixaban. Methods: The chromatographic separation was performed on aluminium plates precoated with silica gel 60 F254 using toluene: ethyl acetate: methanol (3:6:1 v/v/v) as mobile phase followed by densitometric scanning at 279 nm. Results: The chromatographic condition shows sharp peak of apixaban at Rf value of 0.38±0.03. Stress testing was carried out according to international conference on harmonization (ICH)Q1A (R2) guidelines and the method was validated as per ICH Q2(R1) guidelines. The calibration curve was found to be linear in the concentration range of 100-500 ng/band for apixaban. The limit of detection and quantification was found to be 11.66ng/bandand35.33ng/band, respectively. Conclusion: A new simple, sensitive, stability indicating high performance thin layer chromatographic (HPTLC) method has been developed and validated for the determination of apixaban

Management of pseudohypoparathyroidism in pregnancy: a rare entity

Pseudohypoparathyroidism is a very rare genetic disorder and during pregnancy poses multiple challenges related to its monitoring and management. Authors present the case of a  30year old primigravida who was a diagnosed case of pseudohypoparathyroidism since 22 yrs of age, presented to our obs/gynae OPD at 5+5 wks of POG. She was managed by serial monitoring of serum calcium, phosphate and vitamin D throughout  pregnancy with careful dose modification of calcium from 1gm to 3.5gm daily and vitamin D from  0.5mcg  to 1.5mcg daily. During her course of pregnancy, she developed gestational hypothyroidism, gestational diabetes mellitus, intrahepatic cholestasis of pregnancy and gestational hypertension which were controlled and managed successfully. She had an elective caesarean section at 37+6 wks POG for transverse lie. Both maternal and perinatal outcome were good. Patient was discharged with advice to continue with her monitoring of serum calcium, phosphate, vitamin D along with supplementation of calcium and vitamin D life long

New insights into retinal circuits through EM connectomics: what we have learnt and what remains to be learned

The retinal neural circuit is intricately wired for efficient processing of visual signals. This is well-supported by the specialized connections between retinal neurons at both the functional and ultrastructural levels. Through 3D electron microscopic (EM) reconstructions of retinal neurons and circuits we have learnt much about the specificities of connections within the retinal layers including new insights into how retinal neurons establish connections and perform sophisticated visual computations. This mini-review will summarize the retinal circuitry and provide details about the novel insights EM connectomics has brought into our understanding of the retinal circuitry. We will also discuss unresolved questions about the retinal circuitry that can be addressed by EM connectomics in the future

Direct antigen capture by soluble scFv antibodies - A method for detection, characterization, and determination of affinity

For ease of detection, soluble forms of phage-displayed scFv antibodies are usually expressed with a tag, e.g., c-myc or His (Histidine). The binding is then assayed by a monoclonal antibody to the tag. In this article, we describe the use of biotinylated antigen for detecting soluble scFv antibodies without utilizing the peptide tag detection system. The scFv antibodies were against the oncoplacental antigen heat-stable alkaline phosphatase (HSAP). The method essentially consisted of either reverse Western or antigen capture enzyme-linked immunosorbent assay (ELISA). In the reverse Western, periplasmic extract was electrophoresed, and binding to biotinylated antigen was detected by the detection system based on streptavidin-horseradish peroxidase. The antigen capture ELISA utilized the binding of periplasmic extract to a polystyrene plate. We have also demonstrated the use of antigen capture ELISA for studying specificity and affinity of the selected clones. Although these techniques have been developed for antibodies to HSAP, they have general utility for phage expression systems without a peptide tag

Development and Validation of Stability Indicating RP-HPLC Method for Voriconazole

This study describes the development and validation of stability indicating HPLC method for voriconazole, an antifungal drug. Voriconazole was subjected to stress degradation under different conditions recommended by International Conference on Harmonization. The sample so generated was used to develop a stability-indicating high performance liquid chromatographic method for voriconazole. The peak for voriconazole was well resolved from peaks of degradation products, using a Hypersil C18 (250×4.6 mm) column and a mobile phase comprising of acetonitrile: water (40:60, v/v), at flow rate of 1 ml/min. Detection was carried out using photodiode array detector. A linear response (r > 0.99) was observed in the range of 5-25 μg/ml. The method showed good recoveries (average 100.06%) and relative standard deviation for intra and inter-day were ≤ 1.5 %. The method was validated for specificity and robustness also

Phage displayed antibodies to heat stable alkaline phosphatase: framework region as a determinant of specificity

Human antibodies against specific targets of tumor cells are the most desirable molecules for possible immunotherapy. They could be developed by using the combinatorial antibody library displayed on a phage. We selected four human antibody fragments (scFv) binding to the oncoplacental antigen Heat Stable Alkaline Phosphatase (HSAP, the placental isozyme of alkaline phosphatase) from a synthetic human antibody library. Characterization of these scFvs showed they bound HSAP with moderate affinity but did not have isozyme specificity, as determined by binding to cell lines exhibiting differential expression of isozymes of alkaline phosphatase. The VH sequences of two of these scFvs were similar and although both bound to HSAP only one was cross-reactive with albumin. The sequences revealed a difference in the framework region (FR1) of these antibodies, indicating a role for this region in the determination of specificity. This is also significant considering that the heavy chains generated the diversity of the synthetic library used in this study, and only a single light chain showing binding to BSA was used for the entire library