Isolation of placental-type alkaline phosphatase associated with human syncytiotrophoblast membranes using monoclonal antibodies

Webb, Paul D.; McLaughlin, Paul; Risk, Janet M.; Johnson, Peter

doi:10.1016/s0143-4004(86)80028-5

Cited by 17 publications

(13 citation statements)

References 28 publications

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“…This was identical to the [3H]mannose control and the known molecular mass of monomeric PLAP [17,201. Transferrin receptor immunoprecipitates gave a major band at 95 kDa; its monomeric molecular mass [30].…”

Section: Immunoprecipifates Of' Placental-type Alkaline Phosphatasesupporting

confidence: 66%

“…The release was not the result of non-specific proteolytic activity. B. cereus phospholipase C released soluble PLAP, which gave a single 66-kDa band on SDS-PAGE, the same molecular mass as intact PLAP released with detergents [17,201. In contrast, soluble PLAP released with bromelain ran at 64 kDa.…”

Section: Phospholipase C and Bromelain Treatment Of Tumour Cellsmentioning

confidence: 92%

“…Triton X-100 or 1% (mass/ vol.) sodium deoxycholate [17] in Tris/NaCl, pH 8.2 (1 mg protein/ml), were similarly applied to the column as controls. The column was washed with buffer and eluted with 8 M urea, pH 7.5.…”

Section: Immunoaflinity Chromatographymentioning

confidence: 99%

“…The column was washed with buffer and eluted with 8 M urea, pH 7.5. Fractions were tested for alkaline phosphatase [17] and the tube with peak enzymatic activity was lyophilised after exchanging the buffer for 30 mM ammonium hydrogen carbonate on a Sephadex G-25 column (PD-10, Pharmacia). The samples were run on SDS-PAGE, as described [20].…”

Section: Immunoaflinity Chromatographymentioning

confidence: 99%

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Attachment of human placental‐type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes

Webb

Todd

1988

European Journal of Biochemistry

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Phosphatidylinositol anchors human placental-type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes. PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus. This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol-specific phospholipase C in the preparation. Soluble PLAP, released with B. cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS-PAGE as a 66-kDa band. This corresponded to intact PLAP molecules. The protease bromelain cleaved lower-molecular-mass PLAP (64 kDa) from the membranes. Flow cytometry demonstrated that B. cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell-surface molecules. This was in contrast to the non-specific proteolytic action of bromelain or Clostridium perfringens phospholipase C , which had no effect on membrane PLAP expression. Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both [3H]myristic and [3H]palmitic acid. This fatty-acid -PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an 0-ester linkage.

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Section: Immunoprecipifates Of' Placental-type Alkaline Phosphatasesupporting

confidence: 66%

Section: Phospholipase C and Bromelain Treatment Of Tumour Cellsmentioning

confidence: 92%

Section: Immunoaflinity Chromatographymentioning

confidence: 99%

Section: Immunoaflinity Chromatographymentioning

confidence: 99%

See 2 more Smart Citations

Attachment of human placental‐type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes

Webb

Todd

1988

European Journal of Biochemistry

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“…Placental alkaline phosphatase (PLAP) is normally expressed on the foetal membrane after the ninth week of gestation, but the level of expression diminishes after birth to trace amounts present in the testis [1][2][3]. In recent years it has become increasingly evident that PLAP can also be seen in some human malignancies including endometrial [4,5], ovarian [6] and testicular malignancies, particularly in seminomas.…”

Section: Introductionmentioning

confidence: 99%

Standardization and Potential Use of HPLC for Detection of Cellular Placental Alkaline Phosphatase Using Established Tumour Cell Lines and Fresh Tumour Biopsies

Torabi‐Pour¹,

Nouri²,

Chineguwndo³

et al. 1999

Urol Int

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Placental alkaline phosphatase (PLAP) is one of the cellular phosphatases (ALP) expressed in patients with testis cancers, particularly in seminomas. Using various techniques including Western blot and high-performance liquid chromatography (HPLC) systems and ATC2, a newly developed specific anti-PLAP monoclonal antibody (Mab), the presence of active form of PLAP in lysates prepared from testis tumour fragment and tumour cell lines, was studied. This was carried out following isolation of PLAP from biological samples using CNBr Sepharose-conjugated ATC2 beads. The results showed that: (1) The target for the newly developed Mab ATC2 was PLAP. (2) The ATC2-conjugated bead system was an efficient method for isolating pure PLAP. (3) Diethylamine (DEA), in contrast to urea and glycine, was the most efficient for separation of PLAP from ATC2-conjugated beads, as the isolated molecule did not lose any phosphatase activity and there was very little uncoupling of the ATC2 Mab from the beads. (4) ATC2-conjugated CNBr beads could pick up PLAP from a solution containing standard PLAP and lysates prepared from tumour cell lines or testis tissue fragments positive for the PLAP. (5) HPLC profile of testis tumour lines and testis tumours showed two distinct peaks with ALP activity, one at retention time 7– 8 min (corresponding to 95 kDa molecule) and one at 12–13 min corresponding to 70 kDa molecule). These data demonstrated the potential use of various biochemical methods in combination with HPLC for isolation of the fully functional molecules with ALP activity from different samples including lysates prepared from patients with testis cancer. The nature of ALP activity at 95 kDa is being investigated as no such molecule has been reported previously. These techniques might have an important implication for an early detection of germ cell tumours, particularly in patients with equivocal ultrasound.

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The combined use of high performance liquid chromatography and immuno‐biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins

Torabi‐Pour

Nouri

Perrett

et al. 2000

Biomedical Chromatography

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HPLC was used in combination with immuno-bead separation technique for identification of an individual protein from a pool of proteins. This was carried out using an in-house monoclonal antibody (ATC2) specific for placental alkaline phosphatase (PLAP) as a primary antibody for conjugation to CNBr beads. The phosphatase activity (ALP) of PLAP was measured by colorimetric assay (MEDC). The data from this study has so far indicated that: 1. HPLC analysis of molecules following isolation with ATC2-conjugated beads showed high degree of purity. This could be achieved using protein mixtures prepared from lysates of tumour cell lines or tumour fragments. 2. HPLC-isolated PLAP maintained phosphatase activity. 3. Out of the four dissociation reagents used, diethyl amine (DEA) was found to be the best reagent for dissociation of antigen, ie PLAP, but not mAb from CNBr beads. 4. The profile of ALP activity was different for samples prepared from testis and kidney fragments, both in terms of the HPLC peak profile as well as the sensitivity. These data confirmed that the immuno-bead separation technique in conjunction with HPLC were powerful tools for identifying an individual protein from a pool of proteins. These approaches are being used for the identification of PLAP molecules, as a tumour marker in patients suspected of testicular malignancies with equivocal ultrasound.

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Isolation of placental-type alkaline phosphatase associated with human syncytiotrophoblast membranes using monoclonal antibodies

Cited by 17 publications

References 28 publications

Attachment of human placental‐type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes

Attachment of human placental‐type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes

Standardization and Potential Use of HPLC for Detection of Cellular Placental Alkaline Phosphatase Using Established Tumour Cell Lines and Fresh Tumour Biopsies

The combined use of high performance liquid chromatography and immuno‐biochemical techniques for protein isolation: a new approach for identification of an individual protein from a pool of proteins

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