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Production and Characterization of Monoclonal Antibodies against Human Prostate Specific Antigen.

Bayat AA, Ghods R, Shabani M, Mahmoudi AR, Yeganeh O, Hassannia H, Sadeghitabar A, Balay-Goli L, Noutash-Haghighat F, Sarrafzadeh AR, Jeddi-Tehrani M - Avicenna J Med Biotechnol (2015 Jan-Mar)

Bottom Line: All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC.Besides, all mAbs could detect a protein band around 33 kDa in human seminal plasma in western blot.These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.

View Article: PubMed Central - PubMed

Affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.

ABSTRACT

Background: Prostate Specific Antigen (PSA) is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies (mAbs) against PSA have been presented.

Methods: Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer (PCa), Benign Prostatic Hyperplasia (BPH) and brain cancer tissues by Immunohistochemistry (IHC).

Results: Five anti-PSA mAbs (clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/К) and clones (2C8-E9, 2G3-E2, IgG2a/К) were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kDa in human seminal plasma in western blot.

Conclusion: These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.

No MeSH data available.


Related in: MedlinePlus

Detection of PSA in human semen using anti-PSA mAbs by western blot analysis. All produced anti-PSA mAbs could recognize a ∼33 kDa band related to free PSA in the seminal fluids. Two other bands, 90 kDa and 28 kDa, were also found in WB. That can represent PSA-PCI complex and endoproteolytic cleavage product of PSA, respectively.
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Figure 0004: Detection of PSA in human semen using anti-PSA mAbs by western blot analysis. All produced anti-PSA mAbs could recognize a ∼33 kDa band related to free PSA in the seminal fluids. Two other bands, 90 kDa and 28 kDa, were also found in WB. That can represent PSA-PCI complex and endoproteolytic cleavage product of PSA, respectively.

Mentions: First of all, the isotypes of all specific mAbs were determined by home-made ELISA as described in methods section. Our results showed that the isotypes of 2G2-B2, 2F9-F4 and 2D6-E8 were IgG1/k and those of 2G3-E2 and 2C8-E9 were IgG2a/k (Tables 1 and 2). In the next step, reactivities of the purified mAbs were assessed by an indirect ELISA on purified PSA (Figure 3A) and also seminal fluids (Figure 3B). Our findings showed that all produced mAbs specifically recognized PSA in both purified PSA and crude mixture of seminal fluids. The highest reaction was observed for clone 2C8-E9 which recognized the target at lowest concentration of the mAb (9.7 ng/ml). Besides, reactivities of the mAbs were checked in western blot on human semen samples. In this regard, all anti-PSA mAbs detected a 33 kDa protein band in human semen corresponding to free PSA. Two distinct bands were also observed at 28 and 90 kDa (Figure 4). For more characterization, the mAbs were applied for staining of PSA expressing tissues, PCa and BPH, in IHC experiments. Although all clones except 2F9-F4 strongly reacted with PCa and BPH tissues, no reactivity was observed in brain cancer sections as PSA-negative control tissue (Figure 5).


Production and Characterization of Monoclonal Antibodies against Human Prostate Specific Antigen.

Bayat AA, Ghods R, Shabani M, Mahmoudi AR, Yeganeh O, Hassannia H, Sadeghitabar A, Balay-Goli L, Noutash-Haghighat F, Sarrafzadeh AR, Jeddi-Tehrani M - Avicenna J Med Biotechnol (2015 Jan-Mar)

Detection of PSA in human semen using anti-PSA mAbs by western blot analysis. All produced anti-PSA mAbs could recognize a ∼33 kDa band related to free PSA in the seminal fluids. Two other bands, 90 kDa and 28 kDa, were also found in WB. That can represent PSA-PCI complex and endoproteolytic cleavage product of PSA, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4388886&req=5

Figure 0004: Detection of PSA in human semen using anti-PSA mAbs by western blot analysis. All produced anti-PSA mAbs could recognize a ∼33 kDa band related to free PSA in the seminal fluids. Two other bands, 90 kDa and 28 kDa, were also found in WB. That can represent PSA-PCI complex and endoproteolytic cleavage product of PSA, respectively.
Mentions: First of all, the isotypes of all specific mAbs were determined by home-made ELISA as described in methods section. Our results showed that the isotypes of 2G2-B2, 2F9-F4 and 2D6-E8 were IgG1/k and those of 2G3-E2 and 2C8-E9 were IgG2a/k (Tables 1 and 2). In the next step, reactivities of the purified mAbs were assessed by an indirect ELISA on purified PSA (Figure 3A) and also seminal fluids (Figure 3B). Our findings showed that all produced mAbs specifically recognized PSA in both purified PSA and crude mixture of seminal fluids. The highest reaction was observed for clone 2C8-E9 which recognized the target at lowest concentration of the mAb (9.7 ng/ml). Besides, reactivities of the mAbs were checked in western blot on human semen samples. In this regard, all anti-PSA mAbs detected a 33 kDa protein band in human semen corresponding to free PSA. Two distinct bands were also observed at 28 and 90 kDa (Figure 4). For more characterization, the mAbs were applied for staining of PSA expressing tissues, PCa and BPH, in IHC experiments. Although all clones except 2F9-F4 strongly reacted with PCa and BPH tissues, no reactivity was observed in brain cancer sections as PSA-negative control tissue (Figure 5).

Bottom Line: All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC.Besides, all mAbs could detect a protein band around 33 kDa in human seminal plasma in western blot.These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.

View Article: PubMed Central - PubMed

Affiliation: Monoclonal Antibody Research Center, Avicenna Research Institute, ACECR, Tehran, Iran.

ABSTRACT

Background: Prostate Specific Antigen (PSA) is an important laboratory marker for diagnosis of prostatic cancer. Thus, development of diagnostic tools specific for PSA plays an important role in screening, monitoring and early diagnosis of prostate cancer. In this paper, the production and characterization of a panel of murine monoclonal antibodies (mAbs) against PSA have been presented.

Methods: Balb/c mice were immunized with PSA, which was purified from seminal plasma. Splenocytes of hyperimmunized mice were extracted and fused with Sp2/0 cells. By adding selective HAT medium, hybridoma cells were established and positive clones were selected by ELISA after four times of cloning. The isotypes of produced mAbs were determined by ELISA and then purified from ascitic fluids using Hi-Trap protein G column. The reactivities of the mAbs were examined with the purified PSA and seminal plasma by ELISA and western blot techniques. Furthermore, the reactivities of the mAbs were assessed in Prostate Cancer (PCa), Benign Prostatic Hyperplasia (BPH) and brain cancer tissues by Immunohistochemistry (IHC).

Results: Five anti-PSA mAbs (clones: 2G2-B2, 2F9-F4, 2D6-E8, IgG1/К) and clones (2C8-E9, 2G3-E2, IgG2a/К) were produced and characterized. All mAbs, except 2F9-F4 detected the expression of PSA in PCa and BPH tissues and none of them reacted with PSA in brain cancer tissue in IHC. Besides, all mAbs could detect a protein band around 33 kDa in human seminal plasma in western blot.

Conclusion: These mAbs can specifically recognize PSA and may serve as a component of PSA diagnostic kit in various biological fluids.

No MeSH data available.


Related in: MedlinePlus