Obtaining Specific Hybridomas for Ki-67 Protein Immunodetection

BACKGROUND: Active proliferation is specific property of a tumor cells. However, the cost of the analysis is high due to commercial anti-Ki-67 mAbs used as the main immunoreagent for reliable identification of proliferating cells. In this study, recombinant protein was used to obtain specific mAbs for Ki-67 biomarker immunodetection.METHODS: Codon optimized fragment of ki-67 gene was cloned into the pET28c(+)vector. The recombinant protein was purified by immobilized metal affinity chromatography (IMAC) and confirmed by liquid chromatography–mass spectrometry (LC-MS)/MS. Hybridoma cells were obtained by fusing myeloma cells with mouse spleen cells immunized with recombinant antigen. The specificity and activity of mAbs was determined by enzyme-linked immunosorbent assay (ELISA), Western blot and immunocytochemistry.RESULTS: The pET-28c(+)/ki-67 plasmid, which encodes 355 amino acid protein, was obtained. Analysis by LC-MS/MS of the recombinant antigen showed that 77.5% of the amino-acid sequence belonged to Ki-67 protein. Recombinant fragment of Ki-67 protein was used to obtain specific hybridoma strains. ELISA and Western blot demonstrated high affinity and the specificity of obtained mAbs against Ki-67 protein. Newly generated anti-Ki67 mAbs detected target protein in proliferating cells of MCF-7 cell line by immunocytochemistry.CONCLUSION: Newly developed mAbs are potentially useful as an immunodiagnostic tool for assessing the proliferative activity of breast tumor cells using immunocytochemistry.KEYWORDS: breast cancer, Ki-67, monoclonal antibodies, nuclear antigen, recombinant antigen, tumor cells

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Paralogs of Atlantic salmon myoblast determination factor genes are distinctly regulated in proliferating and differentiating myogenic cells

AJP Regulatory Integrative and Comparative Physiology ◽

10.1152/ajpregu.00114.2010 ◽

2010 ◽

Vol 298 (6) ◽

pp. R1615-R1626 ◽

Cited By ~ 30

Author(s):

Neil I. Bower ◽

Ian A. Johnston

Keyword(s):

Cell Cycle ◽

Amino Acids ◽

Amino Acid ◽

Atlantic Salmon ◽

Antigen Expression ◽

Nuclear Antigen ◽

Quiescent State ◽

Mrna Levels ◽

Proliferating Cells

The mRNA expression of myogenic regulatory factors, including myoD1 (myoblast determination factor) gene paralogs, and their regulation by amino acids and insulin-like growth factors were investigated in primary cell cultures isolated from fast myotomal muscle of Atlantic salmon ( Salmo salar). The cell cycle and S phase were determined as 28.1 and 13.3 h, respectively, at 18°C. Expression of myoD1b and myoD1c peaked at 8 days of culture in the initial proliferation phase and then declined more than sixfold as cells differentiated and was correlated with PCNA (proliferating cell nuclear antigen) expression ( R = 0.88, P < 0.0001; R = 0.70, P < 0.0001). In contrast, myoD1a transcripts increased from 2 to 8 days and remained at elevated levels as myotubes were formed. mRNA levels of myoD1c were, on average, 3.1- and 5.7-fold higher than myoD1a and myoD1b, respectively. Depriving cells of amino acids and serum led to a rapid increase in pax7 and a decrease in myoD1c and PCNA expression, indicating a transition to a quiescent state. In contrast, amino acid replacement in starved cells produced significant increases in myoD1c (at 6 h), PCNA (at 12 h), and myoD1b (at 24 h) and decreases in pax7 expression as cells entered the cell cycle. Our results are consistent with temporally distinct patterns of myoD1c and myoD1b expression at the G1 and S/G2 phases of the cell cycle. Treatment of starved cells with insulin-like growth factor I or II did not alter expression of the myoD paralogs. It was concluded that, in vitro, amino acids alone are sufficient to stimulate expression of genes regulating myogenesis in myoblasts involving autocrine/paracrine pathways. The differential responses of myoD paralogs during myotube maturation and amino acid treatments suggest that myoD1b and myoD1c are primarily expressed in proliferating cells and myoD1a in differentiating cells, providing evidence for their subfunctionalization following whole genome and local duplications in the Atlantic salmon lineage.

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MCM2 Is an Independent Predictor of Survival in Patients With Non–Small-Cell Lung Cancer

Journal of Clinical Oncology ◽

10.1200/jco.2001.19.22.4259 ◽

2001 ◽

Vol 19 (22) ◽

pp. 4259-4266 ◽

Cited By ~ 77

Author(s):

Nithya Ramnath ◽

Francisco J. Hernandez ◽

Dong-Feng Tan ◽

Joel A. Huberman ◽

Nachimuthu Natarajan ◽

...

Keyword(s):

Lung Cancer ◽

Small Cell Lung Cancer ◽

Tumor Cells ◽

Cell Lung Cancer ◽

Small Cell ◽

Response To Therapy ◽

Positive Staining ◽

Small Cell Lung ◽

Proliferating Cells ◽

Ki 67

PURPOSE: Minichromosome maintenance protein 2 (MCM2) is a component of the prereplicative complex. It is essential for eukaryotic DNA replication and is only expressed in proliferating cells. The prognostic utility of MCM2 compared with Ki-67, another marker of proliferating cells, on survival of patients with non–small-cell lung cancer (NSCLC) was studied. PATIENTS AND METHODS: We examined the immunohistochemical expression of MCM2 and Ki-67 in primary pathologic tumor specimens from 221 NSCLC patients. For each marker, the fraction of tumor cells with positive staining was assessed as a percentage and categorized into four groups: 0% to 24%, 25% to 49%, 50% to 74%, and ≥ 75%. MCM2 and Ki-67 immunoreactivities were compared with each other, and associations with pathologic and clinical parameters predictive of survival were analyzed with the χ2 test. Cox regression models were used to assess associations between MCM2 and Ki-67 and survival while controlling for confounders. RESULTS: Independent variables significantly associated with survival were tumor stage, performance status, and staining category. Patients with less than 25% MCM2 immunoreactivity had a longer median survival time than patients with ≥ 25% MCM2 immunoreactivity (46 v 31 months; P = .039) and a lower relative risk (RR) of death (RR, 0.55, 95% confidence interval, 0.34 to 0.88). There was no significant association between survival and Ki-67 expression. CONCLUSION: Immunostaining of tumor cells for MCM2 is an independent prognostic parameter of survival for patients with NSCLC. Interpretable results can be obtained on more than 96% of paraffin-embedded specimens, and approximately 35% will be in the favorable subgroup, with less than 25% positively stained tumor cells. Whether MCM2 is predictive of response to therapy needs to be studied.

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Molecular Expression of a Cysteine Proteinase of Clonorchis sinensis and Its Application to an Enzyme-Linked Immunosorbent Assay for Immunodiagnosis of Clonorchiasis

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.2.411-416.2004 ◽

2004 ◽

Vol 11 (2) ◽

pp. 411-416 ◽

Cited By ~ 49

Author(s):

Isao Nagano ◽

Fuquan Pei ◽

Zhiliang Wu ◽

Jun Wu ◽

Huier Cui ◽

...

Keyword(s):

Recombinant Protein ◽

Adult Worm ◽

Immunosorbent Assay ◽

Fasciola Hepatica ◽

Cysteine Proteinase ◽

Clonorchis Sinensis ◽

Recombinant Antigen ◽

Enzyme Linked Immunosorbent Assay ◽

Predicted Amino Acid Sequence ◽

Crude Extracts

ABSTRACT We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.

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Knockout of MMP3 Weakened Solid Tumor Organoids and Cancer Extracellular Vesicles

10.20944/preprints202003.0371.v1 ◽

2020 ◽

Author(s):

Eman A. Taha ◽

Chiharu Sogawa ◽

Yuka Okusha ◽

Hotaka Kawai ◽

May Wathone Oo ◽

...

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Extracellular Vesicles ◽

Fluorescent Proteins ◽

Three Dimensional ◽

3D Culture ◽

Proliferating Cells ◽

Ki 67 ◽

Additional Release

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (300-1000 nm) and small EVs (50-200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 leads to a significant reduction in tumoroid size and to the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. These weakened phenotypes by MMP3 KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated into recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3 resulting in increasing the proliferation and CD9+ tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

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Ki-67 Labeling Index in Breast Cancer

Tumori Journal ◽

10.1177/030089168907500608 ◽

1989 ◽

Vol 75 (6) ◽

pp. 557-562 ◽

Cited By ~ 5

Author(s):

Sergio Crispino ◽

Ambrogio Brenna ◽

Daniela Colombo ◽

Bajardo Flores ◽

Silvestro D'Amico ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Monoclonal Antibody ◽

Menopausal Status ◽

Mitotic Rate ◽

Labeling Index ◽

Nuclear Antigen ◽

Proliferating Cells ◽

Cell Cycle Kinetics ◽

Ki 67

Measurements of cell cycle kinetics have been found to correlate with the clinical course of patients with breast cancer. However, the thymidine labeling index and more rapid methods like flow cytometry remain complicated and costly. We assessed cell proliferation of 67 breast carcinomas by an immunoperoxidase procedure using a monoclonal antibody, Ki-67, which reacts with a nuclear antigen in proliferating cells. The percentage of Ki-67 positive cells ranged from 2% to 70 %. Tumors with high mitotic rate, high nuclear grade, high histologic grade, and negative estrogen receptors had statistically higher Ki-67 labeling rates. We found no significant differences between the Ki-67 labeling rate and other clinical (age at diagnosis, menopausal status) or pathologic (necrosis, fibrosis, vascular invasion, lymphatic invasion, cellular reaction, tumor size, lymph node metastases) features assessed. These results parallel previously reported data, and confirm that this immunohistochemical staining of breast carcinoma by Ki-67 monoclonal antibody can be considered a rapid and convenient method for assessing cell cycle kinetics. However, further studies, evaluating the correlation between Ki-67 labeling rate and prognosis are needed to better define the real usefulness of this analysis in clinical practice.

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Proliferation Activity in Pituitary Adenomas: Measurement by Monoclonal Antibody Ki-67

Neurosurgery ◽

10.1227/00006123-198912000-00012 ◽

1989 ◽

Vol 25 (6) ◽

pp. 927-930 ◽

Cited By ~ 106

Author(s):

Engelbert Knosp ◽

Klaus Kitz ◽

Axel Perneczky

Keyword(s):

Monoclonal Antibody ◽

Pituitary Adenomas ◽

Direct Determination ◽

Nuclear Antigen ◽

Proliferating Cells ◽

Ki 67 ◽

Histological Techniques ◽

Cytological Smear ◽

Significant Difference ◽

Proliferation Activity

Abstract The monoclonal antibody (MAb) Ki-67 detects a nuclear antigen expressed by proliferating cells during the entire cell cycle. In contrast to conventional histological techniques, the use of MAb Ki-67 on frozen sections or cytological smear preparations allows direct determination of the growth rate of tumors routinely. Sixty-two pituitary adenomas were investigated by use of the MAb Ki-67 in a two-step avidin-biotin-peroxidase complex technique. The proliferation activity ranged from 0.1 to 2.8%. There was no significant difference between the proliferation and hormonal state of the adenomas. Adenomas for which there was histological evidence of dural infiltration, however, showed a statistically significant higher proliferation activity (P> 0.05) compared to noninvasive adenomas.

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Identification of Human Hepatocyte Proliferation Related Gene C2orf69

Infection International ◽

10.1515/ii-2017-0066 ◽

2014 ◽

Vol 3 (1) ◽

pp. 1-9

Author(s):

Ren-wen Zhang ◽

Yong Qiao ◽

Xiao-hua Hao ◽

Hong-min Li ◽

Hui Ren ◽

...

Keyword(s):

Cell Proliferation ◽

Recombinant Protein ◽

Western Blot ◽

Polyclonal Antibody ◽

Prokaryotic Expression ◽

Liver Cells ◽

Hepatocyte Proliferation ◽

Enzyme Linked Immunosorbent Assay ◽

Gene Fragment

Abstract Objective To construct the prokaryotic expression vector pET-32a(+)-C2orf69 and induce the expression of recombinant proteins in vitro. Then the possible effects of recombinant protein on cell proliferation was observed and rabbit-anti-C2orf69 protein polyclonal antibodies was obtained. Methods Gene fragment of C2orf69 was amplified by PCR and then prokaryotic expression plasmid pET-32a(+)-C2orf69 was constructed. Recombinant protein C2orf69 expression was identified by SDS-PAGE and Western blot. The white-ear rabbits were immunized with purified recombinant protein C2orf69, and the potency and specificity of polyclonal antibody were evaluated by enzyme-linked immunosorbent assay (ELISA) and Western blot. Also, different liver cells were incubated with recombinant protein C2orf69 in vitro. Results C2orf69 gene fragment was successfully amplified, results of gene sequencing were consistent with the sequence in GenBank. Recombinant protein of C2orf69 was successfully induced and expressed. The polyclonal antibody titer was up to 1︰1 280 000 through enzyme-linked immunosorbent assay. Results of cell proliferation showed that the recombinant protein could inhibit the proliferation of different liver cells. Conclusions The recombinant protein C2orf69 could inhibit the proliferation of different liver cells, and we speculated that it may be a widely roled inhibitor of hepatocyte proliferation. Our experiment showed that the proliferation inhibition of cells may be realized by G1 phase extending and S phase shortening.

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Novel Insights on the Corpus Luteum Function: Role of Vaspin on Porcine Luteal Cell Angiogenesis, Proliferation and Apoptosis by Activation of GRP78 Receptor and MAP3/1 Kinase Pathways

International Journal of Molecular Sciences ◽

10.3390/ijms21186823 ◽

2020 ◽

Vol 21 (18) ◽

pp. 6823 ◽

Cited By ~ 1

Author(s):

Patrycja Kurowska ◽

Ewa Mlyczyńska ◽

Joelle Dupont ◽

Agnieszka Rak

Keyword(s):

Growth Factor ◽

Corpus Luteum ◽

Cell Function ◽

Control Level ◽

B Cell Lymphoma ◽

Nuclear Antigen ◽

Luteal Cell ◽

Enzyme Linked Immunosorbent Assay ◽

Proliferating Cells ◽

Proliferation And Apoptosis

Formation and limited lifespan of corpus luteum (CL) are important for proper ovarian periodicity and fertility. Failed vascularization, imbalance between proliferation and apoptosis leads to luteal phase deficiency and infertility. The aim of this study was to examine the effect of vaspin on angiogenesis, apoptosis and proliferation as well as the involvement of 78-kDa glucose-regulated protein receptor (GRP78) and mitogen-activated kinase (MAP3/1) in these processes. Porcine luteal cells were incubated with vaspin (0.1–10 ng/mL) for 24 h to 72 h and then mRNA and protein expression of angiogenesis: vascular endothelial growth factor (VEGFA), fibroblast growth factor 2 (FGF2), angiopoietin 1 (ANGPT1), VEGFA receptors (VEGFR1, VEGFR2), apoptosis: caspase 3, bcl-2-like protein 4 (BAX), B-cell lymphoma (BCL2), and proliferation: proliferating cells nuclear antigen (PCNA), cyclin A factors as well as secretion of VEGFA, FGF2, ANGT1 were measured by real-time polymerase chain reaction (PCR), immunoblotting and enzyme-linked immunosorbent assay (ELISA), respectively. Moreover, apoptosis was assessed by caspase activity using the Caspase-Glo 3/7 assay, while proliferation was by alamarBlue. We found that vaspin enhanced luteal cell angiogenesis, proliferation, and significantly decreased apoptosis. Additionally, using GRP78 siRNA and the pharmacological inhibitor of MAP3/1 (PD98059), we observed that the effect of vaspin was reversed to the control level in all investigated processes. Taken together, our results suggest that vaspin is a new regulator of female fertility by direct regulation of CL formation and maintenance of luteal cell function.

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Knockout of MMP3 Weakens Solid Tumor Organoids and Cancer Extracellular Vesicles

Cancers ◽

10.3390/cancers12051260 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1260 ◽

Cited By ~ 4

Author(s):

Eman Taha ◽

Chiharu Sogawa ◽

Yuka Okusha ◽

Hotaka Kawai ◽

May Oo ◽

...

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Extracellular Vesicles ◽

Fluorescent Proteins ◽

Three Dimensional ◽

3D Culture ◽

Proliferating Cells ◽

Ki 67 ◽

Additional Release

The tumor organoid (tumoroid) model in three-dimensional (3D) culture systems has been developed to reflect more closely the in vivo tumors than 2D-cultured tumor cells. Notably, extracellular vesicles (EVs) are efficiently collectible from the culture supernatant of gel-free tumoroids. Matrix metalloproteinase (MMP) 3 is a multi-functional factor playing crucial roles in tumor progression. However, roles of MMP3 within tumor growth and EVs have not unveiled. Here, we investigated the protumorigenic roles of MMP3 on integrities of tumoroids and EVs. We generated MMP3-knockout (KO) cells using the CRISPR/Cas9 system from rapidly metastatic LuM1 tumor cells. Moreover, we established fluorescent cell lines with palmitoylation signal-fused fluorescent proteins (tdTomato and enhanced GFP). Then we confirmed the exchange of EVs between cellular populations and tumoroids. LuM1-tumoroids released large EVs (200–1000 nm) and small EVs (50–200 nm) while the knockout of MMP3 resulted in the additional release of broken EVs from tumoroids. The loss of MMP3 led to a significant reduction in tumoroid size and the development of the necrotic area within tumoroids. MMP3 and CD9 (a category-1 EV marker tetraspanin protein) were significantly down-regulated in MMP3-KO cells and their EV fraction. Moreover, CD63, another member of the tetraspanin family, was significantly reduced only in the EVs fractions of the MMP3-KO cells compared to their counterpart. These weakened phenotypes of MMP3-KO were markedly rescued by the addition of MMP3-rich EVs or conditioned medium (CM) collected from LuM1-tumoroids, which caused a dramatic rise in the expression of MMP3, CD9, and Ki-67 (a marker of proliferating cells) in the MMP3-null/CD9-low tumoroids. Notably, MMP3 enriched in tumoroids-derived EVs and CM deeply penetrated recipient MMP3-KO tumoroids, resulting in a remarkable enlargement of solid tumoroids, while MMP3-null EVs did not. These data demonstrate that EVs can mediate molecular transfer of MMP3, resulting in increasing the proliferation and tumorigenesis, indicating crucial roles of MMP3 in tumor progression.

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Strongyloides-Specific IgE Phage cDNA Clones and Development of a Novel ELISA for Strongyloidiasis

Diagnostics ◽

10.3390/diagnostics11060985 ◽

2021 ◽

Vol 11 (6) ◽

pp. 985

Author(s):

Hussain Ahmad ◽

Norsyahida Arifin ◽

Thomas J. Nolan ◽

James B. Lok ◽

Nor Suhada Anuar ◽

...

Keyword(s):

Recombinant Protein ◽

Western Blot ◽

Rapid Test ◽

Strongyloides Stercoralis ◽

Specific Ige ◽

Diagnostic Value ◽

Epidemiological Studies ◽

Enzyme Linked Immunosorbent Assay ◽

Cdna Clones ◽

Diagnostic Potential

Strongyloidiasis, caused mainly by the nematode Strongyloides stercoralis, is prevalent worldwide and potentially fatal in immunosuppressed patients. We report on a new IgE biomarker to diagnose Strongyloides infection. Sera from two groups infected with Strongyloides served as positive samples: Group 1A, in which infection was confirmed by stool-microscopy and/or stool-polymerase chain reaction (PCR) and was seropositive by an IgG-enzyme linked immunosorbent assay (ELISA) and an IgG4 rapid test, and Group 1B in which infection was confirmed by stool-PCR but was seronegative. Negative samples (controls) comprised infections with other parasites (Group II) and healthy donors (Group III). Immunoscreenings of an S. stercoralis complementary DNA (cDNA) library were performed, and the cDNA clone with the highest diagnostic potential (clone A133) was selected for recombinant protein production and then evaluated using IgE Western blot and ELISA. The Western blot showed that the recombinant protein (rA133) was 100% reactive with Group IA (n = 10) and Group IB (n = 5), and 96% non-reactive with Groups II and III (n = 25). Subsequently, the IgE-ELISA was developed and showed 100% diagnostic sensitivity in Groups IA (n = 32) and IB (n = 11); and 99.3% specificity in Groups II and III (n = 144). In conclusion, this study has identified rA133 as a novel recombinant protein with potential diagnostic value, and that the IgE-ELISA incorporating this protein may be useful for patient diagnosis and epidemiological studies.

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