May 22, 2022

Preparation and Characterization of a New Monoclonal Antibody Specific Against Lawsonia intracellularis and Its Application in Indirect Immunofluorescence and Immunocytochemistry Assay

Proliferative enteropathy (PE) is an infectious enteric disease caused by Lawsonia intracellularis (L. intracellularis) and is endemic in pig herds worldwide. However, a L. intracellularis-specific monoclonal antibody plays an important role in the evaluation of L. intracellularis infection in vitro. Therefore, the objective of this study was to produce and identify the characteristics of a new monoclonal antibody against the outer membrane protein (Omp2) of L. intracellularis and apply it in an indirect immunofluorescence assay (IFA) and immunocytochemistry (IHC).
The results indicated that three highly specific monoclonal antibodies against the Omp2 protein (4D9, 3G2, and 7G5) of L. intracellularis were obtained by using purified Omp2 as an immunogen, the titers of ascitic fluids of 4D9, 3G2, and 7G5 cells were 1:2,048,000, 1:512,000, and 1:256,000, respectively. IFA analysis showed that the 4D9, 3G2, and 7G5 have no cross-reactivity with other enteric bacteria commonly found in the ilea of pigs or closely related to L. intracellularis, such as Desulfovibrio, Bilophila wadsworthia (B. wadsworthia)Salmonella choleraesuis (S. choleraesuis), Salmonella typhimurium (S. typhimurium), Escherichia coli (E. coli), and Brachyspira hyodysenteriae (B. hyodysenteriae). IFA and IHC results indicated that the monoclonal antibodies can be successfully used as primary antibodies to detect L. intracellularis in infected cells and in the crypt of the ileum from infected tissues of PE. Our findings suggested that the new monoclonal antibody specific against L. intracellularis will be useful for the evaluation of L. intracellularis infection in vivo and in vitro.

Availability of immunocytochemistry using cocktail antibody targeting p63/cytokeratin14 for the differential diagnosis of fibroadenoma and ductal carcinoma in situ in fine needle aspiration cytology of the breast.

The differential diagnosis of fibroadenoma (FA) and ductal carcinoma in situ (DCIS) has been problematic in fine needle aspiration biopsy (FNAC) because it has been difficult to differentiate between the “large epithelial clusters” associated with FA and those associated with DCIS. The purpose of this study was to prospectively validate the usefulness of immunocytochemical staining using cocktail antibody targeting p63/CK14 in the differential diagnosis of FA and DCIS.
METHODS
Twenty patients diagnosed as having an uncertain malignant potential (indeterminate) for breast cancer on the basis of a FNAC finding were selected randomly: ten patients with FA and ten with DCIS. The cover glass on a specimen stained with the Papanicolaou stain on a glass slide was peeled off, and the specimen was restained by immunocytochemical staining of cocktail antibody targeting p63 and CK14.
RESULTS
Six of the twenty patients were CK14-immunopositive: FA, 6; DCIS, 0. The remaining patients were CK14-immunonegative: FA, 4; DCIS, 10. The number of CK14-immunopositive DCIS patients was significantly different from that of FA patients (P=.0054). Eight out of the twenty patients were p63-immunopositive: FA, 8; DCIS, 0. The remaining patients were p63-immunonegative: FA, 2; DCIS, 10. The number of p63-immunopositive DCIS patients was significantly different from that of FA patients (P=.0004).
CONCLUSIONS
Immunocytochemical staining using cocktail antibody targeting p63/CK14 was useful for the differential diagnosis of FA and DCIS in FNAC of the breast.

Usefulness of immunocytochemistry using a Breast Marker antibody cocktail targeting P63/cytokeratin7/18/cytokeratin5/14 for fine needle aspiration of the breast: a retrospective cohort study of 139 cases.

The Breast Marker Cocktail from Biocare Medical comprises five antibodies recognising p63, and cytokeratins (CKs) 7, 18, 5 and 14. Immunohistochemistry using this cocktail is useful for diagnosing proliferative intraductal breast lesions. However, cytology using the cocktail has not been reported.
METHODS
We report 139 cases of mammary samples collected by fine needle aspiration (FNA) for which histological diagnoses were available. After cell transfer, immunocytochemistry was performed using the cocktail, and clusters of cells were classified. A cluster with no or limited CK5/14 expression (<20% of cells) was classified as a monotonous cluster. One with more than 20% of cells showing CK5/14 expression was defined as a mosaic cluster. When at least one p63-positive cell was present, we defined it as a cluster with p63. We also evaluated background p63-positive myoepithelial cell densities.
RESULTS
The diagnostic sensitivity and specificity for carcinomas were 97.8% (89/91) and 91.7% (11/12), respectively, using the criterion of two or more monotonous clusters lacking p63. Two false-negative cases were triple-negative cancers; one false-positive was an apocrine papilloma. The numbers of monotonous clusters with p63 differed significantly between benign lesions, ductal carcinoma in situ (DCIS)/lobular carcinoma in situ (LCIS) and invasive carcinomas (P < 0.001). The background myoepithelial cell density was significantly higher in fibroepithelial tumours than in other lesions (P < 0.001).
CONCLUSIONS
Immunocytochemistry using this antibody cocktail showed good sensitivity and specificity for diagnosing breast cancers. Thus, this method is useful for mammary cytology using FNA.

Sperm Antibodies ELISA kit

55R-IB79154 Fitzgerald 96 wells 440 EUR

Sperm Antibodies ELISA kit

55R-IB79155 Fitzgerald 96 wells 482 EUR

Sperm Antibodies ELISA kit

55R-IB79156 Fitzgerald 96 wells 440 EUR

Bovine mycobacterium bovis antibodies

QY-E60132 Qayee Biotechnology 96T 426 EUR

Human Cathepsin Antibodies ELISA kit

E01C0695-192T BlueGene 192 tests 1270 EUR

Human Cathepsin Antibodies ELISA kit

E01C0695-48 BlueGene 1 plate of 48 wells 520 EUR

Human Cathepsin Antibodies ELISA kit

E01C0695-96 BlueGene 1 plate of 96 wells 685 EUR

Mouse Cathepsin Antibodies ELISA kit

E03C0695-192T BlueGene 192 tests 1270 EUR

Mouse Cathepsin Antibodies ELISA kit

E03C0695-48 BlueGene 1 plate of 48 wells 520 EUR

Mouse Cathepsin Antibodies ELISA kit

E03C0695-96 BlueGene 1 plate of 96 wells 685 EUR

Rabbit Cathepsin Antibodies ELISA kit

E04C0695-192T BlueGene 192 tests 1270 EUR

Rabbit Cathepsin Antibodies ELISA kit

E04C0695-48 BlueGene 1 plate of 48 wells 520 EUR

Rabbit Cathepsin Antibodies ELISA kit

E04C0695-96 BlueGene 1 plate of 96 wells 685 EUR

Rat Cathepsin Antibodies ELISA kit

E02C0695-192T BlueGene 192 tests 1270 EUR

Rat Cathepsin Antibodies ELISA kit

E02C0695-48 BlueGene 1 plate of 48 wells 520 EUR

Rat Cathepsin Antibodies ELISA kit

E02C0695-96 BlueGene 1 plate of 96 wells 685 EUR

Monkey Cathepsin Antibodies ELISA kit

E09C0695-192T BlueGene 192 tests 1270 EUR

Monkey Cathepsin Antibodies ELISA kit

E09C0695-48 BlueGene 1 plate of 48 wells 520 EUR

Monkey Cathepsin Antibodies ELISA kit

E09C0695-96 BlueGene 1 plate of 96 wells 685 EUR

Pig Cathepsin Antibodies ELISA kit

E07C0695-192T BlueGene 192 tests 1270 EUR

Pig Cathepsin Antibodies ELISA kit

E07C0695-48 BlueGene 1 plate of 48 wells 520 EUR

Pig Cathepsin Antibodies ELISA kit

E07C0695-96 BlueGene 1 plate of 96 wells 685 EUR

Fixation effect of SurePath preservative fluids using epidermal growth factor receptor mutation-specific antibodies for immunocytochemistry.

BACKGROUND
Cytological diagnosis of respiratory disease has become important, not only for histological typing using immunocytochemistry (ICC) but also for molecular DNA analysis of cytological material. The aim of this study was to investigate the fixation effect of SurePath preservative fluids.
METHODS
Human lung cancer PC9 and 11-18 cell lines, and lung adenocarcinoma cells in pleural effusion, were fixed in CytoRich Blue, CytoRich Red, 15% neutral-buffered formalin, and 95% ethanol, respectively. PC9 and 11-18 cell lines were examined by ICC with epidermal growth factor receptor (EGFR) mutation-specific antibodies, the EGFR mutation DNA assay, and fluorescence in situ hybridization. The effect of antigenic storage time was investigated in lung adenocarcinoma cells in pleural effusion by ICC using the lung cancer detection markers.
RESULTS
PC9 and 11-18 cell lines in formalin-based fixatives showed strong staining of EGFR mutation-specific antibodies and lung cancer detection markers by ICC as compared with ethanol-based fixatives. DNA preservation with CytoRich Blue and CytoRich Red was superior to that achieved with 95% ethanol and 15% neutral-buffered formalin fixatives, whereas EGFR mutations by DNA assay and EGFR gene amplification by fluorescence in situ hybridization were successfully identified in all fixative samples. Although cytoplasmic antigens maintained high expression levels, expression levels in nuclear antigens fell as storage time increased.
CONCLUSIONS
These results indicate that CytoRich Red is not only suitable for ICC with EGFR mutation-specific antibodies, but also for DNA analysis of cytological material, and is useful in molecular testing of lung cancer, for which various types of analyses will be needed in future.

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