Sensitive Monoclonal Antibody Research Articles

Novel hapten design, highly sensitive monoclonal antibody production, and immunoassay development for rapid screening of illegally added chloramphenicol in cosmetics

Hapten design and synthesis have been regarded as the key factor to generate high-quality antibodies. In the present study, a novel hapten of chloramphenicol was synthesized, characterized and compared with two conventional haptens. The new hapten generated mAb 4B5 showed higher sensitivity and titer than the other two haptens-based mAbs. The haptens synthesized with the structure of chloramphenicol base generated more sensitive antibodies than the hapten with chloramphenicol succinate, and the spacer arm linked to the phenyl group hapten elicited the strongest antibody response. After optimization, a direct competitive enzyme-linked immunosorbent assay (dcELISA) and a lateral flow immunoassay (LFIA), both based on the mAb 4B5, were developed. The dcELISA had a half maximum inhibition concentration of 0.23 ng/mL and the LFIA showed a cutoff value of 5–10 ng/mL. The LFIA was applied to detect illegally-added chloramphenicol samples in anti-acne cosmetics, five out of 19 samples were tested chloramphenicol containing within 10 min, which result was confirmed with the dcELISA and HPLC. The LFIA has an adequate sensitivity and can be used as a point of care diagnostic device for rapidly screening chloramphenicol in cosmetics.

A Sensitive and Specific Monoclonal Antibody Based Enzyme-Linked Immunosorbent Assay for the Rapid Detection of Pretilachlor in Grains and the Environment.

Pretilachlor is a chloroacetamide herbicide mainly used for weed and broadleaf weed control in rice, that is widely utilized in China. In order to detect the residue of pretilachlor in the environment and food, a highly sensitive and specific monoclonal antibody (mAb) against pretilachlor was prepared, and the half maximum inhibitory concentration (IC50) of the monoclonal antibody was validated to be 31.47 ± 2.35 μg/L. An indirect competitive ELISA (ic-ELISA) based on the antibody with a linear range of 6.25~100 μg/L was developed. The specificity of the antibody was explained by computer simulations and experimental validation. The mAb exhibited negligible cross-reactivity towards alachlor, acetochlor, propisochlor, butachlor, and metalaxyl, and the limits of detection (LOD) for pretilachlor in lake, rice, and soil samples were 4.83~5.23 μg/L. The recoveries of all samples were 78.3~91.3%. The reliability of the ic-ELISA method for residue detection of pretilachlor in the environment and grains was confirmed using high performance liquid chromatography.

Development and characterization of anti-G12V mutated K-Ras monoclonal antibodies using hybridoma technology

Background. Kirsten rat sarcoma oncogene, KRAS, is a gene that encodes for K-Ras protein, a membrane-anchored protein which is involved in intracellular signal transduction. Cells harbouring KRAS mutations have been reported to progress toward cancer development and several studies have suggested the importance of KRAS mutational screening prior to cancer treatment. However, currently available diagnostic methods are costly, time consuming and laborious. Thus, monoclonal antibodies are proposed as an alternative in K-Ras mutational testing. Objective. This study aims to generate and characterise anti-K-Ras monoclonal antibodies based on their specificity and sensitivity towards G12V-mutated K-Ras proteins, which is one of the most commonly mutated KRAS isoforms. Methods. Mice were first immunised with G12V-mutated K-Ras peptides and spleens were harvested. Hybridoma cells which secreted K-Ras-specific antibodies were generated by fusing splenocytes with X63-Ag 8.6539 myeloma cells. Hybridoma polyclonal wells secreting G12V-mutated K-Ras antibodies were subcloned into a single clone producing monoclonal antibodies. The specificity and sensitivity of monoclonal antibodies were evaluated by ELISA and the reactivity of monoclonal antibodies were tested using immunoblotting and immunocytochemistry. Results. The cross-reactivity results indicated that anti-G12V monoclonal antibodies developed in this study is highly specific to G12V mimotopes with a cross-reactivity of 4.2-16.7% towards wild-type and other mutated K-Ras isoforms. The limit of detection of this monoclonal antibody was determined as 3.28 μg/mL. It was also found to be reactive in immunocytochemical assays and native G12V-mutated K-Ras in immunoblotting. Conclusion. A highly specific and sensitive monoclonal antibody was successfully developed, characterised and applied to several assays such as indirect ELISA, Western Blot and immunocytochemistry. These data indicate the potential for this anti-G12V KRAS monoclonal antibody to be further developed for use in various research, diagnostic and therapeutic applications.

Development and application of monoclonal antibody-based dot-ELISA and colloidal gold immunochromatographic strip for rapid, specific, and sensitive detection of tomato brown rugose fruit virus

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus that has become a great concern to tomato production industry. Due to the lack of resistant cultivars, precise detection of ToBRFV is essential to prevent the spread of ToBRFV. In this study, we produced highly sensitive and specific monoclonal antibodies against ToBRFV and established dot-enzyme-linked immunosorbent assay (dot-ELISA) and colloidal gold immunochromatographic strip (CGICS)-based methods for ToBRFV detection. These two methods could specifically detect ToBRFV without cross-reaction with seven tested tobamoviruses and three frequently occurring tomato-infecting viruses. Sensitivity analysis showed that the limit of detection of the established dot-ELISA and CGICS methods reached up to 1:6400 and 1:10,000 (w/v, g/mL) dilution of ToBRFV-infected tomato tissue, respectively. Further analyses using field-collected tomato foliar and fruit samples showed that the results obtained by dot-ELISA and CGICS were consistent with those obtained by reverse transcription polymerase chain reaction. The established methods here allow for specific, sensitive, and robust detection of ToBRFV, and will be helpful for precise monitoring and early warning of ToBRFV.

Immunochromatographic assay for rapid detection of flupyradifurone in grape, blueberry, and tomato samples

Flupyradifurone (FPF) is a neonicotinoid insecticide that effectively controls the spread of various pests. In this study, we established an immunochromatographic assay based on a highly specific and sensitive anti-FPF monoclonal antibody (mAb) to screen for FPF residues in grapes, blueberries, and tomatoes. The cut-off value for the immunochromatographic assay was 5 mg/kg for grapes and 10 mg/kg for blueberries and tomatoes. The calculated limit of detection of the immunochromatographic assay was 0.009 mg/kg, 0.033 mg/kg, and 0.040 mg/kg for grapes, blueberries, and tomatoes, respectively. The recovery rates of the immunochromatographic assay were 97.0–108.2 % in grape samples, 90.9–105.1 % in blueberry samples, and 94.0–103.7 % in tomato samples, and the detection results were highly consistent with LC-MS/MS results. Therefore, this immunochromatographic assay was an effective and rapid tool for screening for FPF in grapes, blueberries, and tomatoes.

Rapid and sensitive detection of cyhalofop-butyl in foods using a gold nanoparticle-based lateral-flow strip

In this study, we prepared a highly sensitive monoclonal antibody (mAb) against Cyhalofop-butyl (Cyh) by indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Based on this mAb, an immunochromatographic strip (ICA) was established to detect Cyh in corn and brown rice samples. Through screening different coating antigens and antibodies, the half-maximum inhibitory concentration (IC50) of the highly specific Cyh mAb 1A3 was 2.2 ng/mL, and the detection linear range (IC20-IC80) was 0.5–5.2 ng/mL. When Cyh in corn samples was detected by ICA, the detection limit of the T line elimination value was 100 μg/kg, and the detection limit for colorimetric value was 25 μg/kg. When the detection sample was brown rice, the detection limit of the T line elimination value was 100 μg/kg, and the detection limit for colorimetric value was 12 μg/kg. The results of recovery experiments for both ic-ELISA and LC-MS/MS were consistent with those of the test results when using the ICA. The ICA test strip met the requirements for on-site rapid detection of Cyh in corn and brown rice samples. Therefore, this method is suitable for real-time detection and monitoring of agricultural products as well as during plant growth, harvesting, transportation, storage, and sales, thereby guaranteeing the safety of consumers.

Development of Highly Sensitive Anti-Mouse HER2 Monoclonal Antibodies for Flow Cytometry

Overexpression of human epidermal growth factor receptor 2 (HER2) in breast cancer is an important target of monoclonal antibody (mAb) therapy such as trastuzumab. Due to the development of trastuzumab–deruxtecan, an antibody-drug conjugate, the targetable HER2-positive breast cancer patients have been expanded. To evaluate the developing modalities using anti-HER2 mAbs, reliable preclinical mouse models are required. Therefore, sensitive mAbs against mouse HER2 (mHER2) should be established. This study developed anti-mHER2 mAbs using the Cell-Based Immunization and Screening (CBIS) method. The established anti-mHER2 mAbs, H2Mab-300 (rat IgG2b, kappa) and H2Mab-304 (rat IgG1, kappa), reacted with mHER2-overexpressed Chinese hamster ovary-K1 (CHO/mHER2) and endogenously mHER2-expressed cell line, NMuMG (a mouse mammary gland epithelial cell) via flow cytometry. Furthermore, these mAbs never recognized mHER2-knockout NMuMG cells. The kinetic analysis using flow cytometry indicated that the dissociation constant (KD) values of H2Mab-300 and H2Mab-304 for CHO/mHER2 were 1.2 × 10−9 M and 1.7 × 10−9 M, respectively. The KD values of H2Mab-300 and H2Mab-304 for NMuMG were 4.9 × 10−10 M and 9.0 × 10−10 M, respectively. These results indicated that H2Mab-300 and H2Mab-304 could apply to the detection of mHER2 using flow cytometry and may be useful to obtain the proof of concept in preclinical studies.

Novel Monoclonal Antibodies Specific for Human Ki67 and P53 Tumor Markers in Breast Cancer Tissue Samples.

Ki67 and P53 are important diagnostic and prognostic biomarkers expressed in several cancers. The current standard method for evaluating Ki67 and P53 in cancer tissues is immunohistochemistry (IHC), and having highly sensitive monoclonal antibodies against these biomarkers is necessary for an accurate diagnosis in the IHC test. To generate and characterize novel monoclonal antibodies (mAbs) against human Ki67 and P53 antigens for IHC purposes. Ki67 and P53-specific mAbs were produced by the hybridoma method and screened by enzyme-linked immunosorbent assay (ELISA) and IHC techniques. Selected mAbs were characterized using Western blot and flow cytometry, and their affinities and isotypes were determined by ELISA. Moreover, using the IHC technique in 200 breast cancer tissue samples, we assessed the specificity, sensitivity, and accuracy of the produced mAbs. Two anti-Ki67 (2C2 and 2H1) and three anti-P53 mAbs (2A6, 2G4, and 1G10) showed strong reactivity to their target antigens in IHC. The selected mAbs were also able to recognize their targets by flow cytometry as well as Western blotting using human tumor cell lines expressing these antigens. The specificity, sensitivity, and accuracy calculated for clone 2H1 were 94.2%, 99.0%, and 96.6%, and for clone 2A6 were 97.3%, 98.1%, and 97.5%, respectively. Using these two monoclonal antibodies, we found a significant correlation between Ki67 and P53 overexpression and lymph node metastasis in patients with breast cancer. The present study showed that the novel anti-Ki67 and anti-P53 mAbs could recognize their respective antigens with high specificity and sensitivity and therefore can be used in prognostic studies.

Preparation of a broad-specificity antibody against zearalenone and its primary analogues and development of immunoassay of Coicis Semen and related products.

The purpose of this study was to prepare a highly sensitive and specific zearalenone (ZEN) monoclonal antibody, which was then used to develop an indirect enzyme-linked immunosorbent assay (ic-ELISA) and a colloidal gold immunochromatographic assay (GICA). These techniques were used for the detection of Coicis Semen and related products (Coicis Semen flour, Yimigao, and Yishigao). Immunogens were synthesized by oxime active ester techniques and characterized via ultraviolet spectrophotometry. Immunogens were injected subcutaneously into the abdominal cavities and backs of mice. Using the prepared antibodies, we developed ic-ELISA and GICA rapid detection methods, which were then applied for the rapid detection of ZEN and its analogues from Coicis Semen and related products. For ic-ELISA, the half maximal inhibitory concentration (IC50 ) values for ZEN, α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL), and β-zearalanol (β-ZAL) were determined to be 1.13, 1.69, 2.06, 0.66, 1.20, and 0.94ng•mL-1 , respectively. For GICA, the cutoff values of ZEN, α-ZEL, β-ZEL, α-ZAL, and β-ZAL on test strips were 0.5ng•mL-1 in phosphate buffer saline (0.01M, pH 7.4), while ZAN was found to be 0.25ng•mL-1 . Furthermore, the cutoff values of test strips were between 10 and 20µg∙kg-1 in Coicis Semen and related products. The results of these two detection methods were in good agreement with results from liquid chromatography-tandem mass spectrometry. This study provides technical support for the preparation of broad-specificity monoclonal antibodies against ZEN and lays the foundation for the simultaneous detection of multiple mycotoxins from food and herbal medicines.

Ultrasensitive immunochromatographic strip for fast screening of piperacillin in milk based on a monoclonal antibody

We developed a lateral flow immunoassay (LFIA) based on a sensitive and specific monoclonal antibody against piperacillin (PIP) with a half-maximal inhibitory concentration of 0.136 ng/mL for PIP detection in milk and milk powder. After the conditions were optimized, the visual limit of detection of LFIA was 2 ng/mL and 5 ng/g, and the cut-off value was 5 ng/mL and 20 ng/g in milk and milk powder, respectively. In the recovery assay of the spiked samples, the results of the LFIA strips were obtained within 10 min and were consistent with those obtained from indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). Therefore, our developed method is a rapid, sensitive, and reliable tool for the detection of PIP in milk and milk powder.

A Novel Anti-CD44 Variant 9 Monoclonal Antibody C44Mab-1 Was Developed for Immunohistochemical Analyses against Colorectal Cancers.

Cluster of differentiation 44 (CD44) is a type I transmembrane glycoprotein and has been shown to be a cell surface marker of cancer stem-like cells in various cancers. In particular, the splicing variants of CD44 (CD44v) are overexpressed in cancers and play critical roles in cancer stemness, invasiveness, and resistance to chemotherapy and radiotherapy. Therefore, the understanding of the function of each CD44v is indispensable for CD44-targeting therapy. CD44v9 contains the variant 9-encoded region, and its expression predicts poor prognosis in patients with various cancers. CD44v9 plays critical roles in the malignant progression of tumors. Therefore, CD44v9 is a promising target for cancer diagnosis and therapy. Here, we developed sensitive and specific monoclonal antibodies (mAbs) against CD44 by immunizing mice with CD44v3-10-overexpressed Chinese hamster ovary-K1 (CHO/CD44v3-10) cells. We first determined their critical epitopes using enzyme-linked immunosorbent assay and characterized their applications as flow cytometry, western blotting, and immunohistochemistry. One of the established clones, C44Mab-1 (IgG1, kappa), reacted with a peptide of the variant 9-encoded region, indicating that C44Mab-1 recognizes CD44v9. C44Mab-1 could recognize CHO/CD44v3-10 cells or colorectal cancer cell lines (COLO201 and COLO205) in flow cytometric analysis. The apparent dissociation constant (KD) of C44Mab-1 for CHO/CD44v3-10, COLO201, and COLO205 was 2.5 × 10-8 M, 3.3 × 10-8 M, and 6.5 × 10-8 M, respectively. Furthermore, C44Mab-1 was able to detect the CD44v3-10 in western blotting and the endogenous CD44v9 in immunohistochemistry using colorectal cancer tissues. These results indicated that C44Mab-1 is useful for detecting CD44v9 not only in flow cytometry or western blotting but also in immunohistochemistry against colorectal cancers.

A sensitive and specific enzyme-linked immunosorbent assay for the detection of pymetrozine in vegetable, cereal, and meat

Pymetrozine is a neonicotinoid insecticide with high efficacy against aphids and planthoppers, and has been used worldwide. To monitor its residue in food, a highly specific and sensitive monoclonal antibody (McAb) was prepared, and an indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed to detect pymetrozine, with a 50% inhibition value (IC50) of 7.70 μg/L. The McAb showed little affinity for acetamiprid, hexazinone, metamitron, nitenpyram, metribuzin, and imidacloprid. The limits of detection (LOD) calculated from the analysis of broccoli, cabbage, wheat, maize, rice, chicken, fish, and crayfish samples were from 1.56 to 2.72 μg/kg and the average recoveries were from 81.25 to 103.19%. icELISA was confirmed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). These results demonstrated that the optimised icELISA is a convenient and effective analytical tool for monitoring pymetrozine residues in food.

Preparation, Identification and Preliminary Application of the Fenvalerate Monoclonal Antibody in Six Kinds of Dark Tea

Fenvalerate has the advantages of a broad insecticidal spectrum, high efficiency, low toxicity and low cost, and it is widely used in agriculture, especially in tea, resulting in the accumulation of fenvalerate residues in tea and the environment, posing a serious threat to human health. Therefore, the timely monitoring of fenvalerate residue dynamics is vital for ensuring the health of humans and the ecological environment, and it is necessary for establishing a fast, reliable, accurate and on-site method for detecting fenvalerate residues. Based on the methods of immunology, biochemistry and molecular biology, mammalian spleen cells, myeloma cells and mice were used as experimental materials to establish a rapid detection method of an enzyme-linked immunosorbent assay to detect the residues of fenvalerate in dark tea. Three cell lines-1B6, 2A11 and 5G2-that can stably secrete fenvalerate antibodies were obtained by McAb technology, and their sensitivities (IC50) were 36.6 ng/mL, 24.3 ng/mL and 21.7 ng/mL, respectively. The cross-reaction rates of the pyrethroid structural analogs were all below 0.6%. Six dark teas were used to detect the practical application of fenvalerate monoclonal antibodies. The sensitivity IC50 of the anti-fenvalerate McAb in PBS with 30% methanol is 29.12 ng/mL. Furthermore, a latex microsphere immunochromatographic test strip with an LOD of 10.0 ng/mL and an LDR of 18.9-357 ng/mL was preliminarily developed. A specific and sensitive monoclonal antibody for fenvalerate was successfully prepared and applied to detect fenvalerate in dark teas (Pu'er tea, Liupao tea, Fu Brick tea, Qingzhuan tea, Enshi dark tea and selenium-enriched Enshi dark tea). A latex microsphere immunochromatographic test strip was developed for the preparation of rapid detection test strips of fenvalerate.

Multiplex lateral flow immunochromatographic assay for the qualitative and quantitative detection of six steroid hormone residues in chicken and pork

To ensure food safety from animal origins and to prevent potential hazards caused by steroid hormones, there is an urgent need to develop a rapid detection method for steroid hormones in foods from animal origins. Three highly sensitive monoclonal antibodies (mAb) against testosterone propionate (Tp), stazolol (STA) and estradiol benzoate (EB) were prepared, with IC50 values of 0.13, 0.14 and 0.19 ng/mL, respectively. Tp-mAb is a group-selective mAb that simultaneously detects Tp, testosterone (T), methyltestosterone (MT) and testosterone phenylpropionate (TPP), and their IC50 values were 0.13, 0.77, 0.90, and 1.25 ng/mL, respectively. A novel multiplex lateral flow immunochromatographic strip (ICS) was further prepared, which could simultaneously detect Tp, STA, and EB in chicken and pork samples with limits of quantification (LOQ) of 0.32, 0.51, 0.35 μg/kg and 0.36, 0.64, 0.43 μg/kg, respectively. Based on the broad spectrum of Tp-mAb, ICS has been further developed to detect Tp, T, MT and TPP simultaneously. This multiplex ICS rapid detection method was efficient, sensitive, and fast, for the simultaneous detection of multiple steroid hormones in actual samples. Its detection time is only 40 min, which it represents a favorable monitoring tool for the detection of steroid hormones in animal-derived foods from the marketplace.

A Rapid Immunochromatographic Method Based on Gold Nanoparticles for the Determination of Imidacloprid on Fruits and Vegetables.

Imidacloprid (IMP) is toxic and a potential carcinogen that is most widely used as an insecticide for pest control and seed treatment. It is important to produce a rapid and sensitive assay for on-site monitoring. We have developed a novel lateral flow assay (LFA) using a sensitive monoclonal antibody (mAb) for monitoring IMP residues on fruits and vegetables. The 50% inhibition concentration result that was found when using the ELISA method was 0.247 ng mL-1, with the cut-off limits using the LFA method the result was 10 ng mL-1 (0.01 M PBS), and in the samples it was 20 ng mL-1 (with a recovery rate of 96-104.7% for Chinese cabbage, cowpea, apple, and pear samples, respectively). All of the results can be determined within seven minutes. The proposed LFA method is a valid, quick, and stable assay for the on-site detection of IMP in large numbers of samples.

Fluorescent microsphere-based lateral-flow immunoassay for rapid and sensitive determination of eugenols

In this study, a sensitive monoclonal antibody (mAb) 1B5 against eugenols was prepared based on a novel hapten. Based on this mAb, a paper-based lateral-flow immunoassay (LFIA) was developed using Eu-fluorescent microspheres sensor, that could achieve qualitative and quantitative detection of eugenols within 10 min. Results showed colorimetric values observed by the naked eye were 12.3 µg/kg, 12.3 µg/kg, 37 µg/kg and 111 µg/kg for eugenol, isoeugenol, methyl eugenol, and methyl isoeugenol, respectively, in both water and fish samples. For quantitative detection of eugenol, isoeugenol, methyl eugenol and methyl isoeugenol, the detection ranges were 4.49–48.4 µg/kg, 6.02–66.8 µg/kg, 16.5–150 µg/kg and 47.9–710 µg/kg in water, and 3.9–30.9 µg/kg, 5.9–62.6 µg/kg, 16.7–255 µg/kg, and 44.5–890 µg/kg in fish, respectively. The recovery test and detection in fish demonstrated the reliability of the LFIA in real samples. Therefore, the developed LFIA produced a promising alternative tool for the rapid on-site detection of eugenols.

A colloidal gold immunoassay strip assay for cadmium detection in oilfield chemicals.

Cadmium ions (Cd2+) are some of the major pollutants in oilfield chemicals. To reduce the pollution of oilfield chemicals, it is necessary to detect and control the content of Cd2+. In this study, we synthesized a highly sensitive and specific monoclonal antibody against Cd2+ with an IC50 of 1.97 ng mL-1 and no cross-reactivity. Based on this antibody, a colloidal gold immunoassay strip detection assay with an IC50 of 1 mg kg-1 and a detection range of 1.0-20 mg kg-1 in oilfield chemicals was developed. This assay could be completed in 20 min and can be used for Cd2+ on-site testing in oilfield chemicals and improve supervision efficiency in oil exploration and development.

A microfluidic immunosensor based on magnetic separation for rapid detection of okadaic acid in marine shellfish

Okadaic acid (OA) is a marine biotoxin that accumulates in seafood and can cause diarrheic shellfish poisoning if consumed. Accordingly, many countries have established regulatory limits for the content of OA in shellfish. At present, methods used for the detection of marine toxins are time-consuming and labor-intensive. In order to realize rapid, simple, and accurate detection of OA, we developed a novel microfluidic immunosensor based on magnetic beads modified with a highly specific and sensitive monoclonal antibody (mAb) against OA that is used in conjunction with smartphone imaging to realize the rapid detection of OA in shellfish. The method achieves on-site detection results within 1 h with an IC50 value of 3.30 ng/mL for OA and a limit of detection (LOD) of 0.49 ng/mL. In addition, the analysis of real samples showed that the recoveries for spiked shellfish samples ranged from 84.91% to 95.18%, and the results were confirmed by indirect competitive enzyme-linked immunosorbent assay (icELISA), indicating that the method has good accuracy and precision. Furthermore, the results are reported in a specially designed smartphone app. The microfluidic immunosensor has the advantages of simple operation, rapid detection, and high sensitivity, providing a reliable technical solution for detecting OA residues in shellfish.

Establishment of a Sensitive Monoclonal Antibody Against Mouse CCR9 (C9Mab-24) for Flow Cytometry

The CC chemokine receptor 9 (CCR9), also known as CD199, is one of chemokine receptors. The CC chemokine ligand 25 (CCL25) is known to be the only ligand for CCR9. The CCR9-CCL25 interaction plays important roles in chemotaxis of lymphocytes and tumor cell migration. Therefore, CCR9-CCL25 axis is a promising target for tumor therapy and diagnosis. In this study, we established a sensitive and specific monoclonal antibody (mAb) against mouse CCR9 (mCCR9) using N-terminal peptide immunization method. The established anti-mCCR9 mAb, C9Mab-24 (rat immunoglobulin [IgG]2a, kappa), reacted with mCCR9-overexpressed Chinese hamster ovary-K1 (CHO/mCCR9) and mCCR9-endogenously expressed cell line, RL2, through flow cytometry. Kinetic analyses using flow cytometry showed that the dissociation constants (KD) of C9Mab-24 for CHO/mCCR9 and RL2 cell lines were 6.0 × 10-9 M and 4.7 × 10-10 M, respectively. Results indicated that C9Mab-24 is useful for detecting mCCR9 through flow cytometry, thereby providing a possibility for targeting mCCR9-expressing cells in vivo experiments.

Development of a Sensitive Anti-Human CCR9 Monoclonal Antibody (C9Mab-11) by N-Terminal Peptide Immunization.

The C-C chemokine receptor 9 (CCR9) belongs to the G-protein-coupled receptor superfamily, and is highly expressed on the T cells and intestinal cells. CCR9 regulates various immune responses by binding to the C-C chemokine ligand, CCL25, and is involved in inflammatory diseases and tumors. Therefore, the development of sensitive monoclonal antibodies (mAbs) for CCR9 is necessary for treatment and diagnosis. In this study, we established a specific anti-human CCR9 (hCCR9) mAb; C9Mab-11 (mouse IgG2a, kappa), using the synthetic peptide immunization method. C9Mab-11 reacted with hCCR9-overexpressed Chinese hamster ovary-K1 (CHO/hCCR9) and hCCR9-endogenously expressed MOLT-4 (human T-lymphoblastic leukemia) cells in flow cytometry. The dissociation constant (KD) of C9Mab-11 for CHO/hCCR9 and MOLT-4 cells were determined to be 1.2 × 10-9 M and 4.9 × 10-10 M, respectively, indicating that C9Mab-11 possesses a high affinity for both exogenously and endogenously hCCR9-expressing cells. Furthermore, C9Mab-11 clearly detected hCCR9 protein in CHO/hCCR9 cells using western blot analysis. In summary, C9Mab-11 can be a useful tool for analyzing hCCR9-related biological responses.