LAG-3 Antibody (L4-PL33) - BSA Free

Flow Cytometry: LAG-3 Antibody (17B4) [NBP1-97657] - Analysis using the FITC conjugate of NBP1-97657. Tumor infiltrating lymphocytes (TILs) express LAG-3 (detected using LAG-3 (human), mAb (17B4).

Western Blot: LAG-3 Antibody (17B4) [NBP1-97657] - Lane 1: MW marker. Lane 2: LAG-3 (human):Fc (human).

Flow Cytometry: LAG-3 Antibody (17B4) [NBP1-97657] - Analysis using the FITC conjugate of NBP1-97657. LAG-3 staining in resting and PHA activated lymphocytes. Image submitted by a verified customer review.

Flow Cytometry: LAG-3 Antibody (17B4) [NBP1-97657] - Analysis using the FITC conjugate of NBP1-97657. Staining of LAG-3 on CD4+ and CD8+ subpopulations of tumour infiltrating lymphocytes (TILs) detected with LAG-3 (human), mAb (17B4) (FITC).

Immunohistochemistry-Paraffin: LAG-3 Antibody (17B4) [NBP1-97657] - LAG-3 in human tonsil tissue sample. Image submitted by a verified customer review.

Flow Cytometry: LAG-3 Antibody (17B4) [NBP1-97657] - Analysis using the FITC conjugate of NBP1-97657. LAG-3 expression on activated human peripheral blood mononuclear cells (PBMC) detected with LAG-3 (human), mAb (17B4) (FITC).Method: T lymphocytes from human PBMC are stimulated with 1 ug/mL of PHA for three days. Then, after seven days of culture, 3x10^6 three-days PHA-activated human PBMC are treated with LAG-3 (human), mAb (17B4) (FITC) or FITC coupled isotype-matched (IgG1) control MAb (used at a saturating dilution of 1:800 and 1:150 respectively) for 30 min. at 4C in RPMI 1640 and washed twice with 1X PBS. Stained cells are then analysed by FC [4].

Flow Cytometry: LAG-3 Antibody (17B4) [NBP1-97657] - Analysis using the FITC conjugate of NBP1-97657. Method: LAG-3 (human), mAb (17B4) (FITC) (10 ug/mL) (is preincubated with a specific peptide epitope (208b) or a control tetanus toxoid (TT) peptide at different molarities prior to staining of TILs.

• Reactivity: Hu, Pm
• Applications: WB, ELISA, Flow, ICC/IF, IHC, IHC-Fr, IHC-P, IP, B/N
• Clone: L4-PL33
• Clonality: Monoclonal
• Host: Mouse
• Conjugate: Unconjugated
• Format: BSA Free
• Concentration: 1.0 mg/ml

LAG-3 Antibody (L4-PL33) - BSA Free Summary

• Immunogen: This LAG-3 Antibody (L4-PL33) was prepared from synthetic peptide corresponding to 30 aa (GPPAAAPGHPLAPGPHPAAPSSWGPRPRRY) from the first N-terminal D1 domain of human LAG-3 (lymphocyte activation gene-3).
• Specificity: LAG-3 Antibody (L4-PL33) recognizes the 30 aa extra-loop of the first N-terminal D1 domain of human LAG-3.
• Isotype: IgG1 Kappa
• Clonality: Monoclonal
• Host: Mouse
• Gene: LAG3
• Purity: Protein A or G purified

Applications/Dilutions

• Dilutions:
  • Block/Neutralize
  • ELISA
  • Flow Cytometry
  • Immunocytochemistry/Immunofluorescence 1:10 - 1:500
  • Immunohistochemistry 1:10 - 1:500
  • Immunohistochemistry-Frozen 1:150
  • Immunohistochemistry-Paraffin
  • Immunoprecipitation 10 ug/mL
  • Western Blot 5 ug/mL
• Application Notes: Blocking/neutralizing application reported in scientific literature (PMID: 29671649, PMID: 28935468)
• Theoretical MW: 57.5 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Reactivity Notes

Primate reactivity reported in scientific literature (PMID: 32284611).

Packaging, Storage & Formulations

• Storage: Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.
• Buffer: PBS, 10% glycerol
• Preservative: 0.02% Sodium Azide
• Concentration: 1.0 mg/ml
• Purity: Protein A or G purified

Notes

Please note the clone number for this product has changed from 17B4 to L4-PL33

Alternate Names for LAG-3 Antibody (L4-PL33) - BSA Free

• 17b4 lag3
• 17b4 neutralizing
• 17b4
• anti human lag3
• Anti-LAG-3 Antibody
• CD223 Antibody
• CD223 Antigen Antibody
• CD223 antigen
• CD223
• Human LAG3 Antibody
• Human LAG-3 Antibody
• LAG-3 17b4
• LAG-3 blocking
• LAG3
• LAG-3
• lymphocyte activating 3
• lymphocyte activation gene 3 protein
• lymphocyte-activation gene 3
• Secreted lymphocyte activation gene 3 protein
• sLAG-3

Background

Lymphocyte activation gene-3 (LAG-3), also referred to as CD233, is a type I transmembrane protein with a theoretical molecular weight of 70 kDa that is a member of the immunoglobulin superfamily (IgSF) (1, 2). Human LAG-3 cDNA encodes 525 amino acids (aa) that includes a 28 aa signal sequence, a 422 aa extracellular domain (ECD) with four Ig-like domains (D1-D4), a transmembrane region and a highly charged cytoplasmic region. Within the ECD, human LAG-3 shares 70%, 67%, 76%, and 73% aa sequence identity with mouse, rat, porcine, and bovine LAG-3, respectively. The extracellular region of LAG-3 and the CD4 co-receptor share ~20% aa sequence homology but are structurally similar and both bind to major histocompatibility complex class II (MHCII) on antigen-presenting cells (APCs), although LAG-3 has much higher affinity (1, 3). LAG-3 is highly expressed in the lymph node, spleen, ovary, and appendix while expressed at a lower level in a variety of other tissues. More specifically, LAG-3 is expressed on activated CD4+ and CD8+ T cells, natural killer T (NKT) cells, natural killer (NK) cells, plasmacytoid dendritic cells (pDCs), and regulatory T cells (Tregs), but not on naive, or resting, T cells (1, 3).

As mentioned above, LAG-3 binds to MHCII and this occurs via a proline-rich amino acid loop in D1 (1, 3). Another unique feature of LAG-3 is the longer connecting peptide region between the D4 and the transmembrane, which is acted upon and cleaved by metalloproteinases a disintegrin and metallopeptidase domain (ADAM) 10 and ADAM17 to generate a soluble 54 kDa form of LAG-3 (sLAG-3) (1, 3). The interaction of LAG-3 with MHCII prevents the MHC molecule from binding to a T-cell receptor (TCR) or CD4, thereby functioning in an inhibitory role and suppressing the TCR signal (4). When LAG-3 crosslinks with the TCR/CD3 complex, it causes reduced T-cell proliferation and cytokine secretion (4). This negative regulation is important in controlling autoimmunity as one study found Lag3-/- NOD (non-obese diabetic) mice had accelerated diabetes onset and increased T-cell infiltration into islet cells (5). On the other hand, besides being a negative regulator of T-cells, LAG-3 binding to MHCII molecules on APCs induces dendritic cell maturation and cytokine secretion by monocytes (5, 6). In addition to MHCII, other reported ligands for LAG-3 includes fibrinogen-like protein 1 (FGL1), liver endothelial cell lectin (lSECtin), galectin-3 (Gal-3), and alpha-synuclein fibrils (1). Gal-3, for instance, is expressed on stromal cells and CD8+ T-cells in the tumor microenvironment and the interaction with LAG-3 was shown to be crucial for the suppression of secreted cytokine IFN-gamma and may control anti-tumor immune responses (1, 5). Interestingly, a mouse model of Parkinson's disease revealed LAG-3 binding to alpha-synuclein fibrils in the central nervous system, contributing to its pathogenesis (1, 5).

Recent cancer immunotherapeutic approaches have focused on inhibitory receptors such as LAG-3 to revive expression of cytotoxic T-cells to attack tumors (6). LAG-3 has been shown to be co-expressed and have synergy with another immune-checkpoint molecule called programmed-death 1 (PD-1) (1, 4, 5, 6). In a mouse model of colon adenocarcinoma LAG3 blockade alone was largely ineffective, however co-blockade of LAG-3 and PD-1 limited tumor growth and resulted in tumor clearance in 80% of mice, compared to 40% with PD-1 blockade alone (5). Additionally, in a model of fibrosarcoma the LAG-3/PD-1 duel blockade increased survival and the percentage of tumor-free mice (5). Analysis of a variety of human tumor samples (e.g. melanoma, colon cancer, head and neck squamous cell carcinoma) also suggest that LAG3 alone and combinatorial treatment with PD-1 may be a good target for treatment (1, 3-6). To date there are over 10 different agents targeting LAG-3 in clinical trials for cancer either as an anti-LAG-3 blocking antibody monotherapy or as a combination antagonist bispecific antibody, primarily with PD-1 (1, 3-6).

Alternative names for LAG-3 includes 17b4 lag3, 17b4 neutralizing, 17b4, CD223, FDC, LAG-3 17b4, LAG-3 blocking, and LAG3.

References

1. Maruhashi, T., Sugiura, D., Okazaki, I. M., & Okazaki, T. (2020). LAG-3: from molecular functions to clinical applications. Journal for Immunotherapy of Cancer, 8(2), e001014. https://doi.org/10.1136/jitc-2020-001014

2. Triebel, F., Jitsukawa, S., Baixeras, E., Roman-Roman, S., Genevee, C., Viegas-Pequignot, E., & Hercend, T. (1990). LAG-3, a novel lymphocyte activation gene closely related to CD4. The Journal of experimental medicine, 171(5), 1393-1405. https://doi.org/10.1084/jem.171.5.1393

3. Ruffo, E., Wu, R. C., Bruno, T. C., Workman, C. J., & Vignali, D. (2019). Lymphocyte-activation gene 3 (LAG3): The next immune checkpoint receptor. Seminars in immunology, 42, 101305. https://doi.org/10.1016/j.smim.2019.101305

4. Long, L., Zhang, X., Chen, F., Pan, Q., Phiphatwatchara, P., Zeng, Y., & Chen, H. (2018). The promising immune checkpoint LAG-3: from tumor microenvironment to cancer immunotherapy. Genes & cancer, 9(5-6), 176-189.

5. Andrews, L. P., Marciscano, A. E., Drake, C. G., & Vignali, D. A. (2017). LAG3 (CD223) as a cancer immunotherapy target. Immunological reviews, 276(1), 80-96. https://doi.org/10.1111/imr.12519

6. Goldberg, M. V., & Drake, C. G. (2011). LAG-3 in Cancer Immunotherapy. Current topics in microbiology and immunology, 344, 269-278. https://doi.org/10.1007/82_2010_114

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

Publications for LAG-3 Antibody (NBP1-97657)(12)

Publications using NBP1-97657

• Li K, Tandurella JA, Gai J et al. Multi-omic analyses of changes in the tumor microenvironment of pancreatic adenocarcinoma following neoadjuvant treatment with anti-PD-1 therapy Cancer cell Oct 21 2022 [PMID: 36306792] (IHC, Human)
• Rappold PM, Vuong L, Leibold J et al. A Targetable Myeloid Inflammatory State Governs Disease Recurrence in Clear Cell Renal Cell Carcinoma Cancer discovery Jun 27 2022 [PMID: 35758895]
• El Halabi L, Adam J, Gravelle P et al. Expression of the Immune Checkpoint Regulators LAG-3 and TIM-3 in Classical Hodgkin Lymphoma Clinical Lymphoma Myeloma and Leukemia Nov 1 2020 [PMID: 33277223] (IHC, Human)
  
  Details:
  Immunohistochemical analysis of biopsies from patients with classic Hodgkins lymphoma.
• Dummer R, Lebbe C, Atkinson V et al. Combined PD-1, BRAF and MEK inhibition in advanced BRAF-mutant melanoma: safety run-in and biomarker cohorts of COMBI-i Nat Med Oct 1 2020 [PMID: 33020648]
• Harper J, Gordon S, Chan C et Al. CTLA-4 and PD-1 dual blockade induces SIV reactivation without control of rebound after antiretroviral therapy interruption Nat Med. [PMID: 32284611] (Flow, Primate)
• Pericart S, Tosolini M, Gravelle P et al. Profiling Immune Escape in Hodgkin's and Diffuse large B-Cell Lymphomas Using the Transcriptome and Immunostaining Cancers (Basel) 2018 Oct 31 [PMID: 30384489] (IHC, Human)
  
  Details:
  Citation using the FITC form of this antibody.
• Wang J, Ti Y, Wang Y et al. LAG-3 Represents a Marker of CD4+ T Cells with Regulatory Activity in Patients with Bone Fracture. Immunol. Invest. Apr 19 2018 [PMID: 29671649] (Human)
• Ma QY, Huang DY, Zhang HJ et al. Function and regulation of LAG3 on CD4(+)CD25(-) T cells in non-small cell lung cancer Exp. Cell Res. 2017 Sep 19 [PMID: 28935468] (Human)
• Macon-Lemaitre L, Triebel F. The negative regulatory function of the lymphocyte-activation gene-3 co-receptor (CD223) on human T cells. Immunology May 31 2005 [PMID: 15885122]
  
  Details:
  Citation using the FITC format of this antibody.
• Di Carlo E, Cappello P, Sorrentino C et al. Immunological mechanisms elicited at the tumour site by lymphocyte activation gene-3 (LAG-3) versus IL-12: sharing a common Th1 anti-tumour immune pathway. J Pathol. 2005 Jan. [PMID: 15586367]
• Huard B, Gaulard P, Faure F et al. Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand. Immunogenetics. 1994 [PMID: 7506235]
• Baixeras E, Huard B, Miossec C et al. Characterization of the lymphocyte activation gene 3-encoded protein. A new ligand for human leukocyte antigen class II antigens. J Exp Med. 1992 Aug. [PMID: 1380059]

Reviews for LAG-3 Antibody (NBP1-97657) (2)

Average Rating: 4.5

(Based on 2 reviews)

We have 2 reviews tested in 1 species: Human.

reviewed by: Christian Schuerch Immunofluorescence - frozen Human 03/07/2020

Summary

• Application: Other
• Sample Tested: human tonsil
• Species: Human
• Lot: N/A

reviewed by: Alex Bowman IHC-P Human 01/23/2018

Summary

• Application: Immunohistochemistry-Paraffin
• Sample Tested: Tonsil
• Species: Human
• Lot: 02011620

FAQs for LAG-3 Antibody (NBP1-97657). (Showing 1 - 3 of 3 FAQs).

1. What is the concentration of your biotin conjugated LAG-3 antibody?
   • We actually do not determine the concentration of our conjugated antibodies but they are usually 80-100% the concentration of the base product, so the concentration should be at least 0.8 mg/ml.
2. While using NBP1-97657AF750 in TMA with 2 other antibodies for CD163 & CD44, we are having an issue with background "swirls" that we have pinned down to what we think is the antibody leaching off of the stained tissue over time. It seems to be in a swirl, around the tissue, after several washes and then mounting. It seems like it is not staying put. The staining is specific though.  I’m inclined to think that this may have to do with the fluorophore itself not the antibody. Could you make suggestions as to what might be happening?
   • F/P ratios and concentration for the antibody you ordered, NBP1-97657AF750 (lot 012220), are:  F/P 7.356, 0.72mg/ml.  Dilution for WB at 5ug/ml is 1:200, so for IHC I would use 20ug/ml, or suggested 1ug 1:150 (~7ug/ml) for the conjugated antibody. For this AF750 fluorophore, we release conjugated with F/P between 3 and 13. And yes, the starting ratios are the same for all AF750 conjugates and they were established experimentally. I have looked at all these 3 antibodies, the data for unconjugated forms as these have the most validation, and it looks that both CD163 and LAG-3 are both weaker than the CD44 ab (require more antibody to be used), and the targets are also less abundant. When the targets are less abundant, using direct conjugates is usually not recommended as you do not get signal amplification as with the secondary antibody. Unluckily, these two antibodies also had lower F/P ratios than the CD44 antibody, for example. At this point my best guess is that the signal intensity for  CD163 and LAG-3 is so weak that it lets you see the "swirl" from the stronger CD44 signal. So, this may not be the best fluorophore (AF750 has moderate brightness (3 out of 5). For NIR, Dylight 750 might be better (4 out of 5) for your set up. 
3. While using NBP1-97657AF750 in TMA with 2 other antibodies for CD163 & CD44, we are having an issue with background "swirls" that we have pinned down to what we think is the antibody leaching off of the stained tissue over time. It seems to be in a swirl, around the tissue, after several washes and then mounting. It seems like it is not staying put. The staining is specific though.  I’m inclined to think that this may have to do with the fluorophore itself not the antibody. Could you make suggestions as to what might be happening?
   • F/P ratios and concentration for the antibody you ordered, NBP1-97657AF750 (lot 012220), are:  F/P 7.356, 0.72mg/ml.  Dilution for WB at 5ug/ml is 1:200, so for IHC I would use 20ug/ml, or suggested 1ug 1:150 (~7ug/ml) for the conjugated antibody. For this AF750 fluorophore, we release conjugated with F/P between 3 and 13. And yes, the starting ratios are the same for all AF750 conjugates and they were established experimentally. I have looked at all these 3 antibodies, the data for unconjugated forms as these have the most validation, and it looks that both CD163 and LAG-3 are both weaker than the CD44 ab (require more antibody to be used), and the targets are also less abundant. When the targets are less abundant, using direct conjugates is usually not recommended as you do not get signal amplification as with the secondary antibody. Unluckily, these two antibodies also had lower F/P ratios than the CD44 antibody, for example. At this point my best guess is that the signal intensity for  CD163 and LAG-3 is so weak that it lets you see the "swirl" from the stronger CD44 signal. So, this may not be the best fluorophore (AF750 has moderate brightness (3 out of 5). For NIR, Dylight 750 might be better (4 out of 5) for your set up. 

Bioinformatics

• Gene Symbol: LAG3