Keyhole limpet hemocyanin (KLH) is a glycosylated multi-subunit metalloprotein that elicits a strong nonspecific immune activation, thus inducing both cellular and humoral immune responses. The exceptional immunogenicity of this protein can be leveraged to vaccinate mice against self-antigens that otherwise would not induce an autoimmune response.
This protocol describes the covalent conjugation of KLH with acyl-coenzyme A-binding protein (ACBP), the autovaccination of mice with ACBP-KLH conjugate together with a potent adjuvant, and the detection of the produced anti-ACBP autoantibodies. For complete details on the use and execution of this profile, please refer to Bravo-San Pedro et al. (2019c).
Keyhole Limpet Hemocyanin-Conjugated Peptides from Hepatitis C Virus Glycoproteins Elicit Neutralizing Antibodies in BALB/c Mice
Currently, no vaccine to prevent hepatitis C virus (HCV) infection is available. A major challenge in developing an HCV vaccine is the high diversity of HCV sequences. The purpose of immunization with viral glycoproteins is to induce a potent and long-lasting cellular and humoral immune response. However, this strategy only achieves limited protection, and antigen selection plays a crucial role in vaccine design. In this study, we investigated the humoral immune responses induced by intraperitoneal injection of keyhole limpet hemocyanin conjugated with 4 highly conserved peptides, including amino acids [aa]317-325 from E1 and aa418-429, aa502-518, and aa685-693 from E2, or 3 peptides from hypervariable region 1 (HVR1) of E2, including the N terminus of HVR1 (N-HVR1, aa384-396), C terminus of HVR1 (C-HVR1, aa397-410), and HVR1 in BALB/c mice.
The neutralizing activity against HCV genotypes 1-6 was assessed using the cell culture HCV (HCVcc) system. The results showed that the 4 conserved peptides efficiently induced antibodies with potent neutralizing activity against 3 or 4 genotypes. Antibodies induced by aa685-693 conferred potent protection (>50%) against genotypes 2, 4, and 5. Peptide N-HVR1 elicited antibodies with the most potent neutralization activities against 3 HCV genotypes: TNcc(1a), S52(3a), and ED43(4a). These findings suggested that peptides within HCV glycoproteins could serve as potent immunogens for vaccine design and development.
Novel Polyclonal Antibody Raised against Tetrodotoxin Using Its Haptenic Antigen Prepared from 4,9-anhydrotetrodotoxin Reacted with 1,2-Ethaneditiol and Further Reacted with Keyhole Limpet Hemocyanin.
A novel polyclonal antibody against tetrodotoxin (TTX) was raised using its haptenic antigen, where 4,9-anhydroTTX was reacted with 1,2-ethanedithiol and this derivative was further reacted with keyhole limpet hemocyanin (KLH). This newly designed antigen (KLH-TTX) was inoculated into rabbits, resulting in the production of the specific polyclonal antibody, which reacted well with TTX and its analogs, 4-epiTTX, 11-oxoTTX and 5,6,11-trideoxyTTX, except for 4,9-anhydroTTX.
The enzyme-linked immunosorbent assay (ELISA) system using this specific antibody was also developed in the present study. This newly developed polyclonal antibody with analytical procedures using direct one-step ELISA is useful to detect TTX and its analogs in toxic organisms and also disclose the mechanisms involved in their metabolic pathways and accumulation of TTX.
Transfer of natural auto-antibodies via egg yolk in chickens divergently selected for natural antibodies binding keyhole limpet hemocyanin.
Barcodes of natural auto-antibody (NAAb) profiles based on staining intensity of isotypes binding numbers of self-(tissue) antigen fragments were suggested as parameters for immune diversity, and related to genetic background and health status in man, rodents and poultry. Here, hens, eggs and hatchlings from chicken lines divergently selected and bred for high (H line) or low (L line) total natural antibodies (NAb) levels in plasma binding keyhole limpet hemocyanin (KLH) at 16 weeks of age were tested for their NAAb repertoire binding chicken liver homogenate (CLH) fragments using quantitative Western immunoblotting. The aims of this study were 1. to detect line differences between the H and L line adult hens, eggs and hatchlings for the IgM and IgG isotypes binding CLH fragments, 2. study the presence of NAAb of both isotypes in yolk and albumen, as well as in hatchlings to detect a maternal NAAb transfer route via the egg to the hatchling, and 3. study whether new self-antigen binding isotypes and idiotypes are present in the hatchling. NAAb binding CLH fragments were found in plasma of adult hens (both IgM and IgG), in yolk (IgG only), and hatchlings (mostly IgG, but low levels of IgM).
Auto-profiles of IgM showed homogeneity, while IgG profiles were heterogenic between individual hens and individual hatchlings. Significant higher levels as indicated by staining intensity and number of stained CLH fragments were found in plasma of hens genetically selected for high levels of NAb binding KLH. Lines could be clustered based on their auto-profiles indicating that profiles of self-binding IgM and IgG antibodies are genetically based. Visual comparison, clustering and correlation of hens and their hatchlings showed similarities for the IgG, but not the IgM isotype, indicating maternal transfer of IgG NAAb via the yolk. The IgM profile in the hatchlings on the other hand might represent neonatal self-binding antibody formation. As a consequence, hatchlings initially depend for self-binding antibodies on maternal IgG provision during early life.
KLH | Keyhole limpet hemocyanin |
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| AS99-001 | Agrisera AB | 50 µg | 299 EUR |
Keyhole limpet hemocyanin(KLH) |
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| THP-0009 | Creative BioMart | 1mg | 2398.4 EUR |
Keyhole Limpet Hemocyanin (KLH) (PE) |
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| MBS6129427-01mL | MyBiosource | 0.1(mL | 715 EUR |
Keyhole Limpet Hemocyanin (KLH) (PE) |
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| MBS6129427-5x01mL | MyBiosource | 5x0.1mL | 3075 EUR |
Keyhole Limpet Hemocyanin (KLH) (AP) |
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| MBS6126331-01mL | MyBiosource | 0.1(mL | 715 EUR |
Keyhole Limpet Hemocyanin (KLH) (AP) |
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| MBS6126331-5x01mL | MyBiosource | 5x0.1mL | 3075 EUR |
Keyhole Limpet Hemocyanin (KLH) (HRP) |
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| MBS6128807-01mL | MyBiosource | 0.1(mL | 715 EUR |
Keyhole Limpet Hemocyanin (KLH) (HRP) |
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| MBS6128807-5x01mL | MyBiosource | 5x0.1mL | 3075 EUR |
Keyhole Limpet Hemocyanin (KLH) (APC) |
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| MBS6126951-01mL | MyBiosource | 0.1(mL | 715 EUR |
Keyhole Limpet Hemocyanin (KLH) (APC) |
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| MBS6126951-5x01mL | MyBiosource | 5x0.1mL | 3075 EUR |
Keyhole Limpet Hemocyanin (KLH) (FITC) |
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| MBS6128186-01mL | MyBiosource | 0.1(mL | 715 EUR |
Keyhole Limpet Hemocyanin (KLH) (FITC) |
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| MBS6128186-5x01mL | MyBiosource | 5x0.1mL | 3075 EUR |
Keyhole Limpet Hemocyanin (KLH)(FITC) |
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| MBS6507864-01mL | MyBiosource | 0.1mL | 865 EUR |
Keyhole Limpet Hemocyanin (KLH)(FITC) |
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| MBS6507864-5x01mL | MyBiosource | 5x0.1mL | 3745 EUR |
Rat Keyhole limpet hemocyanin ELISA |
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| E01A14249 | BlueGene | 96T | 700 EUR |
Goat Keyhole limpet hemocyanin ELISA |
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| E01A49157 | BlueGene | 96T | 700 EUR |
Keyhole Limpet Hemocyanin (KLH) (Biotin) |
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| MBS6127572-01mL | MyBiosource | 0.1(mL | 715 EUR |
Keyhole Limpet Hemocyanin (KLH) (Biotin) |
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| MBS6127572-5x01mL | MyBiosource | 5x0.1mL | 3075 EUR |
Keyhole Limpet Hemocyanin (KLH)(Biotin) |
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| MBS6507863-01mg | MyBiosource | 0.1mg | 865 EUR |
Keyhole Limpet Hemocyanin (KLH)(Biotin) |
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| MBS6507863-5x01mg | MyBiosource | 5x0.1mg | 3745 EUR |
Human Keyhole limpet hemocyanin ELISA |
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| E01A5501 | BlueGene | 96T | 700 EUR |
Sheep Keyhole limpet hemocyanin ELISA |
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| E01A101450 | BlueGene | 96T | 700 EUR |
Localization of keyhole limpet hemocyanin-like immunoreactivity in the nervous system of Biomphalaria alexandrina.
- Recent years have led to increased effort to describe and understand the peripheral nervous system and its influence on central mechanisms and behavior in gastropod molluscs. This study revealed that an antibody raised against keyhole limpet hemocyanin (KLH) cross-reacts with an antigen(s) found extensively in both the central and the peripheral nervous systems of Biomphalaria alexandrina. The results revealed KLH-like immunoreactive (LIR) neurons in the cerebral, pedal, buccal, left pleural, right parietal, and visceral ganglion within the CNS with fibers projecting throughout all the peripheral nerves.
- Numerous KLH-LIR peripheral sensory neurons located in the foot, lips, tentacles, mantle, esophagus, and penis exhibited a bipolar morphology with long tortuous dendrites. KLH-LIR cells were also present in the eye and statocyst, thus suggesting the labeling of multiple sensory modalities/cell types. KLH-LIR cells did not co-localize with tyrosine hydroxylase (TH)-LIR cells, which have previously been described in this and other gastropods. The results thus provide descriptions of thousands of peripheral sensory neurons, not previously described in detail. Future research should seek to pair sensory modalities with peripheral cell type and attempt to further elucidate the nature of KLH-like reactivity.
- These findings also emphasize the need for caution when analyzing results obtained through use of antibodies raised against haptens conjugated to carrier proteins, suggesting the need for stringent controls to help limit potential confounds caused by cross-reactivity. In addition, this study is the first to describe neuronal cross-reactivity with KLH in Biomphalaria, which could provide a substrate for host-parasite interactions with a parasitic trematode, Schistosoma.