Table 1.

Clinical studies using keyhole limpet haemocyanin (KLH) administered via a trans-cutaneous route to assess the influence of an exposure or agent on immune response in humans (Studies conducted between 1994–2013)

Study and aim	KLH formulation (Source), dose, route and site of administration	Antibody assessment: assay type, target immunoglobulin(s), sampling period relative to immunization	Cell-mediated immunity (Ex vivo and in vivo testing)
Ferbas et al. [40] To assess performance characteristics of immunoassays measuring antigen specific response to KLH immunization in healthy controls and patients with systemic lupus erythematosus	HMW-KLH (ImmuneActivator™, Intracel, Rockville, USA) 1000 μg Intradermal Forearm 2 doses: Day 1 & 29	Flow cytometric bead array KLH IgM and IgG; IgG1-4 Baseline and days 7, 14, 28, 35 and 42 post-immunization	Ex vivo: ELISPOT assay to detect numbers of B cells secreting KLH IgG In vivo: Not evaluated
Gallegos et al. [30] To examine the effects of mindfulness-based stress reduction on immunological outcomes in older adults	KLH formulation not noted 8, 40, 100, 200, 1000 μg KLH Intramuscular Deltoid muscle	ELISA (type not-specified) KLH IgM and IgG Baseline, 3 and 24 weeks post immunization	Not evaluated
Boulton et al. [47] To examine the influence of fingolimod therapy on immune responses to immunization with neo-antigens and recall antigens	Sub-unit KLH (Immucothel®, Biosyn, Carlsbad, USA) 0.5 ml (100 μg) adsorbed to alum Intramuscular Site not specified	Indirect ELISA KLH IgM, IgG Baseline and 1, 2, 3, 5 and 7 weeks post-immunization	Ex vivo: Not evaluated In vivo: 10 μg KLH intradermal 4 weeks post-immunization; Anterior aspect of upper or lower arm; Read at 48 h
Kantele et al. [44] To examine the tissue homing response of lymphocytes following KLH immunization and secretion of KLH antibodies at a cellular level	HMW-KLH (Pacific Biomarine, Venice, USA) 100 μg Subcutaneous Site not specified	Quantification of serum KLH antibodies not performed	Ex vivo: ELISPOT assay to detect number of B cells secreting KLH IgG, IgA and IgM after immune-magnetic sorting of lymphocytes by tissue-specific homing receptors In vivo: Not evaluated
Bingham et al. [36] To examine immunization responses in patients with rheumatoid arthritis treated with rituximab	HMW-KLH (Intracel, Frederick, USA) Dose not specified Subcutaneous Site not specified	ELISA (type not-specified) KLH IgG Baseline and 4 weeks post-immunization	Not evaluated
Spazierer et al. [38] To establish an immunization protocol to induce de novo Th2 responses using immunization with KLH	Sub-unit KLH (Immucothel®, Biosyn Arzneimittel GmbH, Fellbach, Germany) 100 μg KLH with alum (dose not specified) Intramuscular Site not specified; Three doses: day 1, 15 and 29	Indirect and sandwich ELISA KLH IgG1, IgG4, IgE, IgM Baseline and days 14, 28, 42 and 56 post-immunization	Ex vivo: Lymphocyte proliferation assays In vivo: Not evaluated
Grant et al. [32] To examine the effect of aerobic exercise in sedentary older adults on primary immune response to KLH immunization	HMW-KLH (BCI-ImmuneActivator™, Intracel, Rockville, USA) 125 μg Intramuscular Deltoid muscle	Indirect ELISA KLH IgG1, IgG2, IgM Baseline and 2, 3 and 6 weeks post-immunization	Ex vivo: Not evaluated In vivo: 5 μg KLH ID 3-week post-immunization; Timing of reading not specified; Site not specified
Miller et al. [33] To compare the responses to KLH immunization in healthy adults with those in immunosuppressed patients (cancer and bone marrow transplant recipients)	HMW KLH HMW KLH (Intracel, Rockville, USA) Sub-unit (Biosyn Corp, Carlsbad, USA) 1. Sub-unit KLH 1000 μg 2. HMW-KLH 1000 μg 3. Sub-unit KLH 1000 μg with Montanide-ISA-51 adjuvant (0.6 ml) Subcutaneous Site not specified	Indirect and sandwich ELISA KLH IgG1, IgG2, IgM Baseline and 4 weeks post-immunization	Ex vivo: Lymphocyte proliferation assays; ELISPOT assay for cellular responses to KLH In vivo: Not evaluated
Smith et al. [28] To examine the effect of distress on primary KLH immunization response in young adults	Sub-unit KLH (Pierce, Rockford, USA) 100 μg KLH adsorbed to 0.9 mg alum Intramuscular Deltoid muscle	Indirect ELISA; KLH IgG Baseline and 3 weeks post-immunization	Ex vivo: Lymphocyte proliferation assays In vivo: 1 μg intradermal 3 weeks post-immunization; Volar aspect of arm; Read at 48 h
Smith et al. [29] To examine the effect of psychological distress on DTH response following primary KLH immunization in young adults	Sub-unit KLH (Pierce, Rockford, USA) 100 μg KLH adsorbed to 0.9 mg alum Intramuscular Deltoid muscle	Not evaluated	Ex vivo: Not evaluated In vivo: 1 μg intradermal 3 weeks post-immunization; Volar aspect of arm; Read at 48 h
Smith et al. [31] To examine the effect of age and physical activity on primary immune response to KLH immunization	Sub-unit KLH (Pierce, Rockford, USA) 100 μg KLH adsorbed to 0.9 mg alum Intramuscular Deltoid muscle	Indirect ELISA KLH IgG, IgG1, IgG2 and IgM; Baseline and 1, 2, 3 and 4 weeks post-immunization	Ex vivo: Not evaluated In vivo: 1 μg intradermal 21 days post-immunization; Volar aspect of arm; Read at 24, 48, 72, 96 & 120 h
Boelens et al. [45] To examine the effect of severe trauma on early primary immune response to KLH immunization in relation to low plasma glutathione	KLH formulation/source not stated 500 μg Subcutaneous Deltoid region	Indirect ELISA KLH IgM, IgA, IgG, IgG1-4 Baseline and days 8 and 13 post-immunization	Ex vivo: Lymphocyte proliferation assay; Frequency of interferon-γ (Th1) and IL-4 (Th2)-producing T-lymphocytes cells by flow cytometry assays In vivo: 100 μg intradermal 14 days post-immunization; Right gluteal region of leg; Read at 48 h
Rentenaar et al. [46] To examine the cellular and humoral responses to immunization in renal transplant recipients receiving different immunosuppressive regimes	HMW KLH (Source not stated) 1000 μg Subcutaneous Right arm	Indirect ELISA KLH IgG Baseline and day 14 post-immunization	Ex vivo: Lymphocyte proliferation assay; In vivo: 1 and 10 μg intradermal 14 days post-immunization; Lower arm; Read at 24 h
Van der Kolk et al. [34] To assess the influence of rituximab on the humoral immune response to immunization with primary and recall antigens in patients with low grade lymphoma	HMW KLH (Calbiochem, San Diego, USA) 1000 μg Subcutaneous Site not specified	Indirect ELISA KLH IgG Baseline and 14 days post-immunization	Not evaluated
Valdez et al. [37] To assess response to immunization after prolonged anti-retroviral therapy in patients with HIV	HMW KLH (ImmuneActivator™, PerImmune, Rockville, USA) 1000 μg Intradermal Site not stated	Indirect ELISA KLH IgG Baseline and 2, 6, 12 and 18 weeks post-immunization	Ex vivo: Lymphocyte proliferation assays In vivo: Intradermal (dose not specified); 6 and 18 weeks post-immunization; Read at 48–72 h
Kondratenko et al. [35] To evaluate responses to primary KLH immunization in patients with immunodeficiency states	HMW KLH (Calbiochem, San Diego, USA) 200 μg Intradermal Upper arm	Indirect ELISA KLH IgG, IgM Baseline and 2 and 4 weeks post-immunization	Ex vivo: Lymphocyte proliferation assay In vivo: Not evaluated

DTH, delayed type hypersensitivity; ELISPOT, Enzyme-linked immunosorbent spot assay; HMW, high molecular weight.