- Apr 1, 2004
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I have seen this thread on many of the boards. It may be a bogus statement but as of yet I have seen no response from Lion. Take it for what it's worth. I do not agree or support this report. Just trying to look out for everyone untill this is cleared up by Lion. Better safe than sorry.
Warning - stay away from Lion's IGF-1!!!!
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Basically he is using an ingredient that is harmful to humans;
here is the official report;
The purpose of this study was to determine the existence of bovine serum albumin (BSA) in a preparation of Long R3 IGF-1 marketed as a kit, including “1 mg Lyophilized Long R3 IGF-1 Receptor Grade” by Superior-Research.com. Secondarily, the presence of a “binding/stabilizing” protein in the supplied diluent was in question and was therefore evaluated as well. The kit, as received, included a 10 ml plastic package (vial) of saline (Sodium Chloride Injection BP), a small glass vial containing a white, powdery substance labeled as coming from ”Research Unlimited” and an empty glass vial for mixing the two. This product is also offered on lionnutrition.com, research-4life.com, musclesci-research.com, ironcorpsresearch.com and anabolicreview-research.com.
The name “Long™ R3 IGF-1” is registered and trademarked, with the “Long” referring to the clone, not the added amino acid chain on the N-terminus. Additionally, this protein is created from an immature pre-cursor of IGF-1 and is not completely identical to the native protein (the R3 substitution is not withstanding). The fact that this product is provided in one glass vial and intended to be transferred to another on reconstitution would indicate the need for significant protein content to prevent considerable loss of product through nonspecific binding to the glass and syringes (since the provided diluent is intended for intravenous or intramuscular injection, one would assume the final diluted product itself is intended for administration in a similar fashion). When asked how this non-specific binding was avoided, Superior-Research.com provided documentation that strongly implied the presence of BSA. When confronted with the potential immunogenicity of this approach, the response was to back-pedal and substitute a “proprietary mix of human-grade proteins” for “BSA” in the explanation.
Upon receipt from Superior-research.com, the kit was prepared using 4 ml of the provided diluent to give a final IGF-1 concentration of 250 ug/ml and will herein be referred to as the test solution. The test solution and the diluent were colorimetrically evaluated for the presence of protein using the Bicinchoninic acid/Copper II protein assay (BCA Protein Assay Kit by Pierce). Briefly, the presence of protein is indicated by a color change to purple from green. The format of the assay was positive/negative with no quantitation. The test solution was a very strong positive while the diluent was completely negative. The conclusion drawn from this result is that the supplied diluent does not contain any protein whatsoever.
The presence of BSA was determined using a modified dot-blot process. The test solution (200 ul per spot, 8 spot grid pattern) was bound to nitrocellulose membranes held within a dot-blot apparatus under vacuum. Positive control spots (200 ul each) of 1 mg/ml of BSA (Sigma-Aldrich) and 1 mg/ml of human serum albumin (HSA, Sigma-Aldrich) were also placed on the membrane. Any protein applied would be bound to the nitrocellulose membrane. After application of the test spots, the membranes were blocked in goat serum. (This methodology uses the goat serum albumin to “fill in” the rest of the space on the membrane, preventing the antibodies from background staining the entire membranes.) After blocking, one membrane was probed with monoclonal antibodies for BSA (mouse anti-BSA unconjugated monoclonal Ab-1, LabVision) and one was probed with monoclonal antibodies for HSA (mouse anti-HSA monoclonal Ab, Biodesign). Both were specific and not cross-reactive with goat serum albumin. The monoclonal antibodies will bind to the BSA or the HSA, if present. The membranes were washed three times and probed with HRP-conjugated rabbit anti-mouse antibodies (ProteoQwest Chemiluminescent Western Blotting Kit, Sigma-Aldrich) and washed three times. The chemiluminescent substrate (same ProteoQwest kit) is then added and the horseradish peroxidase (HRP) enzymatically activates luminol, producing light emissions which are recorded on Kodak film. The presence of a light signal, indicated by the dark blots on the film, indicate the presence of BSA or HSA.
The test for BSA was conclusive. The BSA film showed nine significant, in fact, over-loaded, signals corresponding to the eight test solution spots and the 1 mg/ml BSA spot, as well as one lighter spot at the HSA position on that blot. The HSA film was negative except for the one spot corresponding to the HSA spot. The lighter signal detected for HSA on the BSA blot likely comes from non-specific binding and cross-reactivity. The greatly reduced intensity of the spot as compared to the same spot on the HSA blot would support that supposition. This laboratory cannot confirm the source of the BSA found in the test solution, only that it was present in the sample we received.
Given the positive result for BSA, the identity of the protein as IGF-1 also fell into question. To begin with, authentic receptor grade Long™ R3 IGF-1 protein (DSLaboratories) was evaluated by dot blot (as described above) to choose the antibody (mouse anti-human IGF-1 MoAb or mouse anti-human IGF-1 polyclonal Ab, both from DSLaboratories) which could also identify that version of the protein. Results were questionable with the monoclonal, so a polyclonal antibody to human IGF-1 was chosen to ensure adequate signal. With suitable standards available, polyacrylamide gel electrophoresis, followed by Western Blot was used to evaluate the identity of the protein in the test solution. Besides probing for human versions of the protein, as an additional control, antibodies to sheep, porcine, bovine, murine rabbit and rat IGF-1 were also used. The signal was strong for human IGF-1, but there was some signal from the porcine IGF-1 antibody. This spurious signal was eliminated by diluting the test solution to 100 ug/ml (sample overloading artifact). The results would indicate the protein is, in fact, human IGF-1. However, no definitive statement can be made about Long™ R3 IGF-1 as compared to mature, native recombinant human IGF-1.
Warning - stay away from Lion's IGF-1!!!!
--------------------------------------------------------------------------------
Basically he is using an ingredient that is harmful to humans;
here is the official report;
The purpose of this study was to determine the existence of bovine serum albumin (BSA) in a preparation of Long R3 IGF-1 marketed as a kit, including “1 mg Lyophilized Long R3 IGF-1 Receptor Grade” by Superior-Research.com. Secondarily, the presence of a “binding/stabilizing” protein in the supplied diluent was in question and was therefore evaluated as well. The kit, as received, included a 10 ml plastic package (vial) of saline (Sodium Chloride Injection BP), a small glass vial containing a white, powdery substance labeled as coming from ”Research Unlimited” and an empty glass vial for mixing the two. This product is also offered on lionnutrition.com, research-4life.com, musclesci-research.com, ironcorpsresearch.com and anabolicreview-research.com.
The name “Long™ R3 IGF-1” is registered and trademarked, with the “Long” referring to the clone, not the added amino acid chain on the N-terminus. Additionally, this protein is created from an immature pre-cursor of IGF-1 and is not completely identical to the native protein (the R3 substitution is not withstanding). The fact that this product is provided in one glass vial and intended to be transferred to another on reconstitution would indicate the need for significant protein content to prevent considerable loss of product through nonspecific binding to the glass and syringes (since the provided diluent is intended for intravenous or intramuscular injection, one would assume the final diluted product itself is intended for administration in a similar fashion). When asked how this non-specific binding was avoided, Superior-Research.com provided documentation that strongly implied the presence of BSA. When confronted with the potential immunogenicity of this approach, the response was to back-pedal and substitute a “proprietary mix of human-grade proteins” for “BSA” in the explanation.
Upon receipt from Superior-research.com, the kit was prepared using 4 ml of the provided diluent to give a final IGF-1 concentration of 250 ug/ml and will herein be referred to as the test solution. The test solution and the diluent were colorimetrically evaluated for the presence of protein using the Bicinchoninic acid/Copper II protein assay (BCA Protein Assay Kit by Pierce). Briefly, the presence of protein is indicated by a color change to purple from green. The format of the assay was positive/negative with no quantitation. The test solution was a very strong positive while the diluent was completely negative. The conclusion drawn from this result is that the supplied diluent does not contain any protein whatsoever.
The presence of BSA was determined using a modified dot-blot process. The test solution (200 ul per spot, 8 spot grid pattern) was bound to nitrocellulose membranes held within a dot-blot apparatus under vacuum. Positive control spots (200 ul each) of 1 mg/ml of BSA (Sigma-Aldrich) and 1 mg/ml of human serum albumin (HSA, Sigma-Aldrich) were also placed on the membrane. Any protein applied would be bound to the nitrocellulose membrane. After application of the test spots, the membranes were blocked in goat serum. (This methodology uses the goat serum albumin to “fill in” the rest of the space on the membrane, preventing the antibodies from background staining the entire membranes.) After blocking, one membrane was probed with monoclonal antibodies for BSA (mouse anti-BSA unconjugated monoclonal Ab-1, LabVision) and one was probed with monoclonal antibodies for HSA (mouse anti-HSA monoclonal Ab, Biodesign). Both were specific and not cross-reactive with goat serum albumin. The monoclonal antibodies will bind to the BSA or the HSA, if present. The membranes were washed three times and probed with HRP-conjugated rabbit anti-mouse antibodies (ProteoQwest Chemiluminescent Western Blotting Kit, Sigma-Aldrich) and washed three times. The chemiluminescent substrate (same ProteoQwest kit) is then added and the horseradish peroxidase (HRP) enzymatically activates luminol, producing light emissions which are recorded on Kodak film. The presence of a light signal, indicated by the dark blots on the film, indicate the presence of BSA or HSA.
The test for BSA was conclusive. The BSA film showed nine significant, in fact, over-loaded, signals corresponding to the eight test solution spots and the 1 mg/ml BSA spot, as well as one lighter spot at the HSA position on that blot. The HSA film was negative except for the one spot corresponding to the HSA spot. The lighter signal detected for HSA on the BSA blot likely comes from non-specific binding and cross-reactivity. The greatly reduced intensity of the spot as compared to the same spot on the HSA blot would support that supposition. This laboratory cannot confirm the source of the BSA found in the test solution, only that it was present in the sample we received.
Given the positive result for BSA, the identity of the protein as IGF-1 also fell into question. To begin with, authentic receptor grade Long™ R3 IGF-1 protein (DSLaboratories) was evaluated by dot blot (as described above) to choose the antibody (mouse anti-human IGF-1 MoAb or mouse anti-human IGF-1 polyclonal Ab, both from DSLaboratories) which could also identify that version of the protein. Results were questionable with the monoclonal, so a polyclonal antibody to human IGF-1 was chosen to ensure adequate signal. With suitable standards available, polyacrylamide gel electrophoresis, followed by Western Blot was used to evaluate the identity of the protein in the test solution. Besides probing for human versions of the protein, as an additional control, antibodies to sheep, porcine, bovine, murine rabbit and rat IGF-1 were also used. The signal was strong for human IGF-1, but there was some signal from the porcine IGF-1 antibody. This spurious signal was eliminated by diluting the test solution to 100 ug/ml (sample overloading artifact). The results would indicate the protein is, in fact, human IGF-1. However, no definitive statement can be made about Long™ R3 IGF-1 as compared to mature, native recombinant human IGF-1.
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