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Homeopathy in disguise. Release-Active Forms of Anti-Interferon-Gamma Antibodies – a detailed criticism

Posted by apanchin on 15 Mar 2017 at 21:59 GMT

Major medical organizations including the Australian government's National Health and Medical Research Council [1] and the UK's National Health Service [2] and a number of meta-analysis [3,4,5] suggest that there is no reliable evidence for the efficiency of homeopathy. However, homeopathic papers continue to be published. We were surprised that one such article by Gavrilova et al. «Novel Approach to Activity Evaluation for Release-Active Forms of Anti-Interferon-Gamma Antibodies Based on Enzyme-Linked Immunoassay» [6] passed peer-review in PLOS ONE. A closer investigation revealed critical flaws in the key assumptions and conclusions made by the authors. We also found an alternative and most likely interpretation of their experimental data.

Homeopathy in disguise. First, we need to clarify that this is an article about homeopathy, although the word «homeopathy» is never explicitly mentioned.

1. The authors state: «In the last decade, a number of publications devoted to the so called «release-active forms of drugs» have appeared <…>. It was observed that during the process of decreasing the initial concentration of a drug substance by multiple consecutive dilution or grinding (trituration) with lactose that the end products of such a process have physicochemical and biological properties which are different from the initial substance properties».
This is a claim that homeopathy makes.

Also, note that the «number of publications» about the «so called release active forms of drugs», are exclusively papers published by those affiliated with OOO "NPF "MATERIA MEDICA HOLDING" – a Russian Company that markets a number of drugs which contain active ingredients diluted beyond Avogadro’s limit.

We also note that independent research groups do not use the terms «release active forms of drugs» or «release active forms of antibodies» (RA forms of Abs).

2. The authors state: «RA forms of Abs to IFN-gamma contain release-active dilutions of antibodies to IFN-gamma consisting of a mixture of C12+C30+C50 final dilutions».

Here the object of the study is described using homeopathic terminology. When translated this means the following: a mixture of rabbit polyclonal antibodies to recombinant human interferon gamma diluted to the power of 10-24, 10-60 and 10-100.Or in other words: containing no measurable quantities of antibodies.
Even the 10-24 dilution is indistinguishable from zero. Note that these preparations were eventually diluted even further (at least to ~10-28) during the actual experiments. The claim that dilutions of substances beyond Avogadro’s limit have specific effects is a homeopathic claim.

Therefore, we are dealing with homeopathy in disguise.

The authors, including Oleg Epstein – CEO of the abovementioned Company, are not telling the truth when they state that «There are no patents, products in development or marketed products to declare».

In fact, «RA forms of Abs to IFN-gamma» are the claimed «active ingredient» of one of the Company’s products called «Anaferon for Children». The article by Gavrilova et al. is listed on the Company’s website in the section called «Anaferon for Children», subheading «Articles, Preclinical» [7]. It is also actively self-cited by the Company in contexts such as «Data obtained using ELISA and piezoelectric immunosensors evidences that anaferon is able to modify IFNγ affinity to specific antibodies to IFNγ» [8] and even in statements that the main mechanism of action for «Anaferon» is to improve ligand to receptor binding affinity.

The experiments. The authors performed four experiments using enzyme-linked immunosorbent assay (ELISA).
In experiment 1, they measured the optical density in a standard ELISA test using mouse monoclonal antibodies to human interferon gamma (mAbs) in non-homeopathic concentrations. This experiment seems to be formally correct, although the authors presented a linear relationship only for mAbs dilutions ranged from 1:20000 to 1:80000, while in the following experiments they studied much higher dilutions (up to 1:675000 in experiment 2).
In experiments 2, 3 and 4 the authors added «RA forms of Abs» to mAbs in non-homeopathic concentrations. In other words, they added a mixture of rabbit polyclonal antibodies to recombinant human interferon gamma diluted to the power of 10-24, 10-60 and 10-100 to mAbs diluted non-homeopathically.

The authors add this mixture onto plates coated with IFN-gamma in different concentrations and performed ELISA tests. They used «RA form of buffer» (translation: a buffer homeopathically diluted to non-existence in the same way as the rabbit antibodies) as a control.

To make it clear: the purpose of this study was to show the influence of the addition of 4 parts of antibodies in dilution 10-28 + 10-64 + 10-104 on 1 part of antibodies in dilution 1:25000 (or another non-homeopathic dilution). This makes no sense from a chemical point of view.

The authors however observed differences between samples and controls in experiments 2, 3 and 4. The main problem with the results of the study is that there is a much simpler explanation to the observed differences than the unlikely existence of «RA forms of Abs». Tables S2A, S3A and S4A reveal that the authors used a non-randomized plate layout. This leads to positioning effects or plate effects. Below we provide a citation from a review on the subject recently published by Roselle et al in Analytical and Bioanalytical Chemistry [9].

«A major disadvantage of microtiter plate assays is uneven distribution of variability across the plate resulting in so called plate effects or positioning effects [10], [11], [12], [13], [14], [15], [16], [17]. Because of these positioning effects, test results can vary depending on the relative position of the comparator reference and sample treatments within the plate. In the presence of positioning effects, identical preparations of the same treatment are subjected to slightly different assay conditions resulting in biased assay responses. Depending on the nature of the effects, even small non-random variations in well raw measurements can introduce bias to relative potency results large enough to lead to inaccurate experimental conclusions [18]».

The possible artifacts described above can be dealt with approaches such as spatial randomization [19] or at least by intermitting sample and control plates [20]. Although considering the extraordinary claim made in the article we would also suggest plate replication and proper experimenter blinding to be used.

In other words: the experimental design of the study does not match the further statistical approach that does not consider plate effects. Consistently with the abovementioned and well-known technical artifact, the authors admit that the effect is observed only «when the amount of antigen adsorbed on the plate surface is minimal». They also admit, «These amounts of antigen are so low that they fall close to the sensitivity limit of the ELISA». No effects were observed for antigen concentrations, where ELISA is more reliable (Figures 2A and 2B). Our explanation is also consistent with the high prior probability that any positive test of homeopathy is a false positive [21].

Lack of proper randomization and blinding explained previous failures to reproduce similar homeopathic claims [22]. For example, the claim made by Benveniste et al that human basophiles could be activated by homeopathic dilutions of antibodies [23] could not be reproduced in a more rigorous study that accounted for the abovementioned biases [24].

The authors claim that the effect of RA forms of Abs on the reaction between antibodies and immobilized IFN-gamma is associated «with the competition of RA forms of Abs to IFN-gamma with mAbs for binding to interferon gamma». However, if any homeopathic effect exists, residual «RA influence» on mAbs binding to goat anti-mouse IgG antibodies-horse radish peroxidase conjugate was not excluded. Although we find this hypothesis equally improbable as the one suggested by the authors.

The authors conclude that «With increasing amount of plate-immobilized antigen, the competition becomes insignificant and the effect of RA forms decreases or even becomes undetectable in some experiments» but do not provide any suggestion about the nature of such a speculative phenomenon or any evidence that this is indeed happening.

In addition, in experiment 3 the authors used two different schemas of ingredient mixing (methods 1 and 2) but only one control. They did not specify which experimental modification was used for the control. To obtain proper comparisons each modification should have had its own control (produced by methods 1 and 2 from a control buffer).

The assumptions. For instance, if we assume that the succussion (another homeopathic term, meaning a vigorous shaking and striking on an elastic surface to reduce side-effects and to increase a potency [25]) used by the authors is important, then we need reassurance that this was done in a blinded or automated manner before we can attribute any effect to the substance being diluted, not the way succussion was performed.

On the other hand, the importance of succussion does not have a scientific basis: the authors do not provide a comparison of preparations of Abs to IFN-gamma obtained with and without this procedure.

In addition, it is not clear how the power of dilution equal to 10-24 + 10-60 + 10-100 was chosen for this study, aside from it being used in the drug «Anaferon for Children». These dilutions appear to be arbitrary. Prior to using such dilutions, the authors should have shown that there is a difference between 10-24 + 10-60 + 10-100 and 10-24 which would be itself an incredible result deserving a Nobel Prize in our humble opinion if independently confirmed under strict conditions.

In the introduction, the authors state that: «The main feature of these release-active forms is their ability to exert a modifying influence on the starting material». To exert a modifying influence is not a valid description of a potential mechanism of action of an active drug.

The authors also state that «Antibody-based drugs are widely available among marketed medicinal products». This is true: modern medical practice does involve the use of some antibodies in treatment of different types of cancer, autoimmune diseases or in transplantation. However, the therapeutic value of antibodies against IFN-gamma is still controversial, and, despite a long history of studies, this therapy just reached the level of a few clinical trials [26].

Most importantly, the effects of anti-IFN-gamma antibodies therapy, if any, were anti-inflammatory and immunosuppressive, but not stimulatory or antiviral [27]. It is a homeopathic idea that «like cure likes»: normal antibodies would bind to IFN-gamma while «homeopathic antibodies» are supposed to somehow enhance its antiviral effect («Anaferon for Children» is marketed as a drug against influenza). However, this is not explicitly stated and the homeopathy literature describing these (flawed) principles is not cited.

We also have some minor technical comments:

1. Authors used Microtest Plate 96Well (SARSTEDT AG & Co., Germany, Cat No 82.1582.001) [28]. These are round-bottom plates which is an unusual choice for ELISA (flat bottom plates are preferred) and this should have been explained.

2. It seems unlikely that conventional sandwich-ELISA produces such a strong analytical signal with high optical densities (up to 1.6 in the Experiment 1). Low concentrations usually require amplification system (e.g. streptavidin-biotin). However, authors used 1:1000 dilution of secondary Abs, resulting with 10 times higher concentration than recommended by the manufacturer [29]. This could also introduce high analytical background noise.

3. Different dilutions were used for different experiments. Reasons for this were not explained.

Conclusions

Given all the shortcomings, we conclude that the article by Gavrilova et al. is misleading and deceives its readers. Considering that the actual data consists of just four ELISA runs, we cannot conclude that sufficient effort has been made to investigate such an extraordinary claim as the measurable effects of ultra-low homeopathic dilutions beyond Avogadro’s limit.

Recently we argued that another paper published by a number of the same authors, including Oleg Epstein in journal of Medical Virology, should be retracted due to numerous flaws [30]. Similarly, we recommend a retraction for the article by Gavrilova et al. from PLOS ONE.

Authors: Dueva EV(1) and Panchin AY(2)

1 Chumakov Institute of Poliomyelitis and Viral Encephalitides, Chumakov FSC R&D IBP RAS, Moscow, Russia.
2 Institute for Information Transmission Problems, Russian academy of sciences, Moscow, Russia

Conflict of interest. We have none.

References
1. https://www.nhmrc.gov.au/....
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3. Ernst E (2010) Homeopathy: what does the "best" evidence tell us? Med J Aust 192: 458-460.
4. Shang A, Huwiler-Muntener K, Nartey L, Juni P, Dorig S, et al. (2005) Are the clinical effects of homoeopathy placebo effects? Comparative study of placebo-controlled trials of homoeopathy and allopathy. Lancet 366: 726-732.
5. Ernst E (2002) A systematic review of systematic reviews of homeopathy. Br J Clin Pharmacol 54: 577-582.
6. Gavrilova ES, Bobrovnik SA, Sherriff G, Myslivets AA, Tarasov SA, et al. (2014) Novel approach to activity evaluation for release-active forms of anti-interferon-gamma antibodies based on enzyme-linked immunoassay. PLoS One 9: e97017.
7. http://www.materiamedica.....
8. https://medi.ru/info/1153... (Article in Russian).
9. Roselle C, Verch T, Shank-Retzlaff M (2016) Mitigation of microtiter plate positioning effects using a block randomization scheme. Anal Bioanal Chem 408: 3969-3979.
10. Burt SM, Carter TJ, Kricka LJ (1979) Thermal characteristics of microtitre plates used in immunological assays. J Immunol Methods 31: 231-236.
11. Liang Y, Woodle SA, Shibeko AM, Lee TK, Ovanesov MV (2013) Correction of microplate location effects improves performance of the thrombin generation test. Thromb J 11: 12.
12. Pesce AJ, Michael JG (1992) Artifacts and limitations of enzyme immunoassay. J Immunol Methods 150: 111-119.
13. Oliver DG, Sanders AH, Hogg RD, Hellman JW (1981) Thermal gradients in microtitration plates. Effects on enzyme-linked immunoassay. J Immunol Methods 42: 195-201.
14. Noble JE, Wang L, Cerasoli E, Knight AE, Porter RA, et al. (2008) An international comparability study to determine the sources of uncertainty associated with a non-competitive sandwich fluorescent ELISA. Clin Chem Lab Med 46: 1033-1045.
15. Kricka LJ, Carter TJ, Burt SM, Kennedy JH, Holder RL, et al. (1980) Variability in the adsorption properties of microtitre plates used as solid supports in enzyme immunoassay. Clin Chem 26: 741-744.
16. Harrison RO, Hammock BD (1988) Location dependent biases in automatic 96-well microplate readers. J Assoc Off Anal Chem 71: 981-987.
17. FDA (2013) Guidance for industry: immunogenicity assessment for therapeutic protein products (Draft).
18. Robinson CJ, Sadick M, Deming SN, Estdale S, Bergelson S, et al. (2014) Assay Acceptance Criteria for Multiwell-Plate–Based Biological Potency Assays. Bioprocess International.
19. http://labstats.net/artic....
20. Zimmermann H, Gerhard D, Dingermann T, Hothorn LA (2010) Statistical aspects of design and validation of microtitre-plate-based linear and non-linear parallel in vitro bioassays. Biotechnol J 5: 62-74.
21. Colquhoun D (2014) An investigation of the false discovery rate and the misinterpretation of p-values. R Soc Open Sci 1: 140216.
22. Guggisberg AG, Baumgartner SM, Tschopp CM, Heusser P (2005) Replication study concerning the effects of homeopathic dilutions of histamine on human basophil degranulation in vitro. Complement Ther Med 13: 91-100.
23. Davenas E, Beauvais F, Amara J, Oberbaum M, Robinzon B, et al. (1988) Human basophil degranulation triggered by very dilute antiserum against IgE. Nature 333: 816-818.
24. Maddox J, Randi J, Stewart WW (1988) "High-dilution" experiments a delusion. Nature 334: 287-291.
25. Kayne S (2006) Homeopathic pharmacy: theory and practice (2 ed.). Elsevier Health Sciences: 53.
26. https://clinicaltrials.go....
27. Hommes DW, Mikhajlova TL, Stoinov S, Stimac D, Vucelic B, et al. (2006) Fontolizumab, a humanised anti-interferon gamma antibody, demonstrates safety and clinical activity in patients with moderate to severe Crohn's disease. Gut 55: 1131-1137.
28. https://www.sarstedt.com/....
29. http://www.sigmaaldrich.c....
30. Dueva EV, Panchin AY (2016) Homeopathy in disguise. Comment on Don et al.: Dose-dependent antiviral activity of released-active form of antibodies to interferon-gamma against influenza A/California/07/09(H1N1) in murine model. J Med Virol.

No competing interests declared.

RE: Homeopathy in disguise. Release-Active Forms of Anti-Interferon-Gamma Antibodies – a detailed criticism

GavrilovaES replied to apanchin on 17 Mar 2017 at 19:34 GMT

First, it is important to emphasize that all the AUTHORS’ criticism and skepticism concerning the impossibility of obtaining the results provided in the article are attributed to the fact that Dr. Dueva and Dr. Panchin consider the tested product as homeopathic. Recalling the negative attitude of the Australian government's National Health and Medical Research Council and the UK's National Health Service to homeopathy, the AUTHORS of the ‘READER COMMENTS’ consider our results as a bias and say that they are not possible. This principal statement is incorrect since the product described in the article is not homeopathic and has all the necessary attributes of standard pharmacological medicine (efficacy proved in preclinical and double-blind placebo controlled clinical studies, proved specificity of action, identified pharmacological mechanism of action, manufacturing in accordance with European Pharmacopoeia requirements, etc.). Such an unambiguous conclusion was supported by European and USA government medicine evaluation agencies during official scientific consultations. Detailed description of the product could be found in Don E.S. et al. (2016) [1]. Thus all comments related to homeopathy are not relevant and should not be taken into considerations.

In response to the criticism about terms used for the product description, we would like to underline that the terms ‘released-activity’ and ‘released-active antibodies’ have been introduced by Prof. Epstein O.I. to describe the features of such type of products. At the same time, studies of released-active antibodies have been performed by research teams from numerous well-known organizations and institutes/universities in Russia, Europe, Asia and the USA.

The sections ‘Competing interests’ and ‘Funding’ in the article fully correspond to the journal requirement. Especially we would like to emphasize the following:
1. The article published in PLOS One is dedicated not to the investigation of the effects of drug product containing released-active form of antibodies to interferon-gamma (RA Abs to IFN-g), but possibility to expand the use of ELISA as a method of RA Abs detection where RA Abs to IFN-g were taken as an example. By the time the article was submitted and published, the described ELISA was neither under development for sales in the form of a ready-to-use commercially available ELISA kit, nor commercially available in the form of the corresponding kit. Therefore, there is no contradiction to what we have previously stated in the subsection ‘Competing Interests’.
2. The fact that RA Abs to IFN-g are the Active Pharmaceutical Ingredient of commercially available drug product ‘Anaferon for children’ was not in any way hidden from the readers as it is clearly stated in the article (section ‘Materials and Methods’, subsection ‘Preparation of anti-IFN-gamma release-active dilutions’) that Abs to IFN-g are manufactured as initial substance for commercial drug product ‘Anaferon for children’. Then the main principles of the technology are indicated briefly. Thus, all the information regarding this issue has been initially openly provided in the article without the need to consult any additional sources (e.g., the official website of the company or other publications).
3. The study was sponsored by ‘Materia Medica Holding’ as indicated in the corresponding section of the article. Meanwhile, the research itself was carried out on the research site not owned by ‘Materia Medica Holding’: it is a laboratory at the Department of Molecular Immunology, Palladin Institute of Biochemistry of the National Academy of Sciences of Ukraine (Kiev, Ukraine). All the research works were performed by a specialist in development of immunological methods (more than 90 PubMed indexed research papers dedicated to this and similar subjects).


WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“The experiments. The authors performed four experiments using enzyme-linked immunosorbent assay (ELISA).
In experiment 1, they measured the optical density in a standard ELISA test using mouse monoclonal antibodies to human interferon gamma (mAbs) in non-homeopathic concentrations. This experiment seems to be formally correct, although the authors presented a linear relationship only for mAbs dilutions ranged from 1:20000 to 1:80000, while in the following experiments they studied much higher dilutions (up to 1:675000 in experiment 2).”

WE WOULD LIKE TO STATE THAT:
Experiment 1 described in the article was aimed to demonstrate validity of the developed ELISA method and to provide evidence for the presence of linear range. In the experiments involving ELISA for the assessment of antibodies quantity, it is necessary at first to determine which dilutions are high enough to observe linear relationship between dilution and optical density. That is the approach used in our work. It was established in the previous experiments, that the optimal dilution for the mAbs we used in ELISA measurements are dilutions of 1∶20000 or higher. This dilution is derived from directly proportional relationship between antibody concentrations and color development in the plate wells at this or higher dilutions. Thus, we determined dilution of a sample of antibodies allowing us to obtain direct proportion between color intensity and concentration of antibodies at first, and then this and some other, even higher dilutions were used in the experiments. Thus, in the subsequent experiments several concentrations were tested; in experiment 2, for example, not only antibodies in dilution 1:675000 were tested, but 4 different dilutions, a half of concentrations of which is included in concentration range for the first experiment. However, since experiments 2, 3 and 4 were independent (different concentration of not only antibodies, but also IFN-gamma, different assay patterns (indirect, competitive), etc.), all dilutions used were selected individually for each set of experiments.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“In experiments 2, 3 and 4 the authors added «RA forms of Abs» to mAbs in non-homeopathic concentrations. In other words, they added a mixture of rabbit polyclonal antibodies to recombinant human interferon gamma diluted to the power of 10-24, 10-60 and 10-100 to mAbs diluted non-homeopathically.
The authors add this mixture onto plates coated with IFN-gamma in different concentrations and performed ELISA tests. They used «RA form of buffer» (translation: a buffer homeopathically diluted to non-existence in the same way as the rabbit antibodies) as a control.
To make it clear: the purpose of this study was to show the influence of the addition of 4 parts of antibodies in dilution 10-28 + 10-64 + 10-104 on 1 part of antibodies in dilution 1:25000 (or another non-homeopathic dilution). This makes no sense from a chemical point of view.”

WE WOULD LIKE TO STATE THAT:
The objective of this study was to investigate the ability of ELISA to assess the RA Ab influence on interaction between Ab and its Ag and not to examine chemical nature of how this influence occurs. This study involved active pharmaceutical ingredient with approved formulation and experimentally and clinically proved efficacy. So the issues raised by AUTHORS of the ‘READER COMMENTS’ are beyond the scope of this study and this article. Nevertheless, as has already been mentioned, not binding of RA Ab to Ag underlies the mechanism of action of RA Ab, but ability of RA Abs to alter (modify) Ab-Ag interaction. This suggestion was supported not only in the course of the current study focused on possibility to expand ELISA application as a method for RA Ab detection, where RA Ab to IFN-g were taken as example, but also in other studies. In the article by Pschenitza M et al (2014), the ability of applying ELISA for assessment of RA influence on Ab-Ag interaction was demonstrated, but in the case of RA of non-antibody nature [2]. Moreover, this suggestion is supported by another immunochemical method, namely “piezoelectric immunosensor assay” [3].

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“The authors however observed differences between samples and controls in experiments 2, 3 and 4. The main problem with the results of the study is that there is a much simpler explanation to the observed differences than the unlikely existence of «RA forms of Abs». Tables S2A, S3A and S4A reveal that the authors used a non-randomized plate layout. This leads to positioning effects or plate effects. Below we provide a citation from a review on the subject recently published by Roselle et al in Analytical and Bioanalytical Chemistry [9].

«A major disadvantage of microtiter plate assays is uneven distribution of variability across the plate resulting in so called plate effects or positioning effects [10], [11], [12], [13], [14], [15], [16], [17]. Because of these positioning effects, test results can vary depending on the relative position of the comparator reference and sample treatments within the plate. In the presence of positioning effects, identical preparations of the same treatment are subjected to slightly different assay conditions resulting in biased assay responses. Depending on the nature of the effects, even small non-random variations in well raw measurements can introduce bias to relative potency results large enough to lead to inaccurate experimental conclusions [18]».

The possible artifacts described above can be dealt with approaches such as spatial randomization [19] or at least by intermitting sample and control plates [20]. Although considering the extraordinary claim made in the article we would also suggest plate replication and proper experimenter blinding to be used.”

WE WOULD LIKE TO STATE THAT:
To begin with, in case of confirmation of the absence of edge effects and concentration gradient over the plate by the researcher, randomization of the samples will not be a vital part of ELISA performance. Even in the review by Roselle et al. (2016) there is no mention that all ELISA tests based on non-randomized plate layout are incorrect; the authors only consider non-randomization as a factor that might affect the results and offer solutions for minimizing this effect [4]. The approaches proposed include, in particular, performing replicates within and between the plates. In our case, tested samples were analyzed in triplicates; besides, in Experiment 2, compared samples are grouped on the basis of IFN-g concentrations (the authors mention the possible biases when comparing edge rows with central rows, but in our situation all wells are compared with the adjacent, not with the separated ones; that reduces biases as comparison of wells for tested samples and controls is the main goal of the work performed).

Moreover, manufacturers of commercial ELISA kits often either supply the manual with the specific plate layout [5-7], or recommend to test samples at least in duplicates stating that “Well effects have not been observed with this assay” [8]. The same refers to scientific publications on methodology development: in rare cases where authors publish sample layout of plates, they are usually not randomized [9]. Nevertheless, to rule out influence of sample layout, it is recommended to check such influence, and some authors even provide data indicating that layout does not affect the raw data [10]. In the course of our work no layout influence was found during the additional unpublished research but we provided readers not only with raw data, but also with the plate layout in order to all readers to be able to estimate the validity of data.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“In other words: the experimental design of the study does not match the further statistical approach that does not consider plate effects.”

WE WOULD LIKE TO STATE THAT:
We also considered template influence because the target sample and its control were always tested within one plate.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“Consistently with the abovementioned and well-known technical artifact, the authors admit that the effect is observed only «when the amount of antigen adsorbed on the plate surface is minimal». They also admit, «These amounts of antigen are so low that they fall close to the sensitivity limit of the ELISA». No effects were observed for antigen concentrations, where ELISA is more reliable (Figures 2A and 2B). Our explanation is also consistent with the high prior probability that any positive test of homeopathy is a false positive_ENREF_2 [21].
Lack of proper randomization and blinding explained previous failures to reproduce similar homeopathic claims [22]. For example, the claim made by Benveniste et al that human basophiles could be activated by homeopathic dilutions of antibodies [23] could not be reproduced in a more rigorous study that accounted for the abovementioned biases [24].
The authors claim that the effect of RA forms of Abs on the reaction between antibodies and immobilized IFN-gamma is associated «with the competition of RA forms of Abs to IFN-gamma with mAbs for binding to interferon gamma». However, if any homeopathic effect exists, residual «RA influence» on mAbs binding to goat anti-mouse IgG antibodies-horse radish peroxidase conjugate was not excluded. Although we find this hypothesis equally improbable as the one suggested by the authors.”

WE WOULD LIKE TO STATE THAT:
We used RA form of Ab having specificity identical with mAb, so RA form of Ab should compete only with these mAbs for binding to Ag (provided our suggestion is correct). No effect upon binding of other antibodies should be exerted by these RA forms. Goat antibodies conjugate is added to the plate only when it is washed from all reagents and contains only the bound antigen and mAb attached to it. In other words, the plate is already washed clean and RA forms of Ab are not on the plate, so they cannot exert any effect upon such binding.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“The authors conclude that «With increasing amount of plate-immobilized antigen, the competition becomes insignificant and the effect of RA forms decreases or even becomes undetectable in some experiments» but do not provide any suggestion about the nature of such a speculative phenomenon or any evidence that this is indeed happening.”

WE WOULD LIKE TO STATE THAT:
Indeed, after the analysis of obtained data we came to the conclusion about influence on molecule conformation, and we propose the following explanation of the phenomenon described in the article: “It should be noted that if this apparent increase of IFN-gamma concentration (which is actually an increase of the concentration of molecules with conformations that account for the complementary activity of IFN-gamma toward mAbs) is close to a constant value for this particular type of sample containing RA forms of Abs to IFN-gamma and is minimally dependent on the concentration of IFN-gamma in the sample preparations, then the observed increase will be far more pronounced at low concentrations of IFN-gamma than when using high IFN-gamma concentrations. This observation should imply that notable differences between control samples and samples containing RA forms of Abs to IFN-gamma are likely to occur with low antigen concentrations, whereas with high concentrations of IFN-gamma these differences will be almost undetectable. Our results support this observation”.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“In addition, in experiment 3 the authors used two different schemas of ingredient mixing (methods 1 and 2) but only one control. They did not specify which experimental modification was used for the control. To obtain proper comparisons each modification should have had its own control (produced by methods 1 and 2 from a control buffer).”

WE WOULD LIKE TO STATE THAT:
The basic objective of the experiment was to compare the influence of the different methods of RA Ab to IFN-g sample preparation to each other and not to the control. In tested samples, we either mixed antibodies with RA Ab and then added antigen, or mixed RA Ab with antigen and then added antibodies for assessing impact of reagent adding succession in order to test our suggestion. Since comparison with control was not the main objective of this experiment, control was prepared as described in standard protocol and other experiments.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“The assumptions. For instance, if we assume that the succussion (another homeopathic term, meaning a vigorous shaking and striking on an elastic surface to reduce side-effects and to increase a potency [25]) used by the authors is important, then we need reassurance that this was done in a blinded or automated manner before we can attribute any effect to the substance being diluted, not the way succussion was performed.

On the other hand, the importance of succussion does not have a scientific basis: the authors do not provide a comparison of preparations of Abs to IFN-gamma obtained with and without this procedure.

In addition, it is not clear how the power of dilution equal to 10-24 + 10-60 + 10-100 was chosen for this study, aside from it being used in the drug «Anaferon for Children». These dilutions appear to be arbitrary. Prior to using such dilutions, the authors should have shown that there is a difference between 10-24 + 10-60 + 10-100 and 10-24 which would be itself an incredible result deserving a Nobel Prize in our humble opinion if independently confirmed under strict conditions.”

WE WOULD LIKE TO STATE THAT:
The present study was aimed neither at the comparison of initial and released-active forms of Ab, nor at the examination of the theoretical discussion about dilutions and succession stated by AUTHORS of the ‘READER COMMENTS’. We would like to underline once again that the article is focused on the possibility to expand ELISA application as a method for active pharmaceutical ingredients detection, i.e. RA Ab. As an example we used not the variety of experimental products, but the active pharmaceutical ingredient (RA Ab to IFN-g) with a formulation approved by regulators and possessing proved pharmacological efficacy. Therefore, the AUTHORS’ comments are outside the area of focus of this work.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“In the introduction, the authors state that: «The main feature of these release-active forms is their ability to exert a modifying influence on the starting material». To exert a modifying influence is not a valid description of a potential mechanism of action of an active drug.”

WE WOULD LIKE TO STATE THAT:
The phrase is taken out of context. The following paragraph clarifies the mechanism of RA Ab action with RA Ab to IFN-g as an example: “One of the most studied substances used for the preparation of antibody-containing RA drugs is the anti-IFN-gamma antibody. The efficacy and safety of drugs containing RA forms of Abs to interferon gamma have been extensively studied in various experimental models as well as in clinical studies [17–23]. It was shown during these studies that RA forms of Abs change the conformation and binding affinity of interferon gamma (IFNgamma), demonstrated by changes in antigen-antibody interaction.”

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“The authors also state that «Antibody-based drugs are widely available among marketed medicinal products». This is true: modern medical practice does involve the use of some antibodies in treatment of different types of cancer, autoimmune diseases or in transplantation. However, the therapeutic value of antibodies against IFN-gamma is still controversial, and, despite a long history of studies, this therapy just reached the level of a few clinical trials [26]. Most importantly, the effects of anti-IFN-gamma antibodies therapy, if any, were anti-inflammatory and immunosuppressive, but not stimulatory or antiviral [27].”

WE WOULD LIKE TO STATE THAT:
The assertion that there are very few other anti-IFNg drug products bears no relation to the article under discussion. Apart from that, their mechanism of action is based on other principles that differ from RA forms of Ab mechanism of action (see above), and any extrapolation is unsubstantiated and unreasonable.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“It is a homeopathic idea that «like cure likes»: normal antibodies would bind to IFN-gamma while «homeopathic antibodies» are supposed to somehow enhance its antiviral effect («Anaferon for Children» is marketed as a drug against influenza). However, this is not explicitly stated and the homeopathy literature describing these (flawed) principles is not cited.”

WE WOULD LIKE TO STATE THAT:
We wish to emphasize again that the sample described in the article is not a homeopathic product. A fundamental idea of homeopathy is the similarity principle. The similarity principle states that homeopathic remedy induces symptoms consistent with a particular disease. Then, based on a set of such symptoms, a remedy is selected, that is expected to be effective in treatment of the disease characterized by similar symptoms. This requires specifically designed clinical trials involving healthy volunteers. The concept proposed by the AUTHORS of the ‘READER COMMENTS’ is entirely inconsistent with the description of the similarity principle and is misleading, and the attempt to attribute the mechanism of action of RA Ab to the similarity principle paradigm is artificial and incorrect.
WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“We also have some minor technical comments:
1. Authors used Microtest Plate 96Well (SARSTEDT AG & Co., Germany, Cat No 82.1582.001) [28]. These are round-bottom plates which is an unusual choice for ELISA (flat bottom plates are preferred) and this should have been explained.”

WE WOULD LIKE TO STATE THAT:
In accordance with the specification to these plates, the manufacturer designates them as ELISA plates (see [11] “Brochures”) and using of such types of plates could be found in scientific articles [12]. Each researcher could choose the using reagents (if they are suitable for the intended purpose) in accordance with the availability, experience etc so this question cannot be considered as a valid comment to the scientific research article.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“2. It seems unlikely that conventional sandwich-ELISA produces such a strong analytical signal with high optical densities (up to 1.6 in the Experiment 1). Low concentrations usually require amplification system (e.g. streptavidin-biotin). However, authors used 1:1000 dilution of secondary Abs, resulting with 10 times higher concentration than recommended by the manufacturer [29]. This could also introduce high analytical background noise.”

WE WOULD LIKE TO STATE THAT:
All concentrations indicated in the specification are recommended by the manufacturer. There is only one dilution recommended for this reagent when used in direct ELISA and no recommendations regarding competitive or indirect assay are provided by the manufacturer (and this is unnecessary because during ELISA development any researcher selects conjugate dilutions individually depending on other available reagents, e.g. stop reagent (which also influence OD value) and ELISA reader for OD reading. It is a common situation for ELISA testing when optical density reaches 2.5 and even more, and similar cases can be easily found in scientific publications [13] as well as in calibration curves provided by ELISA reagents manufacturers [14-15].

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“3. Different dilutions were used for different experiments. Reasons for this were not explained.”

WE WOULD LIKE TO STATE THAT:
Dilutions for a specific experiment were selected with respect to the results (in particular, optical density) of the previous experiments. As described in the article, these are not isolated experiments, but examples from a series of experiments. This is standard practice for ELISA development. The experiments are described in details, based on different approaches (indirect/competitive ELISA) and involve different concentrations of not only antibodies, but also IFN-gamma, so different selected working concentrations were used in each experiment.

WITH RESPECT TO THE FOLLOWING COMMENTS TO THE ARTICLE:
“Conclusions
Given all the shortcomings, we conclude that the article by Gavrilova et al. is misleading and deceives its readers. Considering that the actual data consists of just four ELISA runs, we cannot conclude that sufficient effort has been made to investigate such an extraordinary claim as the measurable effects of ultra-low homeopathic dilutions beyond Avogadro’s limit.
Recently we argued that another paper published by a number of the same authors, including Oleg Epstein in journal of medical Virology, should be retracted due to numerous flaws [30]. Similarly, we recommend a retraction for the article by Gavrilova et al. from PLOS ONE.”

WE WOULD LIKE TO STATE THAT:
The article clearly states the study objective, namely to assess the ELISA approach as the assay for detection of RA forms of Abs to IFN-gamma. Design of the study and conducted work have fully addressed the objective.

Article has been written as open and clear as possible and the nature of the questions posted by the AUTHORS of the ‘READER COMMENTS’ confirm that the article is fully transparent for the readers so they have an opportunity to familiarize themselves with these results and make their own opinion.

Regarding the another paper [16] mentioned by the AUTHORS of the ‘READER COMMENTS’, we would like to highlight that the AUTHORS’ comment there is no mention that a reply to the criticism was provided which contained detailed substantiation of the points the critics have made mistakes about [1]. After consideration of our reply and its independent review, the editorial board of Journal of Medical Virology did not accept retraction of the article as it had been requested by Dr. E.V. Dueva and Dr. A.Y. Panchin and found our arguments convincing and well-founded.

References
1. Don ES, Emelyanova AG, Yakovleva NN, Petrova NV, Nikiforova MV, Gorbunov EA, Tarasov SA, Morozov SG, Epstein OI (2016a) The phenomenon of released-activity. Reply on comment on Don et al.: Dose-dependent antiviral activity of released-active form of antibodies to interferon-gamma against influenza A/California/07/09(H1N1) in murine model. J Med Virol, doi: 10.1002/jmv.24759. [Epub ahead of print].
2. Pschenitza M, Gavrilova ES, Tarasov SА, Knopp D, Niessner R, Epstein OI (2014) Application of a heterogeneous immunoassay for the quality control testing of release-active forms of diclofenac. Int Immunopharmacol, 21(1), 225-30. doi: 10.1016/j.intimp.2014.04.029.
3. Don E, Farafonova O, Pokhil S, Barykina D, Nikiforova M, Shulga D, Borshcheva A, Tarasov S, Ermolaeva T, Epstein O (2016b) Use of Piezoelectric Immunosensors for Detection of Interferon-Gamma Interaction with Specific Antibodies in the Presence of Released-Active Forms of Antibodies to Interferon-Gamma. Sensors (Basel), 16(1), pii: E96. doi: 10.3390/s16010096.
4. Roselle C, Verch T, Shank-Retzlaff M (2016) Mitigation of microtiter plate positioning effects using a block randomization scheme. Anal Bioanal Chem, 408(15), 3969-3979. doi: 10.1007/s00216-016-9469-0
5. https://www.ucytech.com/p...
6. https://www.merckmillipor...
7. https://worldwide.promega...
8. http://www.abcam.com/huma...
9. Pham PV, Nguyen NT, Nguyen HM, Khuat LT, Le PM, Pham VQ, Nguyen ST, Phan NK (2014) A simple in vitro method for evaluating dendritic cell-based vaccinations. Onco Targets Ther. 7, 1455-1464. doi: 10.2147/OTT.S67057.
10. Allen CD, Robbins MN, Eguchi T, Owens DW, Meylan AB, Meylan PA, Kellar NM, Schwenter JA, Nollens HH, LeRoux RA, Dutton PH, Seminoff JA (2015) First Assessment of the Sex Ratio for an East Pacific Green Sea Turtle Foraging Aggregation: Validation and Application of a Testosterone. PLoS One. 10(10), e0138861. doi: 10.1371/journal.pone.0138861.
11. https://www.sarstedt.com/...
12. He F, Prabakaran M, Tan Y, Indira K, Kumar SR, Kwang J (2013) Development of dual-function ELISA for effective antigen and antibody detection against H7 avian influenza virus. BMC Microbiology. 13, 219. doi: 10.1186/1471-2180-13-219.
13. Stearns NA, Zhou S, Petri M, Binder SR, Pisetsky DS (2016) The Use of Poly-L-Lysine as a Capture Agent to Enhance the Detection of Antinuclear Antibodies by ELISA. PLoS One. 11(9), e0161818. doi: 10.1371/journal.pone.0161818.
14. http://www.abcam.com/oxyt...
15. https://www.thermofisher....
16. Don ES, Emelyanova AG, Yakovleva NN, Petrova NV, Nikiforova MV, Gorbunov EA, Tarasov SA, Morozov SG, Epstein OI (2016c) Dose-Dependent Antiviral Activity of Released-Active Form of Antibodies to Interferon-Gamma against influenza A/California/07/09(H1N1) in Murine Model // Journal of Medical Virology. 89(5), 759-66. doi: 10.1002/jmv.24717.

No competing interests declared.

RE: RE: Homeopathy in disguise. Release-Active Forms of Anti-Interferon-Gamma Antibodies – a detailed criticism

apanchin replied to GavrilovaES on 18 Mar 2017 at 02:21 GMT

In their reponse the authors claim that their product is not homeopathic. Their patent (by Epstein OI) [https://google.com/patent...] states otherwise:

«A medicament based on antibodies contains an activated form of monoclonal, polyclonal, or natural antibodies to interferon in low or ultra-low doses prepared by multiple consecutive dilutions and exposure to external factors, preferably in accordance with HOMEOPATHIC technology. In order to obtain antibodies, human or heterologous interferon alpha, beta, or gamma, including recombinant interferon, is used; a mixture of various, mostly centimal, HOMEOPATHIC dilutions being employed. A method of treating a pathologic syndrome, whose formation is affected by interferon, consists in the use of activated forms of antibodies to interferon alpha, beta, or gamma in low or ultra-low doses obtained by multiple consecutive dilutions and exposure to external factors.

<>

The invention claimed is:
1. A method of treating a viral disease which comprises administering to a subject suffering from the disease, at least one HOMEOPATHICALLY potentized form of an antibody to interferon.
2. The method of claim 1, wherein said at least one HOMEOPATHICALLY potentized form of antibody is administered in the form of one or more HOMEOPATHIC dilutions.
3. The method of claim 2, wherein said one or more of the HOMEOPATHIC dilutions comprises one or more centesimal HOMEOPATHIC dilutions.
4. The method of claim 3, wherein said one or more HOMEOPATHIC dilutions comprises a mixture of C12, C30, and C200 HOMEOPATHIC dilutions.
5. The method of claim 1, wherein said at least one HOMEOPATHICALLY potentized form of antibody is to alpha, beta or gamma interferon.
6. The method of claim 1, wherein said at least one HOMEOPATHICALLY potentized form of antibody is selected from monoclonal, polyclonal, or natural antibodies.
7. The method of claim 1, wherein said at least one HOMEOPATHICALLY potentized form of and antibody is obtained by multiple consecutive dilutions.
8. The method of claim 1 wherein the viral disease is upper respiratory tract viral disease, viral influenza or herpes.
9. The method of claim 8 wherein the viral disease is caused by influenza virus.
10. The method of claim 8 wherein the viral disease is caused by herpes virus.»

The abovementioned contradiction itself should be enough for the readers to make up their own mind on this subject.

Also the authors provided no additional evidence that the observed effects are not bias. Thus our main points of criticism remain unchallenged.

Panchin AY
Dueva EV

No competing interests declared.

RE: RE: RE: Homeopathy in disguise. Release-Active Forms of Anti-Interferon-Gamma Antibodies – a detailed criticism

ppapas replied to apanchin on 29 Mar 2017 at 03:29 GMT

This paper provides compelling and rigorous evidence for the effects of some ultra low doses and its is fascinating misterious behaviour of its paradoxical effects. The "HOMEOPATHIC technology" means dynamization (succused and serially diluted). The terminology is desafortunate, however is very familiar with most readers.

Dr Panchin and Dr. Dueva do not think in the case of James Randi and the lobby campaign organized by Michael Marshall, the "10:23" event. In these, all skeptics tending to identifiy homeopathy as only dynamization process, not with similia principle. Why does Dr Panchin and Dr Dueva do not contradict the Marshall campaign?

It is fascinating to watch the various denialists who epitomize an anti-scientific attitude and who are clearly fearful of change on the basis of witchhunt, censorship and fake "replication" posted on 1988 by Randi and coworkers. Fake skepticism has nothing to do with healthy skepticism in science.

The implications of this "memory of water" are immense in all applications beyond medicine. It's unethical defame the authors and pressure for the retract the studies on the basis of a priori arguments. Rigorous reseach published in peer review journals overwhelming confirms the specific effects of some dilution, on the basis of similia principle or not:

http://www.sciencedirect....

http://www.sciencedirect....

http://www.sciencedirect....

http://www.natureasia.com...

https://www.researchgate....

Luc Montagnier and coworker finally replicate the Benveniste experiment with "digital" information. He is Nobel Price and the team is of the most highest level, and we see "skeptic" with vested interests as Dr Panchi commenting saying that these scientist are hoaxers... Please...

http://journal-neo.org/20...

No competing interests declared.

RE: RE: Homeopathy in disguise. Release-Active Forms of Anti-Interferon-Gamma Antibodies – a detailed criticism

mjvanraaij replied to GavrilovaES on 25 Mar 2017 at 17:02 GMT

What about the "C50" dilution and "succussion" mentioned in the Methods section, are these not clear unscientific practices? I note the authors carefully do not respond to these points...

No competing interests declared.

RE: RE: RE: Homeopathy in disguise. Release-Active Forms of Anti-Interferon-Gamma Antibodies – a detailed criticism

mjvanraaij replied to mjvanraaij on 25 Mar 2017 at 17:07 GMT

where I wrote "unscientific" I probably would have better written "homeopathic".

No competing interests declared.