Developability Assessment of Physicochemical Properties and Stability Profiles of HIV-1 BG505 SOSIP.664 and BG505 SOSIP.v4.1-GT1.1 gp140 Envelope Glycoprotein Trimers as Candidate Vaccine Antigens.

The induction of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an effective vaccine against HIV-1. A soluble, trimeric, germline (gI) bNAb-targeting variant of the HIV-1 envelope glycoprotein (termed BG505 SOSIP.v4.1-GT1.1 gp140, abbreviated to GT1.1) has recently been developed. Here, we have compared this new immunogen with the parental trimer from which it was derived, BG505 SOSIP.664 gp140. We used a comprehensive suite of biochemical and biophysical methods to determine physicochemical similarities and differences between the 2 trimers, and thereby assessed whether additional formulation development efforts were needed for the GT1.1 vaccine candidate. The overall higher order structure and oligomeric states of the 2 vaccine antigens were quite similar, as were their thermal, chemical, and colloidal stability profiles, as evaluated during accelerated stability studies. Overall, we conclude that the primary sequence changes made to create the gl bNAb-targeting GT1.1 trimer did not detrimentally affect its physicochemical properties or stability profiles from a pharmaceutical perspective. This developability assessment of the BG505 GT1.1 vaccine antigen supports using the formulation and storage conditions previously identified for the parental SOSIP.664 trimer and enables the development of GT1.1 for phase I clinical studies.

Assessment of the effect of systemic delivery of sclerostin antibodies on Wnt signaling in distraction osteogenesis.

Sclerostin is a known inhibitor of the Wnt signaling pathway which is involved in osteogenesis and, when inactivated, stimulates bone formation. To our knowledge, this effect has not been studied in the context of distraction osteogenesis (DO). Tibial DO was conducted on a total of 24 wild-type mice, which were then divided into 2 groups-a saline injection group (control) and an anti-sclerostin (Scl-Ab) injection group (treatment). The mice in the treatment group received 100 mg/kg intravenous injections of the antibody weekly until killing. The 12 mice in each group were subdivided into four time points according to post-osteotomy time of killing-11 days (mid-distraction), 17 days (late distraction), 34 days (mid-consolidation) and 51 days (late consolidation), with 3 mice per subgroup. After killing, the tibia specimens were collected for immunohistochemical analysis. Our results show that the group injected with anti-sclerostin had an earlier peak (day 11) in the distraction phase of the osteogenic molecules involved in the Wnt signaling pathway in comparison to the placebo group. In addition, downregulation of the inhibitors of this pathway was noted in the treatment group when compared with the placebo group. Furthermore, LRP-5 showed a significant increase in expression in the treatment group. Sclerostin inhibition has a significant effect on the DO process through its effect on the Wnt pathway. This effect was evident through the decreased effect of sclerostin on LRP-5 and earlier upregulation of the osteogenic molecules involved in this pathway.

Method establishment for discerned immunogenicity assessment of a recombinant glycoprotein containing nonhuman sialic acid Neu5Gc residues.

AIM: Recombinant glycoprotein produced in nonhuman mammalian cell lines can be modified with the immunogenic nonhuman sialic N-glycolylneuraminic acid (Neu5Gc). We describe here a validated method for detection of antidrug antibodies against both protein and Neu5Gc-containing glycan epitopes. RESULTS: An electrochemiluminescent method was established with drug conjugates as capture and detection reagents. Rabbit antidrug polyclonal antibodies were used as the positive control for protein moiety-specific antibodies, while chicken anti-Neu5Gc polyclonal antibodies were used as the positive control for antibodies against Neu5Gc glycan epitope. Specificity to Neu5Gc was verified by signal inhibition with bovine γ-globulin that contains Neu5Gc. CONCLUSION: The assay illustrated here discerns the immunogenicity of the protein backbone and the sialic acid Neu5Gc glycan moiety of a recombinant protein containing Neu5Gc.

Anti-Ebola therapies based on monoclonal antibodies: current state and challenges ahead.

The 2014 Ebola outbreak, the largest recorded, took us largely unprepared, with no available vaccine or specific treatment. In this context, the World Health Organization declared that the humanitarian use of experimental therapies against Ebola Virus (EBOV) is ethical. In particular, an experimental treatment consisting of a cocktail of three monoclonal antibodies (mAbs) produced in tobacco plants and specifically directed to the EBOV glycoprotein (GP) was tested in humans, apparently with good results. Several mAbs with high affinity to the GP have been described. This review discusses our current knowledge on this topic. Particular emphasis is devoted to those mAbs that have been assayed in animal models or humans as possible therapies against Ebola. Engineering aspects and challenges for the production of anti-Ebola mAbs are also briefly discussed; current platforms for the design and production of full-length mAbs are cumbersome and costly.

Development of a Cost-effective Ovine Polyclonal Antibody-Based Product, EBOTAb, to Treat Ebola Virus Infection.

The highly glycosylated glycoprotein spike of Ebola virus (EBOV-GP1,2) is the primary target of the humoral host response. Recombinant EBOV-GP ectodomain (EBOV-GP1,2ecto) expressed in mammalian cells was used to immunize sheep and elicited a robust immune response and produced high titers of high avidity polyclonal antibodies. Investigation of the neutralizing activity of the ovine antisera in vitro revealed that it neutralized EBOV. A pool of intact ovine immunoglobulin G, herein termed EBOTAb, was prepared from the antisera and used for an in vivo guinea pig study. When EBOTAb was delivered 6 hours after challenge, all animals survived without experiencing fever or other clinical manifestations. In a second series of guinea pig studies, the administration of EBOTAb dosing was delayed for 48 or 72 hours after challenge, resulting in 100% and 75% survival, respectively. These studies illustrate the usefulness of EBOTAb in protecting against EBOV-induced disease.

Crosslinking of peanut allergen Ara h 2 by polyphenol oxidase: digestibility and potential allergenicity assessment.

BACKGROUND: Peanut is one of the eight major food allergens. Its allergen, Ara h 2, can be recognized by over 90% of serum IgE samples from peanut-allergic patients. Therefore, reducing the allergenicity of Ara h 2 is especially important. RESULTS: In the present study, polyphenol oxidase (PPO), a protein cross-linking reaction catalyst that acts on tyrosine residue, was used to modify Ara h 2. After crosslinking, the microstructure, digestibility, IgG binding capability and IgE binding capability of Ara h 2 were analyzed. Cross-linking decreased the potential allergenicity of Ara h 2 by masking the allergen epitope, while the antigenicity of Ara h 2 changed slightly. After crosslinking, the apparent diameter of Ara h 2 was altered from 300 to 1700 nm or 220 nm, indicating that polymerization could either be inter- or intramolecular. Regarding digestibility, crosslinked Ara h 2 was relatively more easily digested by gastric fluid compared with the untreated Ara h 2, but much more difficult in the intestinal fluid. CONCLUSION: The crosslinking reaction catalyzed by PPO, as a non-thermal process, may be beneficial for avoiding food allergy. The reaction could mask allergen epitopes, decreasing the allergenicity of Ara h 2. © 2015 Society of Chemical Industry.

Development and evaluation of a recombinant-glycoprotein-based latex agglutination test for rabies virus antibody assessment.

The measurement of neutralizing antibodies induced by the glycoprotein of rabies virus is indispensable for assessing the level of neutralizing antibodies in animals or humans. A rapid fluorescent focus inhibition test (RFFIT) has been approved by WHO and is the most widely used method to measure the virus-neutralizing antibody content in serum, but a rapid test system would be of great value to screen large numbers of serum samples. To develop and evaluate a latex agglutination test (LAT) for measuring rabies virus antibodies, a recombinant glycoprotein was expressed in an insect cell system and purified, and the protein was coated onto latex beads at concentrations of 0.1, 0.25, 0.5, 0.75, and 1 mg/ml to find out the optimal concentration for coating latex beads. It was found that 0.5 mg/ml of recombinant protein was optimal for coating latex beads, and this concentration was used to sensitize the latex beads for screening of dog serum samples. Grading of LAT results was done with standard reference serum with known antibody titers. A total of 228 serum samples were tested, out of which 145 samples were positive by both RFFIT and LAT, and the specificity was found to be 100 %. In RFFIT, 151 samples were positive, the sensitivity was found to be 96.03 %, and the accuracy/concordance was found to be 97.39 %. A rapid field test-a latex agglutination test (LAT)-was developed and evaluated for rabies virus antibody assessment using recombinant glycoprotein of rabies virus expressed in an insect cell system.

[Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate]. / Obtención y evaluación preliminar de un virus canarypox recombinante como candidato a vacuna antirrábica.

Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate. In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 IU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 IU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary interest.

Obtención y evaluación preliminar de un virus canarypox recombinante como candidato a vacuna antirrábica / Development and preliminary assessment of a recombinant canarypox virus as an antirabic vaccine candidate

En la Argentina, la rabia está circunscripta a algunas provincias del norte. La disponibilidad de nuevas vacunas que eliminen la manipulación del virus rábico y que permitan el control de la enfermedad es de importancia estratégica nacional y regional. Las vacunas basadas en poxvirus recombinantes se han utilizado con éxito como vacunas antirrábicas a nivel mundial. SI bien estos sistemas no están disponibles comercialmente, la plataforma de obtención de virus canarypox (CNPV) recombinantes ya ha sido implementada en nuestro laboratorio. El objetivo de este trabajo fue obtener y evaluar un candidato a vacuna antirrábica basado en CNPV recombinantes que expresan la glicoproteína G (RG) del virus rábico (RV). Se construyó un virus recombinante que expresa la secuencia codificante de RG (CNPV-RG). La inoculación de ratones con este virus indujo altos títulos de anticuerpos seroneutralizantes de RV (3,58 y 9,76 Ul/ml después de una o dos inmunizaciones, respectivamente) y protegió al 78 % de los animales desafiados intracerebralmente con RV. Además, se determinó que el CNPV-RG posee una potencia relativa de 3,5 Ul/ml. Los resultados obtenidos constituyen la primera etapa en la evaluación del CNPV-RG como candidato a vacuna antirrábica. Se requerirán nuevos ensayos para confirmar su utilidad en especies de interés veterinario.

In Argentina, rabies is limited to some northern provinces. Availability of new vaccines abolishing the handling of the rabies virus and allowing disease control has regional and national strategic importance. Vaccines based on recombinant poxviruses have been successfully used as antirabic vaccines worldwide. Although these systems are not commercially available, the platform to obtain recombinant canarypox viruses (CNPV) has been previously set up in our laboratory. The aim of this work was the development and evaluation of an antirabic vaccine candidate based on recombinant CNPV expressing the rabies virus (RV) glycoprotein G (RG). A recombinant virus (CNPV-RG) expressing the RG coding sequence was designed. Inoculation of mice with this virus induced high RV seroneutralizing antibodies (3.58 and 9.76 lU/ml after 1 or 2 immunizations, respectively) and protected 78% of intracerebrally RV-challenged animals. In addition, it was determined that CNPV-RG has a relative potency of 3.5 lU/ml. The obtained results constituted the first stage of CNPV-RG evaluation as antirabic vaccine candidate. Further assays will be necessary to confirm its utility in species of veterinary Interest.

Expression and solubilization of insect cell-based rabies virus glycoprotein and assessment of its immunogenicity and protective efficacy in mice.

Rabies is a fatal zoonotic disease of serious public health and economic significance worldwide. The rabies virus glycoprotein (RVG) has been the major target for subunit vaccine development, since it harbors domains responsible for induction of virus-neutralizing antibodies, infectivity, and neurovirulence. The glycoprotein (G) was cloned using the baculovirus expression vector system (BEVS) and expressed in Spodoptera frugiperda (Sf-9) cells. In order to obtain a soluble form of G suitable for experimentation in mice, 18 different combinations of buffers and detergents were evaluated for their ability to solubilize the insect cell membrane-associated G. The combination that involved 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) detergent in lysis buffer 1, formulated with Tris, NaCl, 10% dimethyl sulfoxide (DMSO), and EDTA, gave the highest yield of soluble G, as evidenced by the experimental data. Subsequently, several other parameters, such as the concentration of CHAPS and the duration and temperature of the treatment for the effective solubilization of G, were optimized. The CHAPS detergent, buffered at a concentration of 0.4% to 0.7% (wt/vol) at room temperature (23 to 25°C) for 30 min to 1 h using buffer 1, containing 10% DMSO, resulted in consistently high yields. The G solubilized using CHAPS detergent was found to be immunogenic when tested in mice, as evidenced by high virus-neutralizing antibody titers in sera and 100% protection upon virulent intracerebral challenge with the challenge virus standard (CVS) strain of rabies virus. The results of the mice study indicated that G solubilized with CHAPS detergent retained the immunologically relevant domains in the native conformation, thereby paving the way for producing a cell-free and efficacious subunit vaccine.

Phenylboronic acid immunoaffinity reactor coupled with flow injection chemiluminescence for determination of alpha-fetoprotein.

A reusable and sensitive immunoassay based on phenylboronic acid immunoaffinity reactor in combination with flow injection chemiluminescence (CL) for determination of glycoprotein was described. The reactor was fabricated by immobilizing 3-aminophenylboronic acid (APBA) on glass microbeads with gamma-glycidoxypropyltrimethoxysilane (GPMS) as linkage. The alpha-fetoprotein (AFP) could be easily immobilized on the APBA coated beads through sugar-boronic interaction. After an off-line incubation, the mixture of the analyte AFP with horseradish peroxidase-labeled AFP antibody (HRP-anti-AFP) was injected into the reactor. This led the trapping of free HRP-anti-AFP by the surface coated AFP on glass beads. The trapped HRP-anti-AFP was detected by chemiluminescence due to its sensitizing effect on the reaction of luminol and hydrogen peroxide. Under optimal conditions, the chemiluminescent signal was proportional to AFP concentration in the range of 10-10 0 ng m L(-1). The whole assay process including regeneration of the reactor could be completed within 31 min. The proposed system showed acceptable detection and fabrication reproducibility, and the results obtained with the present method were in acceptable agreement with those from parallel single-analyte test of practical clinical sera. The described method enabled a low-cost, time saving and was potential to detect the serum AFP level in clinical diagnosis.

Peanut allergen exposure through saliva: assessment and interventions to reduce exposure.

BACKGROUND: Exposure to food allergens through saliva (kissing, utensils) can cause local and systemic allergic reactions. OBJECTIVE: To determine the time course of peanut allergen (Ara h 1) persistence in saliva after ingestion of peanut butter and to evaluate mouth cleansing interventions to reduce salivary peanut allergen. METHODS: Thirty-eight individuals ingested 2 tablespoons of peanut butter, and saliva was collected at various time points. At another time, samples were collected after 5 interventions (brushing teeth, brushing and rinsing, rinsing, waiting then brushing, waiting then chewing gum). Detection of Ara h 1 was performed by a monoclonal-based ELISA (detection limit, 15-20 ng/mL). RESULTS: Salivary Ara h 1 varied considerably immediately after ingestion, but included levels expected to invoke reactions (as much as 40 microg/mL). Most (87%) subjects with detectable peanut after a meal had undetectable levels by 1 hour with no interventions. None had detectable levels several hours later after a peanut-free lunch. This result indicates (95% confidence) that 90% would have undetectable Ara h 1 in saliva under these circumstances. All of the interventions reduced salivary Ara h 1, in some cases by >95%, but Ara h 1 remained detectable in approximately 40% of samples (though typically below thresholds reported to induce reactions). CONCLUSION: Patients with peanut allergy require counseling regarding risks of kissing or sharing utensils, even if partners have brushed teeth or chewed gum. Advice to reduce risks, though not as ideal as total avoidance, includes waiting a few hours plus eating a peanut-free meal. CLINICAL IMPLICATIONS: Waiting several hours and ingesting a peanut-free meal were more effective at reducing salivary peanut protein concentration than simple, immediate interventions.

A multilaboratory peer assessment quality assurance program-based evaluation of anticardiolipin antibody, and beta2-glycoprotein I antibody testing.

We evaluated the performance of anticardiolipin (aCL) and beta2-glycoprotein I (beta2-GPI) antibody assays through a large external quality assurance program. Data from the 2002 cycle of the Royal College of Pathologists of Australasia Quality Assurance Program (RCPA QAP) were analyzed for variation in reported numerical values and semiquantitative results or interpretations according to method type or group and in conjunction with available clinical data. High interlaboratory variation in numerical results and notable method-based variation, combined with a general lack of consensus in semiquantitative reporting, continues to be observed. Numerical results from cross-laboratory testing of 12 serum samples (for immunoglobulin G [IgG]-aCL, IgM-aCL, and IgG-beta2-GPI) yielded interlaboratory coefficients of variation (CVs) that were higher than 50% in six of 12 (50%) specimens for IgG-aCL, and 12 of 12 (100%) specimens for IgM-aCL and IgG-beta2-GPI. Semiquantitative reporting also varied considerably, with total (100%) consensus occurring in only four of 36 (11%) occasions. General consensus (where > 90% of participating laboratories agreed that a given serum sample gave a result of either negative or positive) was only obtained on 13 of 36 (36%) occasions. Variation in results between different method types or groups were also present, resulting in potential biasing of the RCPA QAP-defined target results by the large number of laboratories using the dominant aCL assays. Finally, laboratory findings frequently did not agree with the available clinical information. In conclusion, in a large proportion of specimens from the 2002 RCPA QAP cycle, laboratories could not agree on whether a serum sample tested was aCL-positive or aCL-negative, or beta2-GPI-positive or beta2-GPI-negative. Despite prior attempts to improve the standardization of testing and reporting practices, laboratory testing for aCL and anti-beta2-GPI still demonstrates significant interlaboratory and intermethod variation, which needs to be taken into account for the clinical interpretation of test results, especially those from different laboratories.

Assessment of duration of immunity in crossbred cattle immunized with glycoproteins isolated from Hyalomma anatolicum anatolicum and Boophilus microplus.

To develop immunoprophylactic measures against multi-tick infestation, two glycoproteins of 34 and 29 kDa were isolated from the larvae of Hyalomma anatolicum anatolicum and Boophilus microplus, respectively, and assessed for their efficacy against experimental challenge infestations. The synergistic effect of the antigens in the presence of incomplete Freund's adjuvant was found to confer protection (DT%) in animals against 56.48% of larvae and 52% of adults of H. a. anatolicum, while the effect was 40% against adults of B. microplus. The efficacy (E%) of the antigens in combination against larvae and adults of H. a. anatolicum was calculated as 70% and 64.3%, respectively, and 63% against adults of B. microplus. A direct correlation between anti-glycoprotein antibody response and protection against infestation was observed. Western blot analysis detected specific antigen in the sera of animals of group A. The antigens in combination with incomplete Freund's adjuvant could protect animals from H. a. anatolicum and B. microplus infestations for at least 30 weeks. The possibility of employing the vaccination strategy in Indian conditions is discussed.

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies.

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.

Exposure assessment of high molecular weight sensitisers: contribution to occupational epidemiology and disease prevention.

An important group of sensitising agents are so called high molecular weight sensitisers--proteins or glycoproteins with molecular weights in the 5-70 kDa range that can provoke a specific IgE response in workers exposed to these agents. Exposure to high molecular weight sensitisers could only be evaluated indirectly in the recent past. Few measurement techniques existed that made it possible to measure the allergens directly. As a result, few studies focused on establishing exposure-response relations, and exposure standards have not been established for high molecular weight sensitisers, or those that have are of doubtful scientific basis. Recent use of immunoassays changed this perspective dramatically. Antibodies used in the assays can originate from human serum (sensitized workers), serum from sensitised animals (rabbits producing polyclonal antibodies), or animal derived cell cultures producing monoclonal antibodies. Although few comparative studies exist, the available evidence suggests that although the correlation of allergen concentrations obtained with different assays is good, large systematic differences occur. The use of conversion factors to make data from previously performed allergen measurements comparable or exchangeable is limited and thorough standardization of assays is preferred. Validation and comparison of different assays by comparisons between laboratories seem important issues that have not received the attention needed. Epidemiological studies in several industries that used immunoassay for the exposure characterisation have shown that risk of sensitisation increases with increasing exposure to allergens. Several studies have also shown that clear differences in potency seem to exist. Sensitisation to rat urinary allergens and fungal alpha-amylase occurred in the pg/m3 and ng/m3 range. The main research questions of the near future have to focus on the prevention of occupational sensitisation. Standard setting seems possible for some allergenson the basis of the available scientific evidence for the existence of exposure response relations. However, assays for characterising exposure to allergens have to be rigorously standardised before they can be used under field conditions.

Environmental assessment of Dermatophagoides mite-allergen levels in Sweden should include Der m 1.

The major allergen of Dermatophagoides microceras, Der m 1, as well as the allergens of D. pteronyssinus and D. farinae, Der p 1 and Der f 1, were analyzed in the homes of 111 asthmatic children in three climatic regions in Sweden. The numbers and species of mites were determined by microscopy, and circulating IgE antibodies against mites were measured. Der f 1 was the predominant house-dust-mite (HDM) allergen, Der p 1 the least often found, and Der m 1 represented 31% of the allergen load. However, in the Linköping area, Der m 1 was the major HDM allergen (58%). Mite counts and allergen levels correlated well. Current exposure to HDM allergens at home was associated with the serum IgE antibody response to HDM in the children with no threshold level. Of the children with IgE antibodies against HDM, 67% reacted to all three mites. Mite sensitization rates were marginally increased (7%) by the addition of IgE analysis of D. microceras to the routine analysis of IgE antibodies against D. pteronyssinus and D. farinae. Thus, Der m 1 may be an important HDM allergen and should be considered when HDM exposure data are assessed in areas with a climate like that of Sweden.

The effect of serial dilution error on calibration inference in immunoassay.

A common practice in immunoassay is the use of sequential dilutions of an initial stock solution of the antigen of interest to obtain standard samples in a desired concentration range. Nonlinear, heteroscedastic regression models are a common framework for analysis, and the usual methods for fitting the model assume that measured responses on the standards are independent. However, the dilution procedure introduces a propagation of random measurement error that may invalidate this assumption. We demonstrate that failure to account for serial dilution error in calibration inference on unknown samples leads to serious inaccuracy of assessments of assay precision such as confidence intervals and precision profiles. Techniques for taking serial dilution error into account based on data from multiple assay runs are discussed and are shown to yield valid calibration inferences.