Quantitative Complement Fixation Research Articles

The Comparative Value of Serum Angiotensin Converting Enzyme (ACE) and Lysozyme and the Use of Polyclonal Antibody Activation in the Work-up of Ocular Sarcoidosis.

Background: The diagnosis of ocular sarcoidosis (OS) is difficult to establish in the absence of manifest systemic involvement. To help clinicians reach a diagnosis, we convened a group of experts in 2006 (International Workshop on Ocular Sarcoidosis (IWOS)) to set-up clinical criteria for the diagnosis of ocular sarcoidosis. In addition, laboratory investigational tests represent a much-needed adjunct to ascertain the diagnosis. However, many of these tests have low sensitivity and specificity. Purpose: The aim of our study was to evaluate the usefulness of serum ACE, serum lysozyme and polyclonal antibody activation in the diagnosis of ocular sarcoidosis and compare the frequency of increased serum levels of lysozyme and ACE in proven ocular sarcoidosis or in suspected ocular sarcoidosis. Methods: Serum ACE and lysozyme were assessed in these two groups and their means compared to a group of non-granulomatous (i.e., non-sarcoidosis) uveitis patients. The proportion of elevated serum ACE versus lysozyme was compared in the sarcoidosis patients. Polyclonal antibody activation was measured by establishing exposition of patients to four human commensal herpesviruses (EBV, CMV, HSV and VZV) using ELISA or immunofluorescence and in parallel by performing quantitative complement fixation (CF) serologies. The ratio of elevated CF to positive ELISA/immunofluorescence serologies was calculated. The mean of ratios (polyclonal antibody activation) was compared between ocular sarcoidosis and control groups. Results: Thirty-seven patients (F24/M13) were included in our study including 17 patients with IWOS Level 1 and 2 criteria qualifying for Group 1 (proven sarcoidosis) and 20 ocular sarcoidosis suspect patients. Mean age was 54.52 ± 23.74. Mean serum levels of ACE was 49.17± 29 IU/L in the ocular sarcoidosis group versus 27.4 ± 15.34 IU/L (p ≤ 0.00018, student’s t test) in the control group. Mean serum lysozyme levels was 39.92 ± 55.5 mg/L in the ocular sarcoidosis group versus 10.5 ± 5.8 mg/L (p ≤ 0.0013) in the control group (n = 30). Both tests were elevated in 8/37 (21.6%) patients, elevated ACE and normal lysozyme was noted in 2/37 (5.4%) patients, whereas the proportion of normal ACE/elevated lysozyme was much higher, 23/37 (62.2%). In 4/37 (10.8%) patients, both tests were normal. The mean score of polyclonal activation (N of elevated CF serologies divided by number of viruses to which a patient was exposed) was 0.6 ± 0.33 in the ocular sarcoidosis group versus 0.15 ± 0.2 for the control group (n = 42) (p ≤ 0.00001). Sensitivity and specificity of ACE and lysozyme were, respectively, 27%/96.6% and 83.7%/90%. Sensitivity and specificity of polyclonal antibody activation amounted to 70%/90.4% Conclusion: Lysozyme was found to be much more useful than ACE as a laboratory test to support the diagnosis of ocular sarcoidosis. As shown in a previous study, polyclonal antibody activation appears to be another useful laboratory test supportive of the diagnosis of ocular sarcoidosis.

Indirect determination of FMDV 146S component concentration in non-inactivated suspension by comparison of graphs of the second derivative for real-time RT-PCR curves

During reproduction in biological systems, FMD virus forms four variants of components, three of which do not include RNA of the virus. In the process of industrial production of FMD vaccines, special attention is paid to the number of whole virions, which have the most important biological properties of FMD virus and are the main components that determine the immunogenicity of vaccine preparations. Raw materials for vaccines at various stages of the technological process are tested for concentration of FMDV 146S component. The traditional method of determination is quantitative complement fixation test. In recent years, real-time RT-PCR has been used for indirect determination of FMDV 146S component concentration in a virus-containing suspension. The article presents a new approach to indirect determination of FMDV 146S component concentration in a non-inactivated suspension by comparing the maximum extreme points of the graphs of the second derivative of the fluorescence signal accumulation curves and the number of amplification reaction cycles. The dependence between FMDV 146S component concentration and the maximum extreme points of the graphs of the second derivative of the fluorescence signal accumulation curve is presented in the form of a square function: C 146S FMDV = 0.0111( C p ) 2 – 1.0157 C p + 20.446 with a high accuracy of approximation (R 2 = 0.993). The proposed model allows to quantitatively estimate the content of 146S component in virus-containing vaccine raw materials. The presented method allows studying a large number of samples of non-inactivated raw materials for FMD vaccine in 4–5 hours. The main advantage of the proposed method is the capacity to determine the concentration of FMDV 146S component in a suspension with a high level of ballast proteins (more than 7.00 mg/cm 3 ) and complete viral particles (from 0.01 to 5.00 μg/cm 3 ).

SOME BIOCHEMICAL AND HAEMATOLOGICAL STUDIES IN BRUCELLOSIS INFECTED BUFFALOES, COWS AND SHEEP IN EL-SHARKIA GOVERNORATE

Blood samples from 351 buffaloes, 149 cows and 551 sheep were takenfrom a farm at New Salhia, (El-Sharkia Governorate) and examinedfor the incidence of brucellosis by the following serological tests,qualitative buffered acidified plate antigen (BAPAT), Rose BengalPlate test (RBPT), the standard quantitative tube agglutination test(SAT) and the confirmatory quantitative complement fixation test(CFT). Biochemical and haematological studies were done on 60seropositive (20 from each species) as well as 60 seronegtaiveanimals (20 from each species) as control onesThe bioechmiacl analysis revealed a significant increased enzymaticactivities for both, asparatate aminotransferase (AST) and alanineaminotransferase (ALT), and a significant increase in urea level ofinfected animals as compared with the control ones. On the otherhand, a significant decrease was recorded in total serum protein andglucose levels in all infected species. Serum iron level showed asignificant decrease in infected cows only. Whereas, no changes weredetected for both serum calcium and inorganic phosphorus levels ininfected animals comparing with control ones.Concerning haematological profile, we recorded a significant decreasein Hb and PCV% in all infected species accompanied with a generaldecrease in RBCs count, only of significant value in infected cows. TheWBCs count showed insignificant alterations in all infected animalscompared with control ones.

Immunological studies on venereal spirochetosis of rabbits (rabbit syphilis).

White rabbits in a family, which were clinically diagnosed as moderately or severely diseased with spirochetosis, were bacteriologically and immunologically examined. The specimens from the diseased rabbits, including affected prepuces, scrotum, or skins with an occasional presence of the spirochetes, did not, however, result in growth in six conventional culture media. Serological tests, including quantitative complement fixation test, rapid plasma reagin card test, Treponema pallidum hemagglutination test, and microscopic agglutination test for leptospires using sera from diseased rabbits showed no differences when compared with those of pooled normal rabbit sera. Immunoblot analysis of the polypeptides from three human oral treponemes and three non-oral spirochetes demonstrated that antibodies against several treponemal polypeptides were detected.

An enzyme-linked immunosorbent assay (ELISA) for detecting antimitochondrial antibody.

We have developed an enzyme linked immunosorbent assay (ELISA) for antimitochondrial antibody. Polyvinyl microtiter plate wells are coated with partially purified rat kidney mitochondria, and excess protein binding sites are blocked with bovine serum albumin. Human serum, diluted 1:1,000, is incubated for 1 hr. Then beta-galactosidase-goat-anti-human IgG (H + L) is added followed by the substrate, p-nitrophenyl-beta-D-galactopyranoside. The plates are then read at 404 nM in a microelisa autoreader. A positive result was defined as optical density greater than or equal to 0.100, more than 5 standard deviations above the mean of 36 normal individuals. With this technique, 56 of 60 patients with primary biliary cirrhosis were positive for antimitochondrial antibody (93%), mean O.D., 0.456 +/- 0.031 S.E. Seventeen of 17 patients with extrahepatic bile duct obstruction and 14 or 14 patients with alcoholic cirrhosis were negative. Only 1 of 29 patients with chronic active liver disease was positive (4%). Antinuclear antibody and antimicrosomal antibody do not bind in this assay, and activity is absorbed from sera by preincubation with suspensions of rat kidney mitochondria. The ELISA is approximately 20 times more sensitive than a quantitative microtiter complement fixation technique and more convenient than radioimmunoassay. It is rapid, quantitative and uses stable reagents. In contrast to immunofluorescence techniques, it is not affected by observer interpretation.

Generation, characterization and ELISA of monospecific antibodies against the subunits of a Ca 2+-dependent protein kinase and a Ca 2+-transport ATPase from rabbit skeletal muscle

Monospecific precipitating sheep antibodies were generated for the first time against the purified, homogeneous α-, β- and γ-subunits of the Ca 2+-dependent protein kinase, phosphorylase kinase, from rabbit muscle. As reference, antibodies against the holoenzyme and the Ca 2+-transport ATPase of sarcoplasmic reticulum were induced. In all cases antibody titers could be quantitated (standard error 5–10%) by enzyme-linked immunosorbent assay. Differentiation of antibody binding was achieved by quantitative precipitation and complement fixation assays. In general maximal antibody titers were reached 56 days after primary immunization and high titers (5000) were maintained for several weeks. Anti-α, anti-β and anti-γ avidly precipitate the denatured subunits employed as immunogens as well as the native enzyme. No cross-reactivity between antibodies against a specific subunit and any of the other heterologous subunits was demonstrable in double immunodiffusion assays providing no evidence for immunologically identical sites on the α-, β- and γ-subunits. Since anti-α, anti-β and anti-γ strongly inhibit enzyme activity, it is likely that they do so primarily by sterically interfering with the binding of the large substrate phosphorylase b (M r 2.0 × 10 5) to phosphorylase kinase (M r 1.3 × 10 6). It cannot be excluded, however, that anti-β and anti-γ bind to the active sites on these 2 subunits.

Quantitative complement fixation test in sera from patients with schistosomiasis and intestinal parasitosis.

Quantitative complement fixation test in sera from patients with schistosomiasis and intestinal parasitosis.

Ventriculo-lumbar gradient of concentration of total cerebrospinal fluid proteins: 1 - mechanisms of origin

In normal conditions there is a concentration gradient of proteins along the neuraxis. From a low level in the ventricles, ranging from 5 to 15 mg/10C ml, to an intermediate level in the cisterna magna, the protein content reaches its highest level in the lumbar sac, 12 to 44 mg/100 ml. Several mechanisms were considered to elucidate the origin of this gradient but many investigators think that the progressive increase of the protein concentration is best explained by the transfer of proteins from serum to the cerebrospinal fluid due to the relatively raised permeability of blood-cerebrospinal fluid barrier in the spinal subarachnoid space. This paper presents a study of the protein concentrations in cisternal and lumbar cerebrospinal fluid samples of patients with neurocysticercosis in activity. The 11 patients of the first group had free subarachnoid space communication between the cisterna magna and the lumbar sac; the 6 patients of the second group had a complete block of the subarachnoid space between these two levels. In every cerebrospinal fluid specimen the quantitative complement fixation test for cysticercus was performed and the titer determined in order to make an assessment of the central nervous system humoral immune response. The analysis of the data of this investigation shows that the concentration gradient of proteins is evident in the cerebrospinal fluid of patients with patency of the spinal subarachnoid space, and the ratio of concentrations of protein contents in simultaneous cisternal and lumbar samples was similar to that one observed in normal individuals. This gradient is also detected when the intensity of the humoral immune response is determined by quantitative complement fixation test for cysticercus in simultaneous cisternal and lumbar specimens. After the onset of spinal subarachnoid block, the confront of the results of the tests in cerebrospinal fluid samples, obtained before and after the blockage, shows a large increase both in the total protein content as well as the intensity of the humoral immune response, in the lumbar level. The similar increases both in protein concentration and titer of cysticercus complement fixation test in the lumbar fluid, in comparison with the cisternal fluid, in patients with patent spinal subarachnoid space, and the large simultaneous and similar increases in both protein content and titer of the cysticercus complement fixation test in the lumbar fluid of patients with spinal subarachnoid block are in disagreement with the usual explanation of the origin mechanisms of the gradient.(ABSTRACT TRUNCATED AT 400 WORDS)

Physicochemical and immunologic properties of tissue DNA from mice with oncornavirus-induced leukemia

Active antisera containing antibodies to deproteinized DNA preparations of mouse normal and leukemic tissues have been obtained. Examination of antisera by the quantitative complement fixation test with the corresponding test antigens has demonstrated immunological specificity of DNA preparations isolated from the organs of mice with leukemia induced by oncornaviruses. The DNA specificity in question is inherent not only in the native but also in the thermodenatured molecule of DNA.

Demonstrations of the Production of Specific Antibodies to Poly(I) . poly(C) in Rabbits

The rabbit antiserum against poly(I).poly(C) purified by hydroxyapatite column chromatography contained three distinct antibodies. They were fractionated into three antibody populations by a series of precipitations (with poly(A).poly(U), poly(I), and poly(I).poly(C)) and their specificities were examined by quantitative complement fixation, double diffusion tests and radioimmunoassay. The first population was common to the double helical structure of double-stranded RNAs. The second was specific for poly(I) and the third was specific for poly(I).poly(C). These studies demonstrated that specific antibodies exclusively reactive with poly(I).poly(C) existed in the rabbit antiserum against poly(I).poly(C).

Albumin Evolution within New World Squirrels (Sciuridae)

Immunological studies of the Sciuridae using quantitative micros complement fixation indicate that the ground squirrel and chipmunk lineages diverged from tree squirrels during the early Oligocene. Chipmunks and ground squirrels diverged during the early Miocene. Radiation within the ground squirrel lineage appears to have taken place during the early and middle Miocene. These findings are, for the most part, consistent with fossil, karyotypic and other biochemical data.

S100 protein is present in cultured human malignant melanomas.

S100 protein, so called because of its solubility in 100% saturated ammonium sulphate1, is an acidic cytoplasmic protein specific for the nervous system2,3. It is localized primarily to glial elements of the brain, Schwann cells in the peripheral nervous system and satellite cells in sympathetic ganglia4,5. This brain-specific protein shows a close immunological relationship among a wide variety of vertebrates, as measured by a quantitative complement fixation technique6. Although strict serological conservation and tissue localization have been maintained among different species, no function for S100 protein has been determined. However, the appearance of S100 protein in brain is correlated with maturation of the nervous system in both rat and man7,8. Previous in vitro studies on the production and regulation of S100 protein in the C6 cell line derived from a rat astrocytoma indicated that cell contact and cessation of division induced S100 protein9. Neuroblastoma cell lines lack this protein10,11 although it is found in other astrocyte cell lines. We are unaware of any reports of cell lines, other than those of glial origin, which produce S100 protein. Melanocytes are derived from neuroectodermal elements, and this common origin of glial cells and melanocytes prompted us to study whether S100 protein might be present in cell lines from human malignant melanomas. We now report the presence of S100 protein in five of seven continuous cell lines of human malignant melanoma.

Immunological dissimilarity of the calcium pump protein of skeletal and cardiac muscle sarcoplasmic reticulum

Sheep antiserum produced against purified calcium pump protein from rabbit skeletal muscle sarcoplasmic reticulum was used to test for the immunological cross-reactivity of calcium pump proteins of sarcoplasmic reticulum from skeletal and cardiac muscle from several species. The calcium pump protein from rabbit skeletal muscle sarcoplasmic reticulum, solubilized with Triton X-100, gave a single precipitin line on immunodiffusion. There was no cross-reaction of the antiserum with a soluble extract enriched in calcium binding protein and M 55 (55,000 dalton) protein prepared from rabbit skeletal muscle sarcoplasmic reticulum. A reaction of identity was observed by immunodiffusion when rabbit skeletal muscle and rat skeletal muscle sarcoplasmic reticulum were reacted with antiserum, but only partial identity was seen with pigeon skeletal muscle sarcoplasmic reticulum. Cardiac sarcoplasmic reticulum preparations from pigeon, rat, rabbit, and bovine failed to produce precipitin lines by immunodiffusion. Quantitative complement fixation was used to compare the extent of cross-reactivity of sarcoplasmic reticulum preparations of skeletal and cardiac muscle and the results were expressed in terms of the “index of dissimilarity.” Each cross-reacted with the antiserum. The calcium pump proteins of skeletal muscle sarcoplasmic reticulum from each species were more similar to each other than to the cardiac sarcoplasmic reticulum from the same species. Additional dissimilarity in antigenic structure between species was also found.

31] Quantitative micro complement fixation: Serologic properties of pig liver carboxylesterase

Publisher Summary This chapter discusses the quantitative micro complement fixation. Quantitative micro complement fixation is a most sensitive serologic method for detection of conformational changes in macromolecules. Denaturation of DNA and polyribonucleotides, altered conformation of lactic dehydrogenases, hemoglobin, myoglobin, collagen, lysozyme, aspartate transcarbamylase, pepsinogen and pepsin, carboxypeptidase, and S-100 brain protein have been detected and quantified by micro complement fixation. In addition, the extreme sensitivity of the method has been exploited to measure the molecular changes induced by evolutionary processes. This chapter describes the use of micro complement fixation to measure the rates of dissociation and the equilibrium constants for dissociation of pig liver carboxylesterase as a function of pH and salt concentration. The pig liver carboxylesterase preparation and some of its physical properties as described by Barker and Jencks are discussed in this chapter. Estimation of native carboxylesterase molecules is discussed. Equilibrium constants, as measured by complement (C) fixation, for dissociation of pig liver carboxylesterasc as a function of pH are illustrated. Determination of equilibrium constants is also discussed in this chapter in detail.

An Adenosine Triphosphatase Isolated from Chromaffin-Granule Membranes is Closely Similar to F1-Adenosine Triphosphatase of Mitochondria

1 An ATPase purified with high yield from bovine chromaffin granules closely resembles the mitochondrial F1-ATPase from bovine heart or bovine adrenal medulla with respect to the following properties: apparent molecular weight on gel filtration, subunit composition on dodecyl- sulphate/polyacrylamide gel electrophoresis, cross-reaction with an antiserum directed against F1-ATPase from bovine heart mitochondria, and proteolytic fingerprints of the three largest subunits. 2 The chromaffin granule ATPase could, however, be distinguished from the mitochondrial enzyme: it did not enhance the fluorescence of aurovertin and reacted differently from mitochondrial F1-ATPase in quantitative complement fixation. 3 The following three arguments make it unlikely that the ATPase isolated from chromaffin granules has been artifically dislodged from mitochondria during fractionation: (a) mitochondrial contamination of the purified chromaffin granule membranes was bhow 2%; (b) the purification procedure solubilized at least two thirds of the total ATPase activity of these membranes; (c) an antiserum directed against bovine mitochondrial F1-ATPase inhibited the ATP-driven uptake of 5-hydroxytryptamine by resealed chromaffin granule ‘ghosts’. 4 The proton-translocating ATPase of chromaffin granules is thus almost identical to mitochondrial F1-ATPase. This finding raises intriguing questions about the evolution of chromaffin granules and about the mechanisms by which the two enzymes are transported to different locations within the same cell.

Passive immunization against Pseudomonas with a ribosomal vaccine-induced immune serum and immunoglobulin fractions

Passive protection of mice against Pseudomonas aeruginosa using specific antisera and immunoglobulin fractions induced by immunizing rabbits with a ribosomal vaccine is reported. The results demonstrated that protection by the ribosomal vaccine against challenge with live organisms can be serum mediated. Previous work has shown that the vaccine can be separated into two components on the basis of molecular weight and that both higher (peak A)- and lower (peak B)-molecular-weight fractions were capable of inducing active immunity in mice. The present report indicates that both fractions are also capable of eliciting the production of mouse-protective antibody in rabbits. Agar gel diffusion with antisera to peaks A and B or unfractionated vaccine indicated a common antigenic component among them in addition to an extra antigen in unfractionated vaccine not present in peak B. Passive hemagglutination with antisera to peaks A and B demonstrated high-titer agglutinating antibody only with antiserum to peak A when a method of erythrocyte sensitization for lipopolysaccharide antigens was used. Also, passive hemagglutination was greatly inhibited by small amounts of lipopolysaccharide prepared from the same organism from which the vaccine was made. Both antisera to peaks A and B fixed complement with either A or B antigens. Antisera to peaks A and B, when reacted with peak B antigen, had about the same complement fixation titer (as determined by a quantitative complement fixation test). However, when peak A antigen was used, antiserum to peak A had about twice the complement fixation titer that antiserum to peak B had. These results are consistent with previous observations which suggest that the ribosomal vaccine contains lipopolysaccharide in addition to an unidentified immunogenic principle associated with ribosomes. Furthermore, this immunogen was present in both peaks A and B, but detectable amounts of lipopolysaccharide were present only in peak A. The relative importance of the immunoglobulin G (IgG) and IgM classes of antibodies was also compared. The results indicated that both IgG and IgM isolated from immune rabbit serum are protective in mice. Only IgG precipitated with the vaccine in agar gel diffusion, but both IgG and IgM were active in passive hemagglutination and in complement fixation. The passive hemagglutination titer of the IgM was higher than that of the IgG, but the complement fixation titer of the IgG was higher than that of the IgM. The mouse-protective capability of the IgG and IgM was about the same.

Interferon Induction by a 2′‐Modified Double‐Helical RNA, Poly(2′‐azido‐2′‐deoxyinosinic acid) · polycytidylic acid

Although the presence of free 2′‐hydroxyl groups in both strands of a double‐stranded RNA complex has been recognized as one of the major requisites for the interferon‐inducing activity of double‐stranded RNAs, we have found a particular analogue of (I)n· (C)n in which the 2′‐hydroxyls of the purine nucleotide strand were replaced by azido groups, (dIn3)n· (C)n, to be highly effective in inducing interferon. Various other 2′‐azido analogues of (I)n· (C)n and (A)n· (U)n, i.e. (dIn3)n· (br5C)n, (I)n· (dCn3)n, (dAn3)n· (U)n, (A)n· (dUn3)n, (dAn3)n· (rT)n and (dAn3)n· (dUn3)n, were inactive as inducers of interferon. In human fibroblast cultures, the interferon‐inducing activity of (dIn3)n· (C)n equalled that of (I)n· (C)n. In other interferon‐induction systems (primary rabbit kidney cells, mouse L‐929 cells, intact rabbits), (dIn3)n· (C)n was less active than (I)n· (C)n. As assessed by both radio‐chemical and biological means, (dIn3)n· (C)n was more susceptible to degradation by pancreatic ribonuclease and human serum nucleases than was (I)n· (C)n. The tm of (dIn3)n· (C)n was 52.5 °C, as compared to 62.5 °C for (I)n· (C)n, both determined in 0.15 M Na+, pH 7.0. Under the same conditions, (dIn3)n· (br5C)n had a tm of 77 °C and (I)n· (br5C)n had a tm of 87 °C. The reactivity of (dIn3)n· (C)n and (dIn3)n· (br5C)n towards antibodies to double‐stranded RNA was evaluated by quantitative complement fixation, counterimmunoelectrophoresis and competitive radioimmunoassay. In these tests, (dIn3)n· (C)n and (dIn3)n· (br5C)n showed an immunoreactivity pattern comparable to that of (I)n· (C)n and (I)n· (br5C)n.

USE OF SPECIFIC ANTISERA AGAINST LEUKEMIA-ASSOCIATED ANTIGENS IN DIAGNOSIS AND TREATMENT OF CHILDHOOD ALL

Rabbit antisera against the common forms of childhood acute lymphoblastio leukemia (ALL) the O- and T-ALL were produced. Extensive absorptions of the antisera removed the crossreactions with normal mature lymphocyte populations and bone marrow progenitor cells, 1) A characterization of the type of ALL by use of the highly purified antisera is possible by different test systems such as cytotoxicity, immunfluorescence and quantitative complement fixation. The results show that the former test systems such as rosette technique failled to identify some O-ALL. Therefore by use of specific antisera the benefit of chemotherapeutic treatment for the different forms of ALL can be better calculated. 2) Treatment of a patient with O-ALL during his 3rd bone narrow relapse by high doses of Anti-O-ALL globulin revealed a marked decrease in circulating blast cells but no effect on bone marrow blast cells. Our approach aims for an elimination of residual leuoemic cells in bone marrow during haemopoietic remission using the cytotoxic effect of the antisera. The use of A-O-ALL-G for “antileukemic autotransplantation” is proposed.

Evidence for a common tumour-associated antigen in extracts of human bronchogenic carcinoma.

Xeno-antiserum specific for antigenic components of bronchogenic carcinoma was raised in rabbits, by passively immunizing them to normal human lung antigens at the same time as immunization with a tumour extract from a squamous-cell carcinoma. Antiserum so raised contained minimal quantities of anti-normal antibody which could be removed by a single absorption with glutaral-dehyde-insolubilized normal lung extract. When tested by quantitative complement fixation with a panel of tumour extracts from surgical specimens, it was found that the antiserum gave positive complement fixation with all squamous-cell carcinoma extracts tested, and with some of the extracts from bronchogenic carcinomas of differing pathological types. The antiserum was essentially negative for pooled extracts from normal lung, liver and spleen but gave a weak positive reaction with an extract of pooled foetal lung tissue.

Comparison of laboratory techniques for the evaluation of the antigenic potency of foot-and-mouth disease virus cultures and vaccines

Forty-seven suspensions of type A virus grown in surviving bovine tongue epithelium (Frenkel) cultures were compared in a quantitative complement fixation test (CFT) and an antibody combining test (ACT) to evaluate the antigenic mass of the 140 S component, and with three assay methods for infectivity titration. The cultures were then converted into vaccines and the potency of these was measured with a guinea-pig PD 50 test and again with the ACT. The relationship between the results obtained with different methods used for titration of infectivity, i.e. baby mice and plaque counting in BHK cells in monolayer (BHK-M) or suspended in agar (BHK-S) was poor and, with the exception of the BHK-S technique, showed little correlation with the results of the serological tests. The correlation coefficient between values obtained by the CFT and the ACT on virus cultures was 0.82 and that between CFT and ACT on vaccines 0.88. The guinea-pig PD 50's of 19 vaccines were compared with the ACT and with the CFT and ACT of the corresponding cultures yielding r values of 0.74, 0.62 and 0.62 respectively. Regression lines are presented for the different relations. An accurate cattle PD 50 was determined for two vaccines of the series, using groups of 20 animals per vaccine dilution. The relationship between guinea-pig PD 50 ( X) and cattle PD 50 ( Y), expressed by the regression formula Y = 1.05 X + 0.84, showed that differences in cattle PD 50's are of the same magnitude as those observed in the guinea-pig test, provided that sufficient numbers of animals are used for both tests. The results of the quantitative CFT and ACT were in good agreement with the guinea-pig test and as such these tests can provide valuable information on the quality of Frenkel cultures.