Amount Of Antiserum Research Articles

Pipiserpin, a Culex factor Xa inhibitor, affects female reproductive capacity and serves as a potential target for mosquito control.

Mosquito control is still the main prevention and control measure for numerous mosquito-borne diseases causing millions of deaths each year. New strategies for mosquito control are in demand. Proteases play an important role in mosquito physiology, therefore this study explored the inhibition of a serpin (serine protease inhibitor) in mosquitoes and its effect on reproductive capacity. A factor Xa inhibitor homolog (named Pipiserpin) was amplified and identified in Culex pipiens pallens mosquitoes. We expressed a recombinant Pipiserpin protein in vitro against which a mouse antiserum was generated. We found that female mosquitoes expressed more Pipiserpin protein than male mosquitoes. After mating, female mosquitoes were fed with blood mixed with different amounts of antisera and results showed that consumption of Pipiserpin impeded ovary development and decreased eggs hatching rates compared to that of the pre-immune serum group. We identified a Culex mosquito factor Xa inhibitor, Pipiserpin, which affects female reproductive potential. Our results suggest that Pipiserpin may be a novel target for mosquito population control. The conclusions from our study on Cx. pipiens pallens might serve as a reference for the development of control measures for other mosquitoes as well. © 2022 Society of Chemical Industry.

A unique approach to compensation and its verification

The compensation of multiple fluorochromes within the same experiment enables the correct characterization of cell phenotype by the exclusion of any errors associated with fluorochrome spectral overlap. Fluorochrome compensation is far from standardized, from laboratory to laboratory the reagents and rationale to achieve compensation values varies considerably, so an approach is given to standardize the set up procedure, outlining the major reasons for the steps employed. The first step is to recognize that photomultiplier voltages are of equal importance as the compensation controls to achieve correct fluorochrome compensation, therefore we need to adjust the voltages very carefully to minimize amplification of overlapping fluorochromes, hence minimizing the final compensation values, as a consequence the final experiment will be more “stable“. The photomultiplier voltages are adjusted to ensure maximum signal to noise for the antigen under investigation by the use of single fluorochrome, experiment specific stained cells. This will enable the visualization of non-specific antibody binding affects, and also where possible the percentage of the cells under very uncomplicated conditions, these percentages used to verify that under the polychromatic experiment conditions values remain the same. The single stained cells however may not have a high concentration of antigen, therefore we rely on antibody capture beads to “load” the experimental antibodies, singly onto antibody capture beads, this ensures that for all fluorochromes we have a very high fluorochrome signal, thus enabling accurate compensation values, avoiding errors associated with cells that may have low antigen concentration and consequently low signal and consequent inaccuracies when calculating compensation. It should be noted at this point that the verification of instrument photomultiplier linearity is a prerequisite, as is the verification of tandem fluorochrome integrity. With the optimization of photomultiplier voltages the compensation beads can now be run, either using the automatic compensation protocols on the instrument, or if preferred, adjusting the values manually aligning the negative and positive medians for the single discreet fluorochromes. Especially if the automated compensation protocol has been used, a verification step should be performed by appending each compensation bead into a single list mode file to ensure all beads are compensated, this can also be repeated using the single stained cells, although, in the cell scenario some of the antigens may be weak or absent. This step is imperative prior to running the tube where all antibodies are present so to ensure that compensation values are correct and there is no variance between the beads and cells i.e., they must both be correctly compensated. At this point the sample with all antibodies within the one tube is run, you have an immediate check, using previous single color tubes, to ensure that all percentages between the multicolor tube and the single color tubes correlate, also that there is no loss of intensity, this may sometimes occur due to dilution effects of other antisera when using large amounts of antisera. With careful scrutiny of the compensation values achieved you may be able to reduce compensation values by adjusting photomultiplier voltages but care must be taken not to compromise the sensitivity of the assay

자연산 자주새우(Crangon affinis)에서 흰반점바이러스(WSSV)의 neutralization을 위한 인위감염(experimental infection)

White spot syndrome virus (WSSV) is one of the most virulent viral agents threatening the penaeid shrimp culture industry. This study was carried out to evaluate the susceptibility of the sand shrimp, Crangon affinis, to WSSV as an alternative experimental model. WSSV caused 100% mortality in C. affinis within 7 days after experimental infection by immersion. Based on challenge studies, it was confirmed that C. affinis could be a potential host in WSSV transmission. Also, the neutralization of WSSV was carried out using an antiserum raised against recombinant envelop protein rVP466 to evaluate the WSSV infection mechanism. A constant amount of WSSV (at 1x10⁴ diluted stocks) was incubated with various amounts of antiserum and then mixed to 20 l reservoir for the immersion challenge of C. affinis for neutralization. At 5 days post challenge, the shrimp in the positive control immersed in the immersion reservoir containing WSSV stock showed 100% mortality. The shrimps challenged with the 3 different mixtures of WSSV and rVP466 antiserum (1:0.1, 1:0.5 and 1:1) showed 100%, 68.8% and 68.8% mortality at 14 days post challenge, respectively. These results indicated that the antiserum raised against rVP466 could block WSSV infection in C. affinis. Therefore, this study confirmed that C. affinis can be naturally infected by WSSV as another potential host and that C. affinis can be used as an alternative experimental animal instead of penaeid shrimps.

Partial passive protection with two monoclonal antibodies and frequency of feeding of hyperimmune anti-transmissible gastroenteritis virus (TGEV) serum for protection of three-day-old piglets from a TGEV challenge infection.

Passive protection experiments were conducted to determine the frequency and amounts of hyperimmune antiserum needed to block a transmissible gastroenteritis virus (TGEV) challenge infection and to identify monoclonal antibodies that are partially protective against TGEV. Hyperimmune antiserum or monoclonal antibodies were added to milk at each feeding or at selected feedings when the amount of antiserum was reduced. Three-day-old piglets were challenged with virulent virus that had been preincubated with antiserum or monoclonal antibodies. The results indicated that supplementing antiserum every other day was not efficacious for protection. Supplementing even small quantities of hyperimmune antiserum (0.5 ml) at least once a day in most cases was sufficient for piglet survival but did not prevent morbidity. Increasing the amount (>2 ml) and providing antiserum 3 times/day completely blocked the TGEV challenge infection. Two monoclonal antibodies were discovered that also provided passive protection for baby pigs. One monoclonal antibody, 5G1, had a high neutralizing titer, and the other, 6C4, was more effective in neutralizing and binding to virulent TGEV than to attenuated TGEVs. Both of these monoclonal antibodies were partially effective as supplements in milk for passive protection. Furthermore, these monoclonal antibodies were useful for boosting the efficacy of TGEV-neutralizing colostrum, which by itself was ineffective. These results show that other antigenic sites, different from the 4-well characterized epitopes on the S glycoprotein of TGEV, also are important for passive protection.

A micromethod for serotyping Bacillus thuringiensis.

Serotyping of Bacillus thuringiensis is possible using 96-well microplates instead of tubes. The advantages are a reduction on the incubation time from 120 to 75 min and the amounts of antisera and bacterial suspensions needed 10-fold.

Specific biological effects of an anti-rat PMN antiserum intraperitoneally infected into f344/n rats.

Neutropenia can be produced with antimitotic chemicals, but this method lacks specificity. An alternative is to use antibody-dependent cytotoxicity to produce neutropenia; however, this method has not been completely evaluated with respect to efficacy, specificity, and potential collateral damage, especially to constituents of bone marrow. This study used in vitro and in vivo methods to evaluate specific biological effects of a commercially available rabbit anti-rat neutrophil (PMN) antiserum in F344/N rats. The viability of rat pulmonary alveolar macrophages (PAMs), PMNs, and lymphocytes in vitro was quantified using a trypan blue dye exclusion test. Amounts of antiserum in vitro that rendered PMNs 100% nonviable did not decrease the viability or phagocytic ability of the PAMs and did not decrease the viability of the lymphocytes. Intraperitoneal (IP) injection of the antiserum into rats resulted in complete depletion of the PMNs and about a 50% depletion of the lymphocytes in circulating blood within 24 hours. The numbers of both cell types remained lowered for 5 days, but returned to control values by Day 6 after the IP injection. The antiserum had no effect on the numbers of PAMs or lymphocytes in the pulmonary alveolar airspaces, as determined by quantifying the numbers of these cell types in bronchoalveolar lavage fluid (BALF). The numbers of PMNs in BALF, however, decreased on Days 3 and 4 after IP injection of antiserum, but were not different from control values by Day 5. The viability of the PAMs in BALF of treated rats was not different from control values at any time point. There were no morphological indications that the injected antiserum damaged lung tissue or stem cells in bone marrow. Results demonstrate that the anti-rat PMN antiserum administered IP to F344/N rats depletes circulating PMNs and partially depletes lymphocytes for a period of about 6 days without adversely affecting the precursors of red or white blood cells in bone marrow. We concluded that the antiserum is a relatively specific way to temporarily render rats neutropenic without damaging precursor cells in bone marrow.

The PhastSystem equipment used for crossed immunoelectrophoresis combined with immunoblotting of coprecipitated monoclonal antibodies as studied with platelet membrane receptor proteins.

The Pharmacia PhastSystem equipment has been used for crossed immunoelectrophoresis combined with a technique for immunoblotting with monoclonal antibodies. This miniaturized gel system is compared to the conventional approach using platelet membrane receptor proteins as a model. Whereas in the conventional system the electrophoretic procedure takes place within 20 h, 3 h are adequate for the small gel system. Because of the short second-dimensional electrophoresis, and only one overnight incubation, the total electrophoretic and blotting procedure could be reduced from about 48 h to 24 h. The amount of antiserum used during the second-dimensional electrophoresis could be reduced roughly by a factor of 5. The examples with electrophoresis and immunoblotting using platelet extracts in 1% Triton X-100 demonstrate that membrane receptor proteins can be studied even when present as noncovalent complexes. The immunoblotting can be used with monoclonal antibodies that do not function in Western blotting.

Effect of rabbit antimotilin serum on myoelectric activity and plasma motilin concentration in fasting dog.

It is known that a cyclic increase in plasma motilin concentration occurs during the interdigestive state of dog and the increase coincides with migrating myoelectric complexes (MMCs) of the antrum as well as proximal duodenum. The purpose of the present study is to determine the role of endogenous motilin in the occurrence of MMCs in 10 dogs prepared with a gastric cannula and platinum monopolar electrodes in the gastric antrum, duodenum, jejunum, and ileum. After recording at least two consecutive cycles of MMCs from the proximal duodenum, each dog received an intravenous infusion of highly specific rabbit antimotilin sera in varying doses ranging from 3.5 to 15 ml for a period of 60 or 90 min. During the motility recording period ranging from 6 to 30 h following the administration of the antimotilin serum, several changes in the motility were observed. 1) The occurrence of MMCs in the antrum, duodenum, jejunum, and ileum was temporarily interrupted for varying periods depending on the individual dog studied and the amount of antiserum administered. When the higher dose of antimotilin was administered, a more profound and prolonged inhibition occurred. 2) Phase I activity rarely occurred. Instead, a phase II-like activity continued throughout the recording period. 3) MMCs in the jejunum or ileum occurred at irregular intervals without aboral propagation of MMCs from the duodenum or jejunum. 4) The plasma motilin concentration decreased to levels lower than that observed during phase I of the duodenum and exhibited no cyclic increase until the MMCs reappeared in the proximal duodenum.(ABSTRACT TRUNCATED AT 250 WORDS)

27] Enzyme-linked immunoabsorbent assay (ELISA) and radioimmunoassay (RIA) for the vitamin D-dependent 28,000 dalton calcium-binding protein

Publisher Summary This chapter discusses two sensitive and specific assay methods for the quantitation of intestinal calcium-binding protein (CaBP) in the chick intestine: enzyme-linked immunoabsorbent assay (ELISA) and a radioimmunoassay (RIA). Both ELISA and RIA use antisera produced in the rabbit against highly purified chick intestinal CaBP. The primary difference between the ELISA and the RIA is the use of an enzyme-linked second antibody in ELISA rather than 125 I iodination of CaBP in RIA. RIA is based on the competition for antibody binding between radioactive 125 I-labeled CaBP with nonradioactive CaBP. Because the amounts of 125 I-labeled CaBP and antibody are both fixed and limiting and the amount of nonradioactive CaBP varies, the percentage of bound 125 I-labeled CaBP is inversely related to the amount of nonradioactive CaBP present. ELISA is based on the competition for antibody binding between CaBP that is immobilized to polystyrene EIA wells and free reference or sample CaBP. Antisera bound to free CaBP is washed away, while that bound to the immobilized CaBP remains in the well. The second antibody, goat anti-rabbit γ-globulin, to which alkaline phosphatase has been conjugated, is added and binds to the rabbit antiserum remaining in the wells. The addition of the substrate, p -nitrophenyl phosphate, results in the production of yellow color in any wells containing the immobilized CaBP-antisera-second antibody complex. The amount of CaBP coated to the wells and the amount of antisera are both fixed and limiting so that the intensity of the yellow color produced is inversely proportional to the amount of free CaBP.

Trypanosoma cruzi: Antibody-induced mobility of surface antigens

Antibody induces mobility of surface antigens of live blood forms of Trypanosoma cruzi when these cells are incubated with human or animal antisera. Parasites of one strain (Y) showed aggregation of surface antigens to form an anterior or more frequently a posterior cap. The proportion of cap forming trypomastigotes increased with time and was dependent on temperature and amount of antiserum. Cap formation was inhibited by sodium azide or iodoacetamide. Crosslinking of surface antigens by antibodies causing agglutination of trypomastigotes decreased antigenic mobility and capping. Capping was not affected by treatment of the parasites with colchicine or cytochalasin B. Antigen mobility was not related to the presence of antibodies against parasite and host tissue cross-reacting antigens. Aggregation of surface membrane antigens was also observed in parasites which survive after immune lysis. Results of light and electron microscopic studies suggested that at least part of the aggregated antigens was eliminated from the trypomastigote's surface. Cap formation was strain and stage dependent. It was not observed when Y-strain epimastigotes were used and it occurred less frequently in CL than in Y-strain trypomastigotes.

A rapid sensitive assay for specific protein synthesis in cells and in cell-free translations: Use of Staphylococcus aureus as an adsorbent for immune complexes

Formalin-fixed, heat-inactivated Staphylococcus aureus Cowen Type I (SAC) (Kessler, S. W. (1975) J. Immunol. 115, 1617–1624) has been used to isolate antibody-bound tyrosine aminotransferase (EC 2.6.1.5) from radioactively labeled cell-free extracts and translation reactions of polysomes and poly(A)-containing messenger RNA. Under our conditions, SAC immunoprecipitation of cell-free extracts generated a radioactive background in control serum precipitations of 0.1%, an average of fivefold lower than backgrounds obtained by direct and indirect antibody precipitations. It is shown that the major source of the background, which limits sensitivity of the assay, comes from the amounts of antiserum and bacterial adsorbent in the assay. Procedures are presented to provide low backgrounds in more general applications of the precipitation method. From these experiments, SAC immunoprecipitation is shown to be a rapid, economical method to assay for specific protein synthesis under a variety of experimental conditions with noise levels much lower than with other standard methods.

Antibody-dependent cell-mediated antibacterial activity of human mononuclear cells. I. K lymphocytes and monocytes are effective against meningococi in cooperation with human imune sera.

In cooperation with human heat-inactivated antisera from adults immunized with group C meningococcal polysaccharide, normal human peripheral blood mononuclear cells significantly decreased the viability of group C meningococci (Mgc) in vitro. K lymphocytes (Null cells) and monocytes, (but not T or B lymphocytes) were capable of effecting antibody-dependent cell-mediated (ADC) antibacterial activity in this system. The degree to which meningococcal viability was decreased was a function of the length of the test incubation, the concentration of effector cells, and the amount of antiserum used in the assay. When specific antibodies directed against Mgc were adsorbed from the antiserum, cell-mediated antibacterial activity was abolished. ADC antibacterial activity was also abrogated by performing the assay at 4 degrees C or by heating effector cells to 46 degrees C for 15 min before the assay, Similarities between the ADC antibacterial system and previously described ADCC assays are discussed. The data suggest the K cells (as well as monocytes) may play a role in host immune defense against pathogenic bacteria.

QUANTIFICATION OF TOTAL IgM AND IgG AND SPECIFIC IgM AND IgG TO A THYMUS-INDEPENDENT (LPS) AND A THYMUS-DEPENDENT (TETANUS TOXOID) ANTIGEN IN THE RAT BY ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)

Rat IgM and IgG was determined by mechanized "sandwich" enzyme-linked immunosorbent assay (ELISA) using peroxidase labeled anti-rat-IgM and -IgG. Linear ranges in standard curves of a reference rat serum had a slope similar to the slopes found with sera of 25 rats of various age. IgM and IgG measurements by ELISA in these sera correlated well with results obtained by single radial immuno-diffusion (SRID). In addition, the precision of the enzyme immunoassay was the same as obtained with the SRID. Compared with SRID, ELISA is less time consuming and the amount of antiserum used in the macro-ELISA is one order of magnitude lower; and again 10 times lower in the mechanized micro-ELISA that is currently being developed. In conclusion, the ELISA is a specific, reliable, sensitive, and economic method for routine measurement of rat serum IgG and IgM e.g. in toxicity studies. In the second part of this study, ELISA and the passive hemagglutination test were compared to determine the primary and secondary antibody response to E. coli lipopolysaccharide (LPS) and tetanus toxoid in rats. In the ELISA, the antigens were bound to the wells of polystyrene microplates. Tetanus toxoid was coated directly, LPS after complexing with methylated bovine serum albumin. After incubation with dilutions of the rat sera, the amount of antibody bound to the solid phase was quantified by means of peroxidase-labeled antiimmunoglobulin. The specificity of the enzyme immunoassay was tested by absorption of the sera with the respective antigens. ELISA proved to be more sensitive than the hemagglution reaction, except when titers were determined during the secondary response to tetanus toxoid. Besides its specificity and sensitivity, ELISA is a convenient method for measuring both IgM and IgG antibodies. Finally, evidence is presented that in the rat, the humoral immune response to LPS is a thymus-independent phenomenon. Thus, by using the antibody response to LPS and tetanus toxoid in function studies of the immune system of the rat, insight can be obtained in the thymus-independent and thymus-dependent humoral immune response.

The production of COFAL test antiserum in hamsters

Suitable quantities of antiserum for routine COFAL testing may be readily obtained at relatively low cost by the inoculation of newborn hamsters with a chorioallantoic membrane suspension of Schmidt-Ruppin strain of avian sarcoma virus. Serum from hamsters which do not develop easily observable cutaneous tumours within 3 months may contain as much group specific complement-fixing antibody as serum from animals which do develop such tumours.

Induction of citrate synthase by aldosterone in the rat kidney

The possible induction of renal citrate synthase (E.C. 4.1.3.7) by aldosterone was evaluated in the adrenalectomized rat. Three hours after administration of aldosterone (0.8 microgram/100 g body wt), renal cortical and medullary citrate synthase activity was significantly increased as reported previously by Kinne and Kirsten (Kinne, R., Kirsten, R. 1968. Pfleugers Arch. 300:244). In contrast, no change in this activity was detected in the renal papilla or the liver, under the same conditions. Kinetic analysis revealed that injection of aldosterone had no effect on the KmS for acetyl-CoA and oxalacetate but augmented Vmax of renal medullary citrate synthase activity by 40%. The aldosterone-dependent increase in medullary citrate synthase activity was proportionate to the associated increase in the quantity of antiserum (specific for citrate synthase) required for half-maximal immuno-precipitation. The possibility that aldosterone induced the synthesis of citrate synthase was evaluated in two sets of experiments. In the first set, adrenalectomized rats were injected intraperitoneally with either aldosterone (0.8 microgram/100 g body wt) or the diluent, and simultaneously with 3H or 35S methionine (500 muCi/rat). The isotopes were reversed in about half of the experiments. Three hours after the injection, renal citrate synthase was isolated by ATP-sepharose column chromatography and immuno-precipitation with the specific antiserum. Aldosterone augmented methionine incorporation into renal citrate synthase by 55% but had no effect on incorporation into the hepatic enzyme. In the second set, adrenalectomized rats were injected with either aldosterone (0.8 microcram/100 g body wt) or the diluent, the kidneys were removed 1 hr later and medullary slices were incubated in either 3H- or 35S-methionine at 20 degrees for 2 hr. Mitochondrial citrate synthase was isolated either by ATP-sepharose column chromatography and immuno-precipitation, or by polyacrylamide gel electrophoresis. Aldosterone increased methionine incorporation into the immuno-precipitates by 30% and into the enzyme peak resolved by polyacrylamide gel electrophoresis by 43%. The latter increase was eliminated by prior administration of either actinomycin D (70--80 microgram/100 g body wt) or spirolactone (SC-26304) (80 microgram/100 g body wt). An equimolar dose of dexamethasone (0.8 microgram/100 g body wt) had no effect on the isotope ratio associated with citrate synthase activity in the polyacrylamide gels.

ROLE OF ANTISERUM AND COMPLEMENT IN THE ACUTE ANTIBODY-MEDIATED REJECTION OF MOUSE SKIN ALLOGRAFTS IN STRAIN COMBINATIONS WITH INCREASING HISTOINCOMPATIBILITY1

Acute antibody-mediated rejection (AAR) of mouse skin allografts was studied in nine donor-recipient combinations with increasing histoincompatibility ranging from an H-Y to a complete H-2 plus on-H-2 disparity. AAR was induced by the injection of specific alloantiserum along with a heterologous complement on day 7 after grafting. Sera from rabbits, guinea pigs, and from a human volunteer were used as complement sources. The recipients were treated with antilymphocyte serum on days 0, 2, and 4 to postpone cell-mediated rejection. With increasing histoincompatibility the mean survival time of untreated grafts decreased, the in vitro cytotoxic activity of the alloantiserum rose, and AAR could be induced with lower amounts of antiserum. The higher efficiency of rabbit complement compared with guinea pig complement and human complement, that is known to exist in in vitro cytotoxicity, was also found in vivo. Rabbit complement could induce AAR in combination with relatively weak histoincompatibility (H-2K, H-2D, or non H-2 differences), where guinea pig complement and human complement were ineffective. All three complement species elicited AAR if there was a disparity for H-2D plus non-H2 H-2K plus non H-2, H-2, or H-2 plus non-H2. The rules for immunogenicity of the different histocompatibility loci as they have been described for cell-mediated graft destruction also apply to this humorally mediated rejection process.

Passive immunotherapy of established tumors with syngeneic antitumor serum in combination with immunopotentiators.

The possibility of passive therapy of 6 approximately 8-day established tumors with syngeneic antitumor antiserum was tested using (1) combination of the antiserum and immunopotentiators, (2) combination of the antiserum and inflammatory agents, and (3) repeated injections of antiserum. Therapeutic effect on an ascites tumor was seen using antiserum in combination with BCG or lipopolysaccharide, or less clearly with carrageenan. Combinations of antiserum and PS-K, histamine, or croton oil did not have a synergistic therapeutic effect, but repeated injection of a small amount of antiserum did have a therapeutic effect on established peritoneal and subcutaneous tumors.

The effect of antidynein 1 serum on the movement of reactivated sea urchin sperm.

Rabbit antiserum prepared against an ATPase-containing tryptic fragment of dynein by Ogawa and Mohri (J. Biol. Chem. 250: 6476-6483) specifically inhibited the ATPase activity of dynein 1 and not that of dynein 2. Varying amounts of this antidynein 1 serum were added to demembranated sperm while they were swimming in reactivating solution containing 1 mM ATP. The sperm continued to form regularly propagated flagellar bending waves, but the beat frequency decreased gradually with time, the greater part of the change occurring in the first 15 min. The beat frequency after 1 h was a function of the amount of antiserum used, and could be as low as 1 Hz. The waveforms of the treated sperm resembled those of normal reactivated sperm except that the bend angles of both the principal and reverse bends were larger in the proximal portion of flagellum. The ATPase activity and corresponding beat frequency of sperm which had been pretreated with varying amounts of antidynein 1 serum for 15 min at 0 degrees C and then diluted were both decreased as a function of the amount of antiserum added, the ATPase activity of homogenized, nonmotile sperm also decreased upon pretreatment with antiserum, but the percentage decrease was less than for motile sperm. For moderate to low concentrations of antiserum, the rates of reaction with motile and with rigor sperm were almost identical. The overall results suggest that antidynein 1 inhibits the functioning of the dynein arms, probably by blocking the ATPase sites of the dynein 1.

Direct Radioimmunoassay for Unconjugated Estriol in Pregnancy Serum, with Use of a Radioiodinated Derivative of Estriol

We describe a rapid and direct 125I-based radioimmunoassay for quantification of unconjugated (free) estriol in pregnancy serum. Estriol in serum is adsorbed onto a small column of Sephadex, thereby allowing its separation from proteins and interfering materials. A radioiodinated derivative of estriol (6-ketoestriol-6(o-carboxymethyl)oxime-[125I]tyrosine methyl ester) is added to the column, followed by a limiting amount of antiserum. After incubation, the antibody-bound hormone is separated by a buffer wash. The assay exhibits satisfactory recovery and parallelism, and the intra- and inter-assay coefficients of variation are less than 10%. The values obtained by using the assay correlate well (r = 0.97) with those from a comparison method in which solvent extraction and chromatographic purification are used.

PLATELET RESPONSE TO IMMUNOLOGICAL INJURY

Platelets are susceptible to innnunological destruction, but the nature of the immune lesion has not been defined. Biochemistry, physiology, freeze-etching and electron microscopy were used to detect antibody induced injury. Antiserum (AS) was raised in a horse by repeated I.M. injections of glutaraldehyde fixed human platelets. Absorbtion with PPP removed non-specific activity of AS. The AS caused release of serotonin to a dilution of 1:10,000 without producing ultrastructural damage. AS stimulated aggregation in stirred C-PRP to a dilution of 1:1000. Aggregates resembled those produced by collagen. Lactic dehydrogenase was not discharged by dilutions greater than 1:10. Dilutions to 1:100 caused platelet lysis and produced characteristic membrane lesions visible in replicas of freeze-etched cells. The lesions consisted of 80A° particles in semi-circular or circular arrangements located on the inner surface of the outer leaflet, but not on the outer surface of the inside half of the split lipid bilayer. Complement was required for development of the lesions. Anti-lymphocyte serum strongly active against human platelets produced similar lesions, but antisera to rabbit platelets raised in a goat did not. The results indicate that specific, complement-dependent lesions are produced in platelet membranes by lytic concentrations of AS. Smaller amounts of AS or other anti-platelet substances may cause aggregation and release serotonin without producing specific lesions.