A simple progesterone radioimmunoassay without column chromatography

A radioimmunoassay for plasma progesterone without Chromatographie purification was developed. The 11-hemi-succinate of 11 -hydroxy-progesterone conjugated to bovine serum albumin was injected into rabbits to stimulate antibody production. The resulting antisera was used at a final dilution of 1:3500. The mean recovery of labeled progesterone added to 100 samples after ether extraction (88.9 ± 9.1%) was higher than the recovery obtained when column chromatography followed ether extraction (84.8 ± 7.5%). For comparison, plasma pools were assayed for progesterone with and without the use of columns. A female plasma pool (luteal phase) gave a mean of 546.3 ± 26.5 (SD) ng/100 mls (n = 5) without column chromatography and 557.2 ± 20.8 (SD) ng/100 mls (n = 5) with column chromatography. Another female plasma pool (follicular phase) gave a mean of 87.9 ± 9.6 (SD) ng/100 mls (n = 24) with column chromatography and 93.3 ± 8.6 (SD) ng/100 mls (n = 7) without column chromatography. A male plasma pool gave a mean of 22.8 ± 4.4 (SD) ng/100 mls (n = 13) with column chromatography and 21.8 ± 7.7 (SD) ng/100 mls (n = 3) without column chromatography. The intra assay and inter assay precision gave a coefficient of variation of 3.7 for six samples and 10.9 for 24 samples, respectively. The specificity of the antibody was determined by checking cross reactivity with 26 steroids. The sensitivity (25 pg) and accuracy were proven to be highly satisfactory.     

Assessment of DNA damage and its modulation by dietary and genetic factors in smokers using the Comet assay: a biomarker model

Methods are needed to assess exposure to genotoxins in humans and to improve understanding of dietary cancer prevention. The Comet assay was used to detect smoking-related exposures and dietary modulations in target tissues. Buccal scrapings, blood and faeces were collected from 38 healthy male volunteers (smokers and non-smokers) during a dietary intervention study with bread supplemented with prebiotics±antioxidants. GSTM1-genotype was determined with PCR. Buccal and peripheral lymphocytes were analysed for DNA damage using the Comet assay. Genotoxicity of faecal water (FW) was assayed in human colon HT29 clone 19A cells. 'Tail intensity' (TI) was used as a quantitative indicator of DNA damage in the Comet assay. Intervention with bread reduced DNA damage in lymphocytes of smokers (8.3±1.7% TI versus 10.2±4.1% TI, n=19), but not of non-smokers (8.6±2.8% TI versus 8.3±2.7% TI, n=15). Faecal water genotoxicity was reduced only in non-smokers (9.4±2.9% TI versus 18.9±13.1% TI, n=15) but not in smokers (15.5±10.7% TI versus 20.4±14.1% TI, n=13). The Comet assay was efficient in the detection of both smoking-related exposure (buccal cells) and efficacy of dietary intervention (faecal samples). Smokers and non-smokers profited differently from the intervention with prebiotic bread±antioxidants. Stratification of data by genotype enhanced specificity/sensitivity of the intervention effects and contributed important information on the role of susceptibility.     

混合单克隆抗体在固相ELISA双夹心法中的应用研究

在进行固相ELISA双夹心法时,要选择两种配对的单克隆抗体(McAb)殊非易事。本文用不同McAb的混合物与另一种McAb进行配对夹心,获得了较好的效果。实验表明,在心肌肌球蛋白轻链(CM—LC)的固相ELISA双夹心体系中,以抗CM-LCMcAb(1G6)铺底,(2B4及2F6)混合物为后续复盖抗体,最低检出量可低达10ng/mL,其检出率较单独2B4或单独2F6作为后续复盖抗体者高5—10倍。而若反之,以(2B4及2F6)混合物铺底,1G6作为后续复盖抗体,则其最低检出量竟高至200ng/mL,还不如以其中之一铺底为佳。在人绒毛膜促性腺激素(HCG)的检测体系中,用多克隆抗体与单克隆抗体配对的研究中,也获得了类似的实验结果。     

A sensitive and specific two-site, sandwich-amplified enzyme immunoassay for neuropeptide Y

The development of a new enzyme immunoassay for neuropeptide Y (NPY) is reported. Two monoclonal antibodies directed against distinct epitopes of NPY are used, one as a capture antibody (NPY02) and the other one as an indicator antibody (NPY05), this latter antibody being labeled with alkaline phosphatase. The assay calibration curve was performed over concentrations of 1 to 250 pM in a NPY-free plasma. The intra-assay coefficient of variation (CV) ranged from 0.025 to 11.9%, whereas the interassay CV was comprised between 5 and 12%. The limit of detection of this assay was 1 pM (100 amol/well). Neuropeptide Y levels are related to sampling conditions; basal concentrations of NPY with low SEM are found when less than 1.2 ml of blood is taken in EDTA tubes, the sample is centrifuged at 4°C, and immediately frozen. Unanesthetized spontaneously hypertensive rats exhibited higher NPY plasma concentrations than normotensive Wistar-Kyoto controls (53 ± 7 pM and 25 ± 2 pM, respectively, mean ± SEM, p < 0.01). Plasma NPY levels are similar in 16- and 36-week-old animals. In conclusion, this technique makes it possible to assay a large number of samples within 24 h without requiring radioactivity.     

The effects of ovariectomy and progesterone on peripheral aromatization in the female rhesus monkey

We investigated the acute effects of surgery, i.e. ovariectomy, the long-term effects of ovariectomy, and the effects of progesterone on the peripheral aromatization of androstenedione in rhesus monkeys (Macaca mulatta). For the acute effects of surgery, 7 rhesus monkeys were given a pulse of [³H]androstenedione/[¹⁴C]estrone 2 weeks before and immedately after ovariectomy. In each case all urine was collected for 4 days and analyzed for radioactivity as estrone glucuronide and the peripheral aromatization calculated from the isotope ratios. Similarly, 5 monkeys were studied before and 18 months after ovariectomy. The acute effects of surgery resulted in a significant decrease in the peripheral aromatization of androstenedione to estrone from a mean±SE of 0.94 ± 0.26 to 0.61 ± 0.19%, P = 0.0452. Conversely, the long-term effects of ovariectomy resulted in a significant increase in peripheral aromatization from 0.38 ± 0.06 to 0.67 ± 0.12%, P = 0.0207. In 7 monkeys the peripheral aromatization was measured before and 10 days after the administration of progesterone, 100 mg in oil. There was no difference in peripheral aromatization before, 0.62 ± 0.04% and after progesterone, 0.58 ± 0.05%, P = 0.10. We conclude that the acute stress of ovariectomy, or possibly the loss of ovarian aromatizing tissue, results in a decline in peripheral aromatization, but ovariectomy will have the long-term effect of an increase in aromatization, and that the presence or absence of progesterone does not play a role.     

Specific interactions of steroids, arylhydrocarbons and flavonoids with progesterone receptors from the cytosol of the fungus Rhizopus nigricans

Rhizopus nigricans (R. nigricans) transforms fungitoxic progesterone into the less toxic 11-hydroxyprogesterone which is then able to exit the mycelia into the surrounding water. Hydroxylation of progesterone is an inducible process in which cytosolic progesterone receptors could be involved. In the present study, we characterised receptors with respect to ligand specificity and to their involvement in progesterone induction of hydroxylase. EC₅₀ values of different ligands (steroids, xenobiotic arylhydrocarbons and natural flavonoids) were determined by competition studies using 40 nM (³H)progesterone. C21 and C19 3-oxo-4-ene steroids were good competitors (EC₅₀ of progesterone 2.3 ± 0.1 × 10⁻⁷ M, EC₅₀ of androsten-3,17-dione 24 ± 2 × 10⁻⁷ M). The presence of hydroxyl groups in steroids significantly decreased the affinity for receptors. The arylhydrocarbons -naphthoflavone and ketoconazole exhibited EC₅₀ values of 0.3 ± 0.01 × 10⁻⁷ M and 27 ± 5 × 10⁻⁷ M, respectively, whereas β-naphthoflavone and benzo(a)pyrene were not able to displace labelled progesterone completely. The competition curves obtained by natural flavonoids also did not reach the bottom level of non-labelled progesterone, indicating the interaction at some allosteric binding site(s) of progesterone receptors. All ligands were examined for their involvement in progesterone-hydroxylase induction. Steroid agonists induced the enzyme in a dose-dependent manner in accordance with their affinity for receptors, whereas arylhydrocarbons and natural flavonoids did not induce the enzyme. The agonistic action of steroids, together with the antagonistic action of -naphthoflavone, strongly suggests the involvement of progesterone receptors in progesterone signalling resulting in the induction of progesterone-hydroxylase.     

Employment of a turbidimetric assay system to measure heat-induced protein aggregation

1. 1.|We developed a turbidimetric assay system for quantitation of heat-induced protein aggregation which is presumably caused by protein denaturation.

2. 2.|Rhodanese in 6 M guanidinium chloride was employed in the assay system, because this protein recognizes hydrophobic sites on denatured proteins and aggregates.

3. 3.|Turbidity caused by protein-rhodanase aggregation was recorded at 320 nm by using a u.v./VIS spectrophotometer.

4. 4.|When heated, alcohol dehydrogenase (ADH) aggregates with rhodanese. The increase of ADH-rhodanese aggregation was correlated with the loss of enzymatic activity.

5. 5.|These results indicated that the aggregation was proportional to the extent of ADH denaturation which assumingly caused the loss of ADH activity during heating at 45.5°C.

6. 6.|Similar results were observed when cytosolic proteins from CHO cells were heated at 45.5°C. Heated cytosolic proteins promoted aggregation by complex formation with rhodanese. The aggregation increased with increasing heat dose.

7. 7.|Therefore, the rhodanese assay system can be employed usefully to quantitate the protein aggregation after heat stress.

一种快速检测甲型流感病毒的方法

目的 开发一种快速、简便的基于胶体金免疫层析法（GICA）的试剂盒,以用于对甲型流感病毒的检测。方法以柠檬酸三钠还原法制备胶体金颗粒,标记抗甲型流感病毒内部抗原的单克隆抗体。硝酸纤维素膜上包被两种抗甲型流感病毒单克隆抗体的混合液,制成免疫层析试纸。待测样品中的甲型流感病毒首先与胶体金标记抗体结合,后移动至硝酸纤维素上与固定的单克隆抗体发生反应,形成肉眼可见的红色带。结果GICA试纸条与甲1型和甲3型流感病毒共16种毒株均能发生特异性反应,与乙型流感病毒、副流感病毒、腺病毒和呼吸道合胞病毒无交叉反应。用三种不同甲型流感病毒毒株的不同浓度标本与美国同类经过FDA批准的产品比较,灵敏度相同。结论GICA试纸条灵敏度能够达到临床使用的要求,并具有简便快速、无需特殊仪器设备等优点,对甲型流感的诊断和流行病学调查具有十分重要的应用价值。     

Bioluminescent enzyme immunoassay for progesterone using monoclonal antibodies and glucose-6-phosphate dehydrogenase labels

Bacterial luciferase, NAD(P): FMN oxidoreductase and anti-mouse immunoglobulin were co-immobilized on Sepharose 4B. This reagent together with a progesterone glucose-6-phosphate dehydrogenase conjugate and various anti-progesterone monoclonal antibodies was used to develop a non-separation bioluminescent immunoassay for progesterone. This monoclonal antibody based assay was sensitive and reliable and using the tracer progesterone-11-acetate-glucose-6-phosphate dehydrogenase, the majority of the monoclonal antibodies give a better sensitivity with this enzymatic tracer than that obtained with an iodinated tracer. In a second assay design progesterone-glutathione was co-immobilized with bacterial luciferase and NAD(P): FMN oxidoreductase on Sepharose 4B and three monoclonal antibodies were labelled with glucose-6-phosphate dehydrogenase. With aqueous progester-one standards, this assay gave comparable sensitivity to the bioluminescent enzyme immunoassay using the second antibody immunoadsorbant and to an RIA but was unsuitable for plasma samples.     

Cy5 labeled antimicrobial peptides for enhanced detection of Escherichia coli O157:H7

Fluorescently labeled antimicrobial peptides were evaluated as a potential replacement of labeled antibodies in a sandwich assay for the detection of Escherichia coli O157:H7. Antimicrobial peptides naturally bind to the lipopolysaccharide component of bacterial cell walls as part of their mode of action. Because of their small size relative to antibodies peptides can bind to cell surfaces with greater density, thereby increasing the optical signal and improving sensitivity. This method combines the specificity of a capture antibody with the increased sensitivity provided by using a labeled peptide as a detection molecule. The antimicrobial peptides cecropin P1, SMAP29, and PGQ were labeled with the fluorescent dye Cy5 via maleimide linker chemistry. Preliminary screening using a whole-cell solution binding assay revealed that Cy5 cecropin P1 enhanced the detection of E. coli O157:H7 relative to a Cy5 labeled anti-E. coli O157:H7 antibody 10-fold. Detection sensitivity of antibody and peptide were also compared with a prototype immuno-magnetic bead biosensor. Detection using Cy5 cecropin P1 resulted in a 10-fold improvement in sensitivity. Correlation of peptide antimicrobial activity with detection of E. coli O157:H7 indicated that activity was not predictive of the sensitivity of the fluorescent assay.     

Flow cytometric analysis of human breast tumors and assessment of in vitro chemosensitivity by clonogenic assays

We report a double-agar clonogenic system adapted to human breast cancer. We optimized the conditions for cell growth and clonogenicity with respect to hormones (insulin, estradiol, progesterone) and components of the extracellular matrix (collagen, laminin and fibronectin). Using our experimental improvements, 67% of the breast tumor samples received were grown successfully. Tests on 21 tumors with three agents: Doxorubicin, Methotrexate and 5-Fluorouracil permit objective discrimination of the in vitro pharmacosensitivity of human breast tumors. Flow cytometric analysis reveal that 64% of the tumors were diploid and 36% were aneuploid. The aneuploid tumors grew better in the double agar layer system used for the clonogenic assay. The diploid tumors were especially rich in estrogen (ER⁺) and progesterone (PR⁺) receptors whereas the aneuploid tumors were mostly estrogen and progesterone receptors negative (ER^–/PR^–). Finally, we noted no difference in drug responsiveness depending on the tumor ploidy and steroid receptor content.Abbreviations DCC dextran coated charcoal - DI DNA index - DXB Doxorubicin - ECM extracellular matrix component - ER estrogen receptors - FCM flow cytometry - 5-FU 5-Fluorouracil - HTSCA human tumor stem cell assay - MTX Methotrexate - PBC primary breast carcinoma - PI proliferative index - PR progesterone receptors - SPF S phase fraction     

Monoclonal antibody-based enzyme linked immunosorbent assay for the analysis of jasmonates in plants

Methyl jasmonate (MeJA) and its free-acid form,jasmonic acid (JA) are naturally occurring plant growth regulators widely distributed in higher plants.In order to improve the sensitivity for the analysis of MeJA at low levels in small amounts of plant samples,a monoclonal antibody (MAb) (designated as MAb 3E5D7C4B6) against MeJA was derived from a JAbovine serum albumin (BSA) conjugate as an immunogen.The antibody belongs to the IgG1 subclass with a κ type light chain and has a dissociation constant of approximately 6.07 x 10-9 M.MAb3E5D7C4B6 is very specific to MeJA.It was used to develop a direct competitive enzyme-linked immunosorbent assay (dcELISA),conventional and simplified indirect competitive ELISAs (icELISA).JA was derivatized into MeJA for the ELISA analysis.The IC50 value and detection range for MeJA were,respectively,34 and 4-257 ng/mL by the conventional icELISA,21 and 3-226 ng/mL by the simplified icELISA and 5.0 and 0.7-97.0 ng/mL by the dcELISA.The dcELISA was more sensitive than either the conventional or simplified icELISA.The assays were used to measure the content of jasmonates as MeJA in tobacco leaves under drought stress or inoculated with tobacco mosaic virus and tomato leaves inoculated with tomato mosaic virus or Lirioinyza sativae Blanchard as compared with the corresponding healthy leaves.The increased jasmonates content indicated its role in response to the drought stress and pathogens.     

Oligoclonal Enzyme-linked Immunosorbent Assay Capable of Determining the Major Food Allergen, Ovomucoid, Irrespective of the Degree of Heat Denaturation

We have recently established an enzyme-linked immunosorbent assay (ELISA) for total ovomucoid determination, irrespective of the degree of its heat denaturation, by using a monoclonal antibody (mAb) 7D specific to the carbohydrate moiety of ovomucoid (Biosci Biothechnol Biochem, 68, 2490–2497, 2004). Two novel methods have been developed to improve the ELISA. First, its sensitivity was enhanced 100 times by using an oligoclonal cocktail of mAb 7D and two other mAbs with different epitopes as a second antibody. Second, it was shown that usage of denaturing reagents such as SDS and β-mercaptoethanol for extraction was acceptable for ELISA within a range of stability of a first antibody on a solid phase. Properties of the oligoclonal sandwich ELISA system thus constructed were discussed in connection with allergen labeling.     

An improved ELISA method for the detection of Salmonella typhimurium

The applicability of enzyme-linked immunosorbent assay (ELISA) for the detection of salmonellas in foodstuffs was investigated. Several factors affecting the sensitivity of the ELISA, such as the type of protein used for plate post-coating, the method of antibody labelling, and accelerators for antigen-antibody and enzyme-substrate reactions, were studied. Labelling of the antibody with horseradish peroxidase and the use of o -phenylenediamine as substrate in the detection system were demonstrated to be most suitable for the enzyme assay.
Based on these findings, an improved ELISA method was developed for the detection of Salmonella typhimurium. The improved technique was able to detect as few as 5×10⁴-10⁵ cell/ml of salmonellas, and about 24 h were required to enrich the bacteria in food samples and to perform the test. With some modifications, the ELISA assay could reach a very high level of sensitivity and provide excellent repro-ducibility.     

Maternal serum progesterone concentration during pregnancy and lamb birth weight at parturition in Javanese Thin-Tail ewes with different litter sizes

Pregnant ewes, (N=38), with similar body weight and age (19, 13 and 6 carrying 1, 2 and 3 fetuses, respectively) were used to study the relationship between maternal serum progesterone concentration during pregnancy and lamb birth weight at parturition. Average maternal serum progesterone concentration in the ewes carrying 1, 2, and 3 fetuses was: 5.3±0.3, 6.2±0.7 and 6.6±0.5 ng/ml, during weeks 0 to 7 of gestation; 16.9±1.4, 25.3±1.5, and 26.8±2.5 ng/ml, during weeks 8 to 20; and 13.2±1.0, 18.7±1.0, and 19.8±1.7 ng/ml, during the entire gestation period respectively. Total lamb birth weight in ewes carrying 1, 2, and 3 fetuses was 1.9±0.1, 3.2±0.2, and 4.2±0.4 kg, respectively. In the respective litter sizes, ewes with higher mean serum progesterone concentration during the whole pregnancy gave birth to lambs with higher birth weight (r²=0.76, 0.42 and 0.46, for ewes carrying 1, 2 and 3 fetuses, respectively). The results of the study indicated that prenatal growth could probably be improved by increasing endogenous secretion of maternal progesterone during pregnancy.     

单克隆抗体与抗血清联合应用建立血清CMLC测定方法的研究

单克隆抗体(McAb)和抗血清各有特点。本文提纯人心肌肌球蛋白轻链(CMLC)并制备其单克隆抗体和抗CMLC兔血清(下称抗血清),试建立测定血清CMLC的酶联免疫(ELISA)方法。通过比较,McAb和抗血清联合应用可提高测定方法的灵敏度和特异性;并对各反应试剂的工作浓度进行确定,建立了McAb(1C_(11)-D_7株)为铺底抗体,抗血清为覆盖抗体,碱性磷酸酶标记的羊抗兔IgG为第三抗体的三抗体酶联免疫夹心测定血清CMLC的方法(MP-ELISA)。血清CMLC最小可测浓度为2.5ng/mL。     

A direct antigen heterologous enzyme immunoassay for measuring progesterone in serum without using displacer

An antigen heterologous enzyme-linked immunosorbent assay (ELISA) for directly measuring progesterone in serum is described. Six combinations of antigens and enzyme conjugates were tested; the enzyme conjugate 17-alphaOH-progesterone-3-O-carboxymethyloxime-alkalinephosphatase (17-alphaOH-P-3-CMO-ALP) and the immunogen progesterone-3-carboxymethyloxime-bovine serum albumin (P-3-CMO-BSA) were found to be best. Fifty microliters of standard or serum sample and 100 microL of the 17-alphaOH-P-3-CMO-ALP enzyme conjugate were added to the antibody coated wells, and incubated for 1 h at 37 degrees C. Bound enzyme activity was measured by using p-nitrophenyl phosphate as substrate. The sensitivity of the assay was 0.11 ng/mL, and intra- and inter-assay CVs ranged from 5.1% to 9.6%. The analytical recoveries were 97-105%. The serum progesterone values obtained by this method correlated well with those obtained by radioimmunoassay; r=0.97 (n=44). Moreover, in this ELISA no displacing agent was used or special means was required to displace progesterone from corticosteroid binding globulin (CBG). Serum progesterone concentrations of subjects, with histories of recurrent spontaneous abortions were also measured, and correlated well with clinical history.     

The measurement of progesterone in serum by a non-competitive idiometric assay

A novel non-competitive idiometric time-resolved fluoroimmunoassay for the determination of serum progesterone was developed, based on the use of two types of anti-idiotypic antibody that recognize different epitopes within the hypervariable region of the primary antiprogesterone antibody. The first anti-idiotype, the betatype, competes with progesterone for an epitope of the primary antiprogesterone antibody at the binding site. The second anti-idiotype, the alphatype, binds to the antiprogesterone antibody in the presence of progesterone, but does not bind to the betatype antiprogesterone complex due to epitope proximity. In the present configuration, the biotinylated alphatype was captured onto anti-biotin IgG which was immobilized on microtiter wells. Reaction mixtures containing europium-labeled antiprogesterone antibody complexed sequentially with progesterone in standards or serum samples and with the betatype anti-idiotypic antibody were then reacted with the immobilized alphatype anti-idiotypic antibody. After 30 min of incubation, the fluorescence of europium is measured by time-resolved fluorescence and is proportional to the concentration of progesterone over the range 0–320 nmol/mL. The method demonstrates good sensitivity, precision, and comparability with a direct competitive radioimmunoassay. The idiometric assay for progesterone is suitable for dipstick technology and biosensors.     

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.     

Development of an enzyme‐linked immunosorbent assay (ELISA) for Luteinizing Hormone (LH) in the elephant (Loxodonta africana and Elephas maximus)

A simple, rapid enzyme‐linked immunosorbent assay (ELISA) for the measurement of LH in plasma and serum of elephants (Loxodonta africana and Elephas maximus) has been developed, validated, and used for comparative studies. Purified elephant LH (eleLH) diluted in elephant plasma was used as standards (0.78–50 ng/ml). A monoclonal antibody against the β‐subunit of bovine LH (518B₇) was used as the capture antibody. The second antibody (a polyclonal rabbit anti‐human LH antibody), conjugated to horseradish peroxidase, cleaved a substrate (tetramethyl benzidine), resulting in a color change. The total assay time was approximately 2½ hr, with incubations at room temperature. Sensitivity was found to be 1.56 ng/ml. Cross‐reactivities to elephant FSH and TSH were low: 0.9% and 0.15%, respectively. The accuracy of the assay was demonstrated by comparing the ELISA with a validated eleLH radioimmunoassay (RIA), progesterone data, and ultrasound observations. Blood samples from 18 Asian and African elephant cows were analyzed with the ELISA and RIA, and an additional 11 cows were used to describe endocrine parameters for LH and progesterone using only RIA. No difference was found in LH peak concentrations between the ELISA and RIA. The time from the progesterone decline to the first LH peak, and the time between the two peaks were similar between species. Asian cows had higher LH peaks than African cows. Ultrasound confirmed the time of ovulation occurring with the second LH peak. Three cows were inseminated and confirmed to be pregnant using this ELISA as a timing device. Instrumentation is not always required, as LH peaks approximating 3 ng/ml can be visually observed. In conclusion, this ELISA can be used as a field test to determine time of ovulation for artificial insemination (AI) or natural breeding of both species of elephants, and thus is an important tool for the preservation of captive populations worldwide. Zoo Biol 23:65–78, 2004. © 2004 Wiley‐Liss, Inc.